Nuclear Ribosomal ITS Sequences Research Articles

Molecular phylogenetics and classification of Euphorbia subgenus Chamaesyce (Euphorbiaceae)

AbstractEuphorbia subg. Chamaesyce contains around 600 species and includes the largest New World radiation within the Old World–centered genus Euphorbia. It is one of the few plant lineages to include members with C3, C4 and CAM photosynthesis, showing multiple adaptations to warm and dry habitats. The subgenus includes North American–centered groups that were previously treated at various taxonomic ranks under the names of “Agaloma,,, “Poinsettia”, and “Chamaesyce”. Here we provide a well–resolved phylogeny of Euphorbia subg. Chamaesyce using nuclear ribosomal ITS and chloroplast ndhF sequences, with substantially increased taxon sampling compared to previous studies. Based on the phylogeny, we discuss the Old World origin of the subgenus, the evolution of cyathial morphology and growth forms, and then provide a formal sectional classification, with descriptions and species lists for each section or subsection we recognize.

Stipitate stereoid basidiocarps have evolved multiple times

Stipitate stereoid fungi are Basidiomycetes with a stipe, a spathulate-to funnel-shaped pileus, a smooth hymenophore, and hyaline, smooth spores. Representatives of the genera Cotylidia, Cymatoderma, Muscinupta, Podoscypha and Stereopsis were subjected to molecular phylogenetic analyses based on nuclear ribosomal large subunit, 5.8S and ITS sequences. For four of the genera the type species was included in analyses. Stereopsis radicans, the type species of Stereopsis, forms a lineage with the corticioid species Clavulicium globosum but could not be placed in any of the presently accepted orders within Agaricomycotina. Stereopsis vitellina falls within the Atheliales, making it the first pileus- and stipe-forming fungus recovered in this order. Cotylidia and Muscinupta again are shown to be members of the Hymenochaetales, whereas Cymatoderma and Podoscypha belong in the Polyporales. Cymatoderma is polyphyletic and Cymatoderma sensu stricto is separated from other stipitate stereoid fungi in the Polyporales, whereas the remaining Cymatoderma species are nested within a well supported clade holding all Podoscypha species but also Abortiporus biennis.

The correct phylogenetic position of Lotus conimbricensis Brot. (Leguminosae, Loteae) based on nuclear ribosomal ITS sequences

The correct phylogenetic position of Lotus conimbricensis Brot. (Leguminosae, Loteae) based on nuclear ribosomal ITS sequences The nrDNA ITS sequence determined in Lotus conimbricensis in a previous phylogenetic study was unusual, in that it was almost identical to those retrieved from the morphologically distinct species L. subbiflorus. In the present study we sequenced new specimens of both species to reassess the phylogenetic position of L. conimbricensis. We conclude that the ITS sequence of L. conimbricensis used in the earlier analyses was most likely erroneous, and in fact L. conimbricensis is not closely related to L. subbiflorus. Critical reexamination of previously published data indicates that several other similar errors may exist for other Lotus species, and these should be checked before taxonomic conclusions are made.

Origins and close relatives of a semi‐domesticated neotropical fruit tree: Chrysophyllum cainito (Sapotaceae)

Understanding patterns and processes associated with domestication has implications for crop development and agricultural biodiversity conservation. Semi-domesticated crops provide excellent opportunities to examine the interplay of natural and anthropogenic influences on plant evolution. The domestication process has not been thoroughly examined in many tropical perennial crop species. Chrysophyllum cainito (Sapotaceae), the star apple or caimito, is a semi-domesticated species widely cultivated for its edible fruits. It is known to be native to the neotropics, but the precise geographic origins of wild and cultivated forms are unresolved. We used nuclear ribosomal ITS sequences to infer phylogenetic relationships among C. cainito and close relatives (section Chrysophyllum). We employed phylogeographic approaches using ITS and plastid sequence data to determine geographic origins and center(s) of domestication of caimito. ITS data suggest a close relationship between C. cainito and C. argenteum. Plastid haplotype networks reveal several haplotypes unique to individual taxa but fail to resolve distinct lineages for either C. cainito or C. argenteum. Caimito populations from northern Mesoamerica and the Antilles exhibit a subset of the genetic diversity found in southern Mesoamerica. In Panama, cultivated caimito retains high levels of the diversity seen in wild populations. Chrysophyllum cainito is most closely related to a clade containing Central and South American C. argenteum, including subsp. panamense. We hypothesize that caimito is native to southern Mesoamerica and was domesticated from multiple wild populations in Panama. Subsequent migration into northern Mesoamerica and the Antilles was mediated by human cultivation.

Cytogeography of the Humifusa clade of Opuntia s.s. Mill. 1754 (Cactaceae, Opuntioideae, Opuntieae): correlations with pleistocene refugia and morphological traits in a polyploid complex.

Ploidy has been well studied and used extensively in the genus Opuntia to determine species boundaries, detect evidence of hybridization, and infer evolutionary patterns. We carried out chromosome counts for all members of the Humifusa clade to ascertain whether geographic patterns are associated with differences in ploidy. We then related chromosomal data to observed morphological variability, polyploid formation, and consequently the evolutionary history of the clade. We counted chromosomes of 277 individuals from throughout the ranges of taxa included within the Humifusa clade, with emphasis placed on the widely distributed species, Opuntia humifusa (Raf.) Raf., 1820 s.l. and Opuntia macrorhiza Engelm., 1850 s.l. We also compiled previous counts made for species in the clade along with our new counts to plot geographic distributions of the polyploid and diploid taxa. A phylogeny using nuclear ribosomal ITS sequence data was reconstructed to determine whether ploidal variation is consistent with cladogenesis. We discovered that diploids of the Humifusa clade are restricted to the southeastern United States (U.S.), eastern Texas, and southeastern New Mexico. Polyploid members of the clade, however, are much more widely distributed, occurring as far north as the upper midwestern U.S. (e.g., Michigan, Minnesota, Wisconsin). Morphological differentiation, although sometimes cryptic, is commonly observed among diploid and polyploid cytotypes, and such morphological distinctions may be useful in diagnosing possible cryptic species. Certain polyploid populations of Opuntia humifusa s.l. and Opuntia macrorhiza s.l., however, exhibit introgressive morphological characters, complicating species delineations. Phylogenetically, the Humifusa clade forms two subclades that are distributed, respectively, in the southeastern U.S. (including all southeastern U.S. diploids, polyploid Opuntia abjecta Small, 1923, and polyploid Opuntia pusilla (Haw.) Haw., 1812) and the southwestern U.S. (including all southwestern U.S. diploids and polyploids). In addition, tetraploid Opuntia humifusa s.l., which occurs primarily in the eastern U.S., is resolved in the southwestern diploid clade instead of with the southeastern diploid clade that includes diploid Opuntia humifusa s.l. Our results not only provide evidence for the polyphyletic nature of Opuntia humifusa and Opuntia macrorhiza, suggesting that each of these represents more than one species, but also demonstrate the high frequency of polyploidy in the Humifusa clade and the major role that genome duplication has played in the diversification of this lineage of Opuntia s.s. Our data also suggest that the southeastern and southwestern U.S. may represent glacial refugia for diploid members of this clade and that the clade as a whole should be considered a mature polyploid species complex. Widespread polyploids are likely derivatives of secondary contact among southeastern and southwestern diploid taxa as a result of the expansion and contraction of suitable habitat during the Pleistocene following glacial and interglacial events.

Expanded phylogenetic and dating analyses of the apples and their relatives (Pyreae, Rosaceae)

Despite previous efforts to elucidate relationships within the Pyreae (Rosaceae), relationships among the major sub-lineages, generic limits, and divergence times have remained uncertain. The present study greatly expands phylogenetic analyses of the Pyreae by using a combination of 11 chloroplast regions plus nuclear ribosomal ITS sequences from 486 individuals representing 331 species and 27 genera. Maximum likelihood and Bayesian analyses generally support existing generic boundary, although Sorbus, as previously circumscribed, is clearly non-monophyletic. Two significant conflicts were detected between the chloroplast and ITS phylogenies, suggesting that hybridization played a role in the origins of Micromeles and Pseudocydonia. In addition, we provide estimates of the divergence times of the major lineages. Our findings support the view that the major Pyreae lineages were established during the Eocene–Oligocene period, but that most of the modern diversity did not originate until the Miocene. At least five major, early Old World-New World disjunctions were detected and these vicariance events are generally most consistent with movement through the Beringia.

Molecular Systematics of Polygonum minus Huds. Based on ITS Sequences

Plastid trnL-trnF and nuclear ribosomal ITS sequences were obtained from selected wild-type individuals of Polygonum minus Huds. in Peninsular Malaysia. The 380 bp trnL-trnF sequences of the Polygonum minus accessions were identical. Therefore, the trnL-trnF failed to distinguish between the Polygonum minus accessions. However, the divergence of ITS sequences (650 bp) among the Polygonum minus accessions was 1%, indicating that these accessions could be distinguished by the ITS sequences. A phylogenetic relationship based on the ITS sequences was inferred using neighbor-joining, maximum parsimony and Bayesian inference. All of the tree topologies indicated that Polygonum minus from Peninsular Malaysia is unique and different from the synonymous Persicaria minor (Huds.) Opiz and Polygonum kawagoeanum Makino.

Taxonomic position of Pedicularis hallaisanensis Hurusawa, an endemic plant of Mt. Halla

Pedicularis growing at Mt. Halla of Jeju Island is known as an endemic species of P. hallaisanensis Hurusawa. On the other hand, the plant is morphologically similar to P. amoena, P. spicata, and P. verticillata in gross morphology, so the taxonomic treatment of the taxon remains controversial. To clarify the taxonomic position of the plants, we examined external morphological characters and nuclear ribosomal DNA ITS sequences for P. hallaisanensis and its related species. The plants of Mt. Halla are clearly different from P. amoena and P. verticillata in the morphology of calyx lobes, the length of galea and lower lip, density of glandular hairs on plants, presences of the radical leaves after anthesis and molecular data. However, P. hallaisanensis is not clearly separated from P. spicata distributed in N. E. Asia on external morphological characters and DNA sequences of internal transcribed spacers. In this study, the morphological and molecular data suggested that P. hallaisanensis should be merged into the former species.

Identification of traditional medicine "Fructus Arctii" by nuclear ribosomal DNA ITS sequences

To identify the traditional medicine Fructus Arctii from its adulterants by ITS. Twenty-six samples of the different Fructus Arctii materials and 10 samples of the adulterants of the fruits of A. tomentosum, Onopordum acantium, Silybum marianum, and Amorpha fruticosa were collected. The total DNA of the samples were extracted, amplified cloned and sequenced. ITS sequences were obtained from 26 samples respectively, there were Fructus Arctii 641 bp, A. tomentosum 641 bp, Onopordum acantium 639 bp, Silybum marianum 630-631 bp, Amorpha fruticosa 625-626 bp, which were registered in the GenBank. The similarity in ITS sequences between Fructus Arctii and the adulterants was less than 95%. In contrast, the similarity between any pair of Fructus Arctii was greater than 99%. The similarity was 98.29%-99.22% between Fructus Arctii and A. tomentosum. Phylogeny tree reconstruction using UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Fructus Arctii from adulterants. ITS sequences can be used as a reliable molecular marker for the identification of Fructus Arctii.

Corrigendum to: Phylogenetics of hopbushes and pepperflowers (Dodonaea, Diplopeltis - Sapindaceae), based on nuclear ribosomal ITS and partial ETS sequences incorporating secondary-structure models

Hopbushes and pepperflowers (Dodonaea, Diplopeltis ? Sapindaceae) are important components of Australia's arid zone and sclerophyll and temperate forests and woodlands. Phylogenetic analyses of nuclear ribosomal ITS and partial ETS sequences for near-complete sampling of both genera were performed using a Bayesian statistical method and RNA specific models of nucleotide evolution that incorporate secondary structure (separate models for stems and loops). Diplopeltis is paraphyletic. Diplopeltis stuartii is not closer to other species of the genus than it is to species outside the genus. There are also several evolutionary elements in the molecular data that support D. stuartii as distinct from the other members of the genus. The monophyly of Dodonaea as redefined here to include all species of Distichostemon is unequivocally supported by the molecular data and the morphological synapomorphies of petal-less flowers with a highly reduced intrastaminal disk that is absent in staminate flowers. There do not appear to be any obvious evolutionary trends in the morphological characters (leaf and capsule form, presence or absence of aril, or breeding system) that have been previously used to group taxa. However, there are some morphological characters that may be useful to delineate some of the clades recovered in the present molecular study. New combinations in Dodonaea are made for all species of Distichostemon.

<strong><em>Campylopus</em> Brid. (Leucobryaceae) in Macaronesia revisited</strong>

Based on nuclear ribosomal ITS sequence analysis, biogeographic affinities of Campylopus in Macaronesia are inferred with a focus on the Madeira and Azores archipelagos. Molecular phylogenetic reconstructions reveal a complex pattern of biogeographic connections with regions on both sides of the Atlantic Ocean. Old World affinities predominate in C. pilifer, with only one out of the 30 sequenced Macaronesian samples belonging to the New World C. pilifer clade. New World affinities predominate in C. fragilis. For C. flexuosus connections with Europe, Asia and/or the Neotropics are indicated. Specimens of C. subulatus from Madeira are part of a clade of Neo- and Paleotropical species. Campylopus cygneus and C. shawii are resolved as closely related, but inference of their putative Caribbean origin will need further sampling. In C. introflexus the geographic origin of the Macaronesian populations cannot be inferred due to the low intraspecific sequence divergence. Campylopus pilifer var. brevirameus is reported for the first time for the Azores, C. cygneus for the first time for Madeira. An updated distribution of Campylopus in Macaronesia is presented, which includes 11 species.

Molecular phylogeny and systematics of the banana family (Musaceae) inferred from multiple nuclear and chloroplast DNA fragments, with a special reference to the genus Musa

Musaceae is a small paleotropical family. Three genera have been recognised within this family although the generic delimitations remain controversial. Most species of the family (around 65 species) have been placed under the genus Musa and its infrageneric classification has long been disputed. In this study, we obtained nuclear ribosomal ITS and chloroplast (atpB-rbcL, rps16, and trnL-F) DNA sequences of 36 species (42 accessions of ingroups representing three genera) together with 10 accessions of ingroups retrieved from GenBank database and 4 accessions of outgroups, to construct the phylogeny of the family, with a special reference to the infrageneric classification of the genus Musa. Our phylogenetic analyses elaborated previous results in supporting the monophyly of the family and suggested that Musella and Ensete may be congeneric or at least closely related, but refuted the previous infrageneric classification of Musa. None of the five sections of Musa previously defined based on morphology was recovered as monophyletic group in the molecular phylogeny. Two infrageneric clades were identified, which corresponded well to the basic chromosome numbers of x=11 and 10/9/7, respectively: the former clade comprises species from the sections Musa and Rhodochlamys while the latter contains sections of Callimusa, Australimusa, and Ingentimusa.

Endophytic fungus Cladosporium cladosporioides LF70 from Huperzia serrata produces Huperzine A

A strain LF70 endophytic fungus was isolated from the leaves of Huperzia serrata. The fungus was identified as Cladosporium cladosporioides LF70 according to its morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. The strain could produce Huperzine A (HupA) identified through thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with authentic HupA. The amount of HupA produced by this endophytic fungus was quantified to be 56.84 μg/L by HPLC, which was higher than that of other reported endophytic fungi, Acremonium sp., Blastomyces sp., and Botrytis sp. Acetylcholinesterase inhibition activity of HupA produced by strain LF70 was also similar to authentic HupA in vitro. Isolation of such a fungus may provide a promising alternative approach to producing HupA, which is used in treating Alzheimer’s disease and preventing further memory degeneration.

Phylogenetics of hopbushes and pepperflowers (Dodonaea, Diplopeltis - Sapindaceae), based on nuclear ribosomal ITS and partial ETS sequences incorporating secondary-structure models

Hopbushes and pepperflowers (Dodonaea, Diplopeltis – Sapindaceae) are important components of Australia’s arid zone and sclerophyll and temperate forests and woodlands. Phylogenetic analyses of nuclear ribosomal ITS and partial ETS sequences for near-complete sampling of both genera were performed using a Bayesian statistical method and RNA specific models of nucleotide evolution that incorporate secondary structure (separate models for stems and loops). Diplopeltis is paraphyletic. Diplopeltis stuartii is not closer to other species of the genus than it is to species outside the genus. There are also several evolutionary elements in the molecular data that support D. stuartii as distinct from the other members of the genus. The monophyly of Dodonaea as redefined here to include all species of Distichostemon is unequivocally supported by the molecular data and the morphological synapomorphies of petal-less flowers with a highly reduced intrastaminal disk that is absent in staminate flowers. There do not appear to be any obvious evolutionary trends in the morphological characters (leaf and capsule form, presence or absence of aril, or breeding system) that have been previously used to group taxa. However, there are some morphological characters that may be useful to delineate some of the clades recovered in the present molecular study. New combinations in Dodonaea are made for all species of Distichostemon.

The use and limits of ITS data in the analysis of intraspecific variation in Passiflora L. (Passifloraceae)

The discovery and characterization of informative intraspecific genetic markers is fundamental for evolutionary and conservation genetics studies. Here, we used nuclear ribosomal ITS sequences to access intraspecific genetic diversity in 23 species of the genus Passiflora L. Some degree of variation was detected in 21 of these. The Passiflora and Decaloba (DC.) Rchb. subgenera showed significant differences in the sizes of the two ITS regions and in GC content, which can be related to reproductive characteristics of species in these subgenera. Furthermore, clear geographical patterns in the spatial distribution of sequence types were identified in six species. The results indicate that ITS may be a useful tool for the evaluation of intraspecific genetic variation in Passiflora.

Molecular phylogeny of Chrysanthemum, Ajania and its allies (Anthemideae, Asteraceae) as inferred from nuclear ribosomal ITS and chloroplast trnL-F IGS sequences

To better understand the evolutionary history, intergeneric relationships and circumscription of Chrysanthemum and Ajania and the taxonomic position of some small Asian genera (Anthemideae, Asteraceae), the sequences of the nuclear ribosomal internal transcribed spacer (nrDNA ITS) and the chloroplast trnL-F intergenic spacer (cpDNA IGS) were newly obtained for 48 taxa and combined with those already deposited in GenBank. Phylogenies with an emphasis on Chrysanthemum, Ajania and its allies, by both maximum parsimony and Bayesian analysis, were constructed using either the ITS sequence alone, the IGS sequence alone or combined sequences. The IGS sequence was low phylogenetically informative, but some deletions and insertions were informative for interspecific and intergeneric delimitations. The ITS and the ITS/IGS phylogenies both suggested the presence of two major clades. The monophyly of subtribe Artemisiinae (clade A) could be retrieved when the phylogenetic positions of some ambiguous taxa were renewedly considered. Subtribe Artemisiinae was chiefly divided into two groups, (1) one corresponding to Chrysanthemum, Arctanthemum, Ajania, Opisthopappus and Elachanthemum (the Chrysanthemum group), (2) another to Artemisia, Crossostephium, Neopallasia and Sphaeromeria (the Artemisia group). Within the Chrysanthemum group, ChrysanthemumArctanthemum and Ajania were closely related to each other, and the generic circumscription was ambiguous; Phaeostigma was excluded from this group that was also confirmed by the 6-bp insertion in the IGS sequence; radiate or rare discoid Brachanthemum was excluded, and discoid Elachanthemum without ray florets was added to this group; at the same time, Opisthopappus in subtribe Tanacetinae should be transferred to subtribe Artemisiinae and became one of the components of the Chrysanthemum group. Based on the molecular phylogenetic framework, the evolution of pollen and capitulum characters was inferred.

Micromorphology and Character Evolution of Nutlets in Tribe Mentheae (Nepetoideae, Lamiaceae)

Mentheae is an economically important tribe in Lamiaceae with many ornamentals and culinary herbs. Although its monophyly is well supported by molecular data, the subtribal delimitation and the intergeneric relationships are still poorly understood. The nutlet morphology (SEM) of the tribe and the presence/absence of myxocarpy is documented here for 57 out of 65 genera in order to assess the systematic value of nutlet characters and to evaluate the existing molecular phylogenies for this group. Significant variation was found in nutlet shape, morphology of the abscission scar, distribution of trichomes, and surface sculpture. Five different surface types were recognized in Mentheae based on differences in cell outline, anticlinal walls, cell boundary relief, and curvature of the outer periclinal wall. Representative nutlet features proved to be phylogenetically informative in Mentheae mainly at the generic level. Cladistic analyses were performed based on 15 morphological nutlet characters and/or available molecular data (nuclear ribosomal ITS and chloroplast trnL-trnF spacer sequences). The resulting topologies indicate that the addition of nutlet characters to the DNA-data matrix improved the overall resolution and also increased bootstrap support values in comparison with exclusively molecular phylogenetic analyses. The evolution of major nutlet characters within the tribe Mentheae is discussed.

Nuclear and chloroplast DNA variation in Cephalaria squamiflora (Dipsacaceae), a disjunct Mediterranean species

AbstractCephalaria squamiflora is a chamaephyte restricted to rupicolous habitats in islands of the Western (Balearic Islands, Sardinia) and Eastern Mediterranean (Crete and few Aegean islands). Four narrowly distributed races (subspp. squamiflora, mediterranea, ebusitana, balearica) have been described to encompass the morphological variation within the species. We have used nuclear ribosomal ITS and cpDNA sequences to assess how the patterns of molecular differentiation are related to taxonomic and geographic boundaries. Extensive intragenomic ITS variation was detected in samples from all territories, the average sequence divergence among cloned ribotypes was 1.339%. The parsimony network of cloned ITS sequences suggests a split between Eastern and Western Mediterranean accessions. Chloroplast DNA sequences showed five distinct haplotypes, only one of which was shared between islands (Majorca and Sardinia). Both nuclear and cpDNA markers supported the monophyly of the C. squamiflora complex and identified a highly structured pattern of molecular variation composed by sister monophyletic lineages that mirror major biogeographic units (Western and Eastern Mediterranean). The molecular evidence supports the hypotheses that vicariance events linked to the geological history of the region or dispersal across the Mediterranean may explain the distribution of the complex.

Are Lotus creticus and Lotus cytisoides (Leguminosae) closely related species? Evidence from nuclear ribosomal ITS sequence data

Lotus creticus is a potentially important perennial legume for soil management in the Mediterranean climate. This plant is in focus of experimental research in different countries. The so-called Lotus creticus group is taxonomically problematic. Although some authors consider all members of the group as a single variable species, others segregate several species distinct from L. creticus, especially L. cytisoides. Recent morphological studies suggested that L. creticus and L. cytisoides are taxonomically distinct at specific level and not even closely related to each other. Two molecular phylogenetic studies published so far and based on one accession of L. creticus each gave conflicting results on relationships of this species. In this paper, sequences of nuclear ribosomal ITS (nrITS) are produced from multiple accessions of both L. creticus and L. cytisoides. Taxonomic identity of each accession has been tested using morphology. The new nrITS sequences are inserted in a data matrix containing a representative set of Lotus species covering all the geographical range and all major taxonomic groups of Lotus. Phylogenetic analysis of nrITS sequence data showed that L. creticus is related to L. assakensis and other species of the section Pedrosia while L. cytisoides is related to L. longisiliquosus and other species of the section Lotea. These results are important for establishing future germplasm collection, breeding and selection programs of perennial Lotus species.

A geographical pattern of Antirrhinum (Scrophulariaceae) speciation since the Pliocene based on plastid and nuclear DNA polymorphisms

AbstractAim To infer phylogenetic relationships among Antirrhinum species and to reconstruct the historical distribution of observed sequence polymorphism through estimates of haplotype clades and lineage divergence.Location Antirrhinum is distributed primarily throughout the western Mediterranean, with 22 of 25 species in the Iberian Peninsula.Methods Plastid (83 trnS‐trnG and 83 trnK‐matK) and nuclear (87 ITS) sequences were obtained from 96 individuals representing 24 of the 25 Antirrhinum species. Sequences were analysed using maximum parsimony, Bayesian inference and statistical parsimony networking. Molecular clock estimates were obtained for plastid trnK‐matK sequences using the penalized likelihood approach.Results Phylogenetic results gave limited support for monophyletic groups within Antirrhinum. Fifty‐one plastid haplotypes were detected and 27 missing haplotypes inferred, which were all connected in a single, star‐like network. A significant number of species shared both the same haplotypes and the same geographical areas, primarily in eastern Iberia. Furthermore, many species harboured populations with unrelated haplotypes from divergent haplotype clades. Plastid haplotype distribution, together with nucleotide additivity in 59 of the 86 nuclear ribosomal ITS sequences, is interpreted as evidence of extensive hybridization. Lineage divergence estimates indicated that differentiation within Antirrhinum post‐dates the Miocene, when the Mediterranean climate was established.Main conclusions Incongruence between plastid sequences, nuclear sequences and taxonomic delimitation is interpreted as strong evidence of limited cladogenetic processes in Antirrhinum. Rather, extensive nucleotide additivities in ITS sequences in conjunction with haplotype and haplotype‐clade distributions related to geographical areas support both recent and ancient hybridization. This geographical pattern of Antirrhinum speciation, particularly in eastern Iberia, is congruent with isolation–contact–isolation processes in the Pleistocene.