Recent colonization and radiation of North American Lycaeides (Plebejus) inferred from mtDNA

North American Lycaeides populations exhibit remarkable variation in ecological, morphological, and behavioral characters, as well as an established history of introgressive hybridization. We examined mitochondrial DNA variation from 55 Eurasian and North American Lycaeides populations using molecular phylogenetics and coalescent-based methods in order to clarify the evolutionary and demographic history of this polytypic group. Specifically we addressed the following questions: (1) Do mitochondrial alleles sampled from North America form a monophyletic group, which would be expected if North American Lycaeides were descended from a single Eurasian ancestor? (2) When did Lycaeides colonize North America? and (3) What is the demographic history of North American Lycaeides since their colonization? Bayesian maximum likelihood methods identified three major mitochondrial lineages for Lycaeides; each lineage contained haplotypes sampled from both Eurasia and North America. This suggests a complex colonization history for Lycaeides, which likely involved multiple founding lineages. Coalescent-based analyses placed the colonization of North America by Eurasian Lycaeides sometime during or after the late Pliocene. This was followed by a sudden increase in population size of more than an order of magnitude for the North American population of Lycaeides approximately 100,000-150,000 years before the present. These mitochondrial data, in conjunction with data from previous ecological, morphological, and behavioral studies, suggest that the diversity observed in Lycaeides in North America is the result of a recent evolutionary radiation, which may have been facilitated, in part, by hybridization.     

Hybridization between European and Asian dandelions ( Taraxacum section Ruderalia and section Mongolica)2. Natural hybrids in Japan detected by chloroplast DNA marker

Natural hybridization in Taraxacum between native sexual diploids and introduced agamospermous triploids occurring in Japan was studied by means of chloroplast DNA (cpDNA) marker. We first determined the nucleotide sequences between trnT (UGU) and trnF (GAA) of cpDNA for 22 plants obtained from Japan and Europe. The sequences analyzed were about 1,574 base pairs long. Among all accessions, the total numbers of polymorphic characters were 56 nucleotide substitutions, three insertions/deletions (ins/dels), and one repeat number polymorphism of mononucleotide motif. Of these polymorphic characters, four nucleotides and one ins/del were applicable in the discrimination between Japanese and European taxa of dandelions. We selected the ins/del in an intergenic region between trnL (UAA) 3′ exon and trnF (GAA) as a cpDNA marker. Using a newly developed cpDNA marker, 225 plants of putative Taraxacum officinale collected from 11 populations in Niigata City were investigated. Eighty-two percent of them showed a Japanese haplotype of cpDNA, and they were regarded as hybrids. Compared with the previous studies, it is likely that the prevalence of the hybrid plants is a general phenomena at least in urban areas in Japan. The validity of the cpDNA marker for screening Taraxacum hybrids is discussed. Electronic Publication     

Evolution of the Eastern Asian and Eastern North American disjunct genus Symplocarpus (Araceae): insights from chloroplast DNA restriction site data

Chloroplast DNA (cpDNA) variation was surveyed with 20 restriction endonucleases for the eastern Asian and eastern North American disjunct genus Symplocarpus (Araceae). The cpDNA phylogeny reveals a sister group relationship between S. foetidus from eastern North America and S. renifolius from eastern Asia. The cpDNA divergence between the two intercontinental sister species is 0.61%, which suggests an estimated divergence time of 6.1 million years ago during the late Miocene. The Bering land bridge hypothesis is compatible with the estimated time of divergence for the migration of Symplocarpus between eastern Asia and North America. Furthermore, a single origin of the exothermic spadices in Symplocarpus is suggested by the phylogeny. The cpDNA data also provide independent support for the recognition of three species within the genus.     

Genome size variation among common dandelion accessions informs their mode of reproduction and suggests the absence of sexual diploids in North America

Apomixis is the ability of plants to produce asexual seeds, which are clones of the mother plant. The phenomenon of apomixis is tightly linked to ploidy, where diploids lack apomixis and reproduce sexually, while higher ploidy levels can exhibit apomixis. Taraxacum F.H.Wigg. species (dandelions) commonly exhibit apomixis; however, only limited studies have evaluated genome size variation of the iconic weedy dandelion, Taraxacum officinale F.H.Wigg. (common dandelion), particularly in North America. To evaluate the ploidy and reproductive biology of common dandelion, we created a global collection, with an emphasis on North America and determined genome size by evaluating seeds with flow cytometry. A total of 635 accessions were screened, and with the exception of two accessions from Germany and Austria, all were found to exclusively contain polyploid seed. The mode of reproduction of a sample of 96 of these accessions was observed by conducting emasculations. Our results indicate that the diploid, sexual cytotype of common dandelion may be absent in North America, while diploids were found in previously described areas of Central Europe. This suggests that the clonal lineages of common dandelion in North America may largely be derivative from apomictic plants introduced from abroad. Furthermore, if all North American common dandelion is apomictic, it may be unreceptive to pollen, which may isolate it from other dandelion species and inform the potential for gene flow.     

INTRASPECIFIC CHLOROPLAST DNA VARIATION AND BIOGEOGRAPHY OF NORTH AMERICAN LIRIODENDRON L. (MAGNOLIACEAE)

Restriction site variation in chloroplast DNA (cpDNA) was surveyed to analyze population dynamics in Liriodendron tulipifera L., a woody angiosperm found in eastern North America. Two cpDNA haplotypes, differing by the presence or absence of five restriction site changes (nucleotide sequence divergence estimated as approximately 0.15%) are geographically structured; 61 widespread populations possess the “northern” haplotype and three isolated populations of central Florida possess the “southern” haplotype. This geographic break in cpDNA distribution corresponds to patterns of geographic distribution revealed by a previous survey of allozyme variation, with the exception that analyses of allozyme data further divided the populations containing the northern cpDNA haplotype into two groups, a widespread upland group and a coastal intermediate group. Analyses of these two independent data sets together support the hypothesis that L. tulipifera survived the glacial advances of the Pleistocene in two distinct refugia, possibly as different taxa, and the intermediate coastal group was putatively formed from recent hybridizations between these entities.     

Structure and inheritance of ribosomal DNA variants in cultivated and wild hop, Humulus lupulus L.

Genetic variation was assessed among cultivated and wild hop, Humulus lupulus, by restriction fragment length polymorphisms (RFLPs) of the ribosomal RNA genes (rDNA). Two rDNA length variants of 10.3 and 9.3 kbp represented by three phenotypes designated A, B and C were detected with XhoI. Restriction-site mapping showed that hop rDNA is structurally similar to those of most higher plants. A high level of homogeneity existed in rDNA repeat lengths among the diverse hop genotypes. Generally, phenotype A was predominant in wild and cultivated European and Asian genotypes; phenotype B in North American cultivars; while phenotype C was present only in native North American hop, providing a potential molecular marker for the identification of this germ plasm. The rDNA data provided genetic evidence for the separation of native and cultivated American genotypes and supports the hypothesis that North American hop cultivars are of hybrid origin from European and native American genotypes. The segregation of rDNA phenotypes in four F₁ families suggests that a single locus with two co-dominant alleles controls genetic variability for rDNA variants in hop.     

Inferences on the phylogeography of the fungal pathogen Heterobasidion annosum, including evidence of interspecific horizontal genetic transfer and of human-mediated, long-range dispersal

Fungi in the basidiomycete species complex Heterobasidion annosum are significant root-rot pathogens of conifers throughout the northern hemisphere. We utilize a multilocus phylogenetic approach to examine hypotheses regarding the evolution and divergence of two Heterobasidion taxa associated with pines: the Eurasian H. annosum sensu stricto and the North American H. annosum P intersterility group (ISG). Using DNA sequence information from portions of two nuclear and two mitochondrial loci, we infer phylogenetic relationships via parsimony, Bayesian and median-joining network analysis. Analysis of isolates representative of the entire known geographic range of the two taxa results in monophyletic sister Eurasian and North American lineages, with North America further subdivided into eastern and western clades. Genetically anomalous isolates from the Italian presidential estate of Castelporziano are always part of a North American clade and group with eastern North America, upholding the hypothesis of recent, anthropogenically mediated dispersal. P ISG isolates from Mexico have phylogenetic affinity with both eastern and western North America. Results for an insertion in the mitochondrial rDNA suggest this molecule was obtained from the Heterobasidion S ISG, a taxon sympatric with the P ISG in western North America. These data are compatible with an eastern Eurasian origin of the species, followed by dispersal of two sister taxa into western Eurasia and into eastern North America over a Beringean land bridge, a pattern echoed in the phylogeography of other conifer-associated basidiomycetes.     

The importance of sexual and asexual reproduction in the recent evolution of Allium vineale

In the weedy plant species Allium vineale (wild garlic), individuals may simultaneously produce sexually and asexually derived offspring, by seed and bulbils, respectively. In this study, genetic and genotypic diversity was determined in samples from 14 European A. vineale populations using nuclear (RAPD) and cytoplasmic (PCR-RFLP of cpDNA) markers to investigate the importance of the different reproductive modes. In the whole sample, 77 nuclear multilocus genotypes and four chloroplast haplotypes (chlorotypes) were found. Populations exhibited a high degree of subdivision for nuclear and cytoplasmic markers as estimated from hierarchical F-statistics; at the same time, identical chlorotypes could be found in populations separated by large distances. Genotypic diversity was significantly lower than expected under free recombination in almost all populations, indicating that recruitment into populations is mostly by asexually produced offspring. Nevertheless, within each chlorotype, the distribution of markers from pairs of nuclear loci was incompatible with a purely clonal structure, suggesting that many multilocus genotypes have originated by sexual recombination rather than by mutation within asexual lineages. It is argued that the weedy habit of A. vineale is likely to have favored bulbil reproduction, whereas sexually generated genotypes may have facilitated local adaptation during the species' expansion across Europe.     

Mitochondrial DNA variation and reticulate evolution of the genus Abies

The sequences of three regions of mitochondrial DNA (mtDNA) of a total length of 5226 bp were used to study the phylogeography of the genus Abies. The mtDNA haplotype network, comprising 36 studied Abies taxa, consisted of two branches; the first represented all American species plus two Asian, and the second included the remaining Eurasian species. Within these clusters, the haplotypes formed nine major groups, generally corresponding to the clades of the previously obtained phylogeny based on chloroplast DNA (cpDNA), but the relationships of these groups were significantly different; species assignment to the particular mtDNA haplotype group was more in line with its geographical distribution. In addition, the mtDNA haplotype network contains cycles indicating the recombination. It is assumed that the incongruence of cpDNA and mtDNA phylogenies is caused by the introgression capture of alien mtDNA during species hybridization and thus contains information about past migrations. The cases of incongruence of mitochondrial and chloroplast DNA suggesting a migration of Abies between Asia and North America are discussed.     

The role of disjunction and postglacial population expansion on phylogeographical history and genetic diversity of the circumboreal plant Chamaedaphne calyculata

In the last decade a number of studies has illustrated quite different phylogeographical patterns amongst plants with a northern present‐day geographical distribution, spanning the entire circumboreal region and/or circumarctic region and southern mountains. These works, employing several marker systems, have brought to light the complex evolutionary histories of this group. Here I focus on one circumboreal plant species, Chamaedaphne calyculata (leatherleaf), to unravel its phylogeographical history and patterns of genetic diversity across its geographical range. A survey of 29 populations with combined analyses of chloroplast DNA (cpDNA), internal transcribed spacer (ITS) and AFLP markers revealed structuring into two groups: Eurasian/north‐western North American, and north‐eastern North American. The present geographical distribution of C. calyculata has resulted from colonization from two putative refugial areas: east Beringia and south‐eastern North America. The variation of chloroplast DNA (cpDNA) and ITS sequences strongly indicated that the evolutionary histories of the Eurasian/north‐western North American and the north‐eastern North American populations were independent of each other because of a geographical disjunction in the distribution area and ice‐sheet history between north‐eastern and north‐western North America. Mismatch analysis using ITS confirmed that the present‐day population structure is the result of rapid expansion, probably since the last glacial maximum. The AFLP data revealed low genetic diversity of C. calyculata (P = 19.5%, H = 0.085) over the whole geographical range, and there was no evidence of loss of genetic diversity within populations in the continuous range, either at the margins or in formerly glaciated and nonglaciated regions. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 761–775.     

A MOLECULAR REEXAMINATION OF INTROGRESSION BETWEEN HELIANTHUS ANNUUS AND H. BOLANDERI (COMPOSITAE)

Heiser (1949) hypothesized that a weedy race of Helianthus bolanderi had originated by the introgression of genes from H. annum into a serpentine race of H. bolanderi. Although Heiser's investigation of these species is frequently cited as one of the best examples of introgression in plants, definitive evidence of gene exchange is lacking (Heiser, 1973). To determine whether the weedy race of H. bolanderi actually originated via introgression, we analyzed allozyme, chloroplast-DNA (cpDNA), and nuclear-ribosomal-DNA (rDNA) variation. Evidence from enzyme electrophoresis did not support the proposed introgressive origin of weedy H. bolanderi. We detected a total of 37 low-frequency alleles distinguishing the serpentine race of H. bolanderi from H. annuus. Weedy H. bolanderi possessed only four of the 37 marker alleles. Further analysis demonstrated that serpentine H. bolanderi combined seven of the 35 alleles distinguishing H. annuus from weedy H. bolanderi, indicating that serpentine H. bolanderi shares three more marker alleles with H. annuus than does weedy H. bolanderi. These results are similar to expectations for race divergence from a single common ancestor and suggest that, if introgression occurred, the majority of marker alleles were rapidly lost following the initial hybridization event. Even more compelling evidence opposing Heiser's (1949) hypothesis, however, was from restriction-fragment analysis of cpDNA and nuclear rDNA. We detected a total of 17 cpDNA and five rDNA restriction-site mutations among the 19 populations examined. No parallel or back mutations were observed in phylogenetic trees constructed using either cpDNA or rDNA mutations, and both phylogenies were completely congruent regarding the alignment of all three taxa. In addition, the weedy race of H. bolanderi possessed a unique cpDNA, which was outside the range of variation observed among populations of either of the presumed parental species. Mean sequence divergences between the cpDNAs of weedy H. bolanderi and those of serpentine H. bolanderi and H. annuus were 0.30% and 0.35%, respectively. These estimates are comparable to sequence-divergence values observed between closely related species in other plant groups. Given the lack of parallel or convergent mutations in the cpDNA and rDNA phylogenetic trees, the complete congruence of these trees with flavonoid- and allozyme-variation patterns, and the presence of a unique and divergent chloroplast genome in the weedy race of H. bolanderi, we suggest that the weedy race of H. bolanderi was not derived recently through introgression, as hypothesized, but is relatively ancient in origin.     

Morphological and molecular comparisons ofCampsis grandiflora andC. radicans (Bignoniaceae), an eastern Asian and eastern North American vicariad species pair

Morphological and molecular comparisons were made forCampsis grandiflora (Thunb.)K. Schumann (Bignoniaceae) from eastern Asia andC. radicans (L.)Seemann from eastern North America. Chloroplast DNA (cpDNA) variation was surveyed with 20 restriction endonucleases. The cpDNA divergence between the two vicariad species was 2.44%, which is the highest reported among North Temperate disjunct taxa and one of the highest reported for infrageneric taxa. Detailed morphological comparisons also suggest a high level of divergence. Cluster analyses based on 22 morphological characters and 39 OTUs revealed two distinct groups corresponding with the two species. The average taxonomic distance between the two species was 1.806. Analyses of variance (ANOVA) revealed that 12 of the 18 quantitative characters differed significantly ( 0.01) betweenC. grandiflora andC. radicans. Divergence time based on cpDNA data was estimated as 24.4 million years. The Bering land bridge hypothesis was favored over the North Atlantic land bridge hypothesis based on the estimated divergence time and the geological history of the North Temperate region. The high levels of morphological and cpDNA divergence are not consistent with morphological stasis, which has been proposed as a common mode of evolution for North Temperate disjunct taxa.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Chloroplast DNA phylogeny and phylogeography of the North American plums (Prunus subgenus Prunus section Prunocerasus, Rosaceae)

The North American plums are a closely related group that are not easily circumscribed, have overlapping morphologies, and are known to hybridize. We previously showed that the North American plums are a closely related, monophyletic group of taxa with little to no cpDNA sequence divergence between taxa. In that study, we came to the unanticipated conclusion that relationships inferred among the taxa contrast sharply with previously defined groups based on morphological characters. Here the aim was to determine if the primary cpDNA haplotypes identified in our earlier study are confined to the taxa in which they were initially observed. The cpDNA rpL16 intron was sequenced for 207 accessions of the 17 North American plum taxa plus Prunus texana. The results show that many taxa contain more than one of the three primary cpDNA haplotypes. Aside from the results found in sect. Prunocerasus, this study has broader implications for phylogenetics in general. The common practice of choosing a single exemplar to represent a taxon can be profoundly misleading in closely related groups. In hindsight, the possibility existed in our earlier study that we could have chosen a different combination of exemplars, which could have resulted in a different inferred phylogeny.     

Evidence for multiple sources of invasion and intraspecific hybridization in Brachypodium sylvaticum (Hudson) Beauv. in North America

We compared the levels and distribution of genetic diversity in Eurasian and North American populations of Brachypodium sylvaticum (Huds.) Beauv. (false brome), a newly invasive perennial bunchgrass in western North America. Our goals were to identify source regions for invasive populations, determine the number of independent invasion events, and assess the possibility that postinvasion bottlenecks and hybridization have affected patterns of genetic diversity in the invaded range. We tested the hypothesis that this Eurasian grass was accidentally introduced into two areas in Oregon and one site in California by examining nuclear microsatellites and chloroplast haplotype variation in 23 introduced and 25 native populations. In the invaded range, there was significantly lower allelic richness (R(S)), observed heterozygosity (H(O)) and within-population gene diversity (H(S)), although a formal test failed to detect a significant genetic bottleneck. Most of the genetic variation existed among populations in the native range but within populations in the invaded range. All of the allelic variation in the invaded range could be explained based on alleles found in western European populations. The distribution of identified genetic clusters in the North American populations and the unique alleles associated with them is consistent with two historical introductions in Oregon and a separate introduction to California. Further analyses of population structure indicate that intraspecific hybridization among genotypes from geographically distinct regions of western Europe occurred following colonization in Oregon. The California populations, however, are more likely to be derived from one or perhaps several genetically similar regions in the native range. The emergence and spread of novel recombinant genotypes may be facilitating the rapid spread of this invasive species in Oregon.     

Hybridization and morphogenetic variation in the invasive alien Fallopia (Polygonaceae) complex in Belgium

The invasive alien knotweeds, Fallopia spp. (Polygonaceae), are some of the most troublesome invasive species in Europe and North America. Invasive success in Fallopia may be enhanced by multiple hybridization events. We examined the pattern of hybridization and its evolutionary consequences in Belgium with a concerted analysis of ploidy levels (chromosome counts and flow cytometry), morphological variation, and genetic variation (RAPDs). At least four taxa with different ploidy levels were part of the pattern of invasion in Belgium. Hybrid F. ×bohemica with various chromosome numbers restored the genotypic diversity that was lacking in the parental species. Hybrid genotypes were mainly assigned to a specific genetic pool and not to a mixture between the genetic pools of the putative parental species as would be expected for hybrids. Parental species and hexaploid hybrids differed significantly for a set of well-defined morphological characters, enabling future researchers to distinguish these taxa. On the basis of our results, the importance of hybridization has probably been underestimated in large parts of the adventive range of alien Fallopia species, pointing to the need for concerted molecular and morphological analyses in the study of the evolutionary consequences of hybridization.     

A systematic study of North American Angelica species (Apiaceae) based on nrDNA ITS and cpDNA sequences and fruit morphology

Angelica is a taxonomically complex genus widespread throughout the North Temperate Zone. Previous phylogenetic studies of the genus have focused primarily on its East Asian species. The relationships among its North American members, the monophyly of these species, and the value of fruit morphology in circumscribing its taxa have yet to be examined. This study represents the most comprehensive sampling of Angelica to date (100 species) and includes all 26 species in North America. Relationships are inferred using Bayesian inference, maximum likelihood, and maximum parsimony analyses of ITS sequences and, for multiple accessions of each North American species, cpDNA ndhF-rpl32, rpl32-trnL, and psbM-psbD sequences. The fruit morphological characters examined were those considered phylogenetically important in East Asian Angelica. The results revealed that the North American species fell into three major clades: North American Angelica clade, Archangelica clade, and the Eurasian Angelica clade. Angelica dawsonii has affinities with Lomatium brandegeei. Fourteen species within the North American Angelica clade were strongly supported as monophyletic. Two paraphyletic species resulted in new combinations in A. lineariloba and A. venenosa. Conflict between the ITS-derived and cpDNA-derived phylogenies and the lack of resolution in portions of the trees may be due to chloroplast capture and rapid species radiation. Fruit morphology supported some interspecific relationships based on molecular data, and relationships revealed by ITS and cpDNA data were roughly in accordance with fruit classification type and geographic distribution region, respectively. A diagnostic key based on fruit morphology is provided for the identification of the North American Angelica taxa.     

A phylogeny of the damselfly genus calopteryx (Odonata) using mitochondrial 16S rDNA markers

We seek to reconstruct the phylogenetic relationships of the damselfly genus Calopteryx, for which extensive behavioral and morphological knowledge already exists. To date, analyses of the evolutionary pathways of different life history traits have been hampered by the absence of a robust phylogeny based on morphological data. In this study, we concentrate on establishing phylogenetic information from parts of the 16S rDNA gene, which we sequenced for nine Calopteryx species and five outgroup species. The mt 16S rDNA data set did not show signs of saturated variation for ingroup taxa, and phylogenetic reconstructions were insensitive to variation of outgroup taxa. Parsimony, neighbor-joining, and maximum-likelihood reconstructions agreed on parts of the tree. A consensus tree summarizes the significant results and indicates problematic nodes. The 16S rDNA sequences support monophyly of the genera Mnais, Matrona, and Calopteryx. However, the genus Calopteryx may not be monophyletic, since Matrona basilaris and Calopteryx atrata are sister taxa under every parameter setting. The North American and European taxa each appear as monophyletic clades, while the Asian Calopteryx atrata and Calopteryx cornelia are not monophyletic. Our data implies a different paleobiogeographic history of the Eurasian and North American species, with extant Eurasian species complexes shaped by glacial periods, in contrast to extant North American species groups.     

Chloroplast DNA variation and reticulate evolution in sexual and apomictic sections of dandelions

Sequencing of the trnL-trnF intergenic spacer in chloroplast DNA (cpDNA) from 237 sexual and apomictic species of dandelions (genus Taraxacum) from Europe, Asia and arctic North America revealed 46 haplotypes, which differed mainly by a variable number of polymorphic tRNA pseudogenes next to the trnF gene. The haplotypes could be divided into 20 cpDNA lineages, but independent duplications and deletions of the pseudogene copies made it difficult to further reconstruct the phylogeny. Intraspecific cpDNA variation was found in the primitive sexual T. serotinum. However, in contrast to a recent study, no cpDNA variation was detected within 12 apomictic species representing a variety of haplotypes. The cpDNA haplotype may therefore help to define these critical apomicts. On the other hand, the genetic variation may easily be overestimated, if the clones are not correctly identified, because some morphologically similar microspecies carried very different haplotypes. In all, 36 sections of the genus were sampled. Four primitive, mainly sexual, sections only displayed a group of ancient haplotypes, whereas morphologically more advanced sections often exhibited many different haplotypes from up to seven cpDNA lineages. In the latter cases, the lineages were rarely unique to a certain section. For example, the two most widespread haplotypes, belonging to different lineages, were found together in nine sections. This suggests that significant gene flow has occurred among the advanced sections, although sexual reproduction is not currently known in several of them. The result is consistent with the reticulate distribution of morphological characters among the sections.