Chloroplast DNA evidence for reticulate evolution in Eucalyptus (Myrtaceae)

Four highly differentiated chloroplast DNA (cpDNA) lineages were identified in the forest tree species Eucalyptus globulus Labill. (Myrtaceae) in Australia using restriction site polymorphisms from Southern analysis. The cpDNA variation did not conform with ssp. boundaries, yet there was a strong geographical pattern to the distribution of the lineages. One lineage (C) was geographically central and widespread, whereas the other three lineages were found in peripheral populations: Western (W), Northern (N) and Southern (S). Thirteen haplotypes were detected in E. globulus , seven of which belonged to clade C. At least three of the cpDNA lineages (C, N and S) were shared extensively with other species. On the east coast of the island of Tasmania, there was a major north–south difference in cpDNA in the virtually continuous distribution of E. globulus . Northern populations harboured haplotypes from clade C while southeastern populations harboured a single haplotype from clade S. This difference was also reflected in several co-occurring endemic species. It is argued that the extensive cpDNA differentiation within E. globulus is likely to originate from interspecific hybridization and 'chloroplast capture' from different species in different parts of its range. Superficially, this hybridization is not evident in taxonomic traits; however, large-scale common garden experiments have revealed a steep cline in quantitative genetic variation that coincides with the haplotype transition in Tasmania. Our cpDNA results provide the strongest evidence to date that hybridization has had a widespread impact on a eucalypt species and indicate that reticulate evolution may be occurring on an unappreciated scale in Eucalyptus .     

Physical mapping of ribosomal RNA genes in peonies (Paeonia,Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution

Physical maps of the 18S–5.8S–26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of P. rockii and P. tenuifolia, chromosomes 2, 3, 4, and 5 of P. delavayi, and all five pairs of chromosomes of P. emodi and P. veitchii. Combining this information with the previously obtained rDNA maps of P. brownii and P. californica of section Oneapia, we hypothesized that the most recent common ancestor of extant peony species had three rDNA loci located on chromosomes 3, 4, and 5. Increase in number of rDNA loci occurred later in each of the three sections, and the increase from three to four loci represents a parallel gain of an rDNA locus on chromosome 2 in P. delavayi of section Moutan and P. brownii of section Oneapia. The increase in number of rDNA loci likely resulted from the translocation of rDNA repeats from chromosomes bearing rDNA loci to chromosomes without them; such translocation is probably facilitated by the telomeric location of rDNA loci. For allotetraploid peony species lacking polymorphism in sequences of the internal transcribed spacers (ITS) of rDNA, the rDNAs derived from divergent diploid parents may have been homogenized through concerted evolution among at least six rDNA loci in the allotetraploids. Chromosomal location of rDNA loci has a more substantial impact on the tempo of concerted evolution than the number of loci.     

基于多基因序列和形态性状的牡丹组种间关系

牡丹被认为是中国的国花,具有很高的医学、观赏和经济价值.野生牡丹被认为是栽培牡丹的野生祖先,因此弄清牡丹组的种间亲缘关系具有重要的理论和实践意义.由于受到信息量的限制,根据单基凼数据或形态数据往往无法对牡丹组的种间关系得到明确的结果.本研究用12份样品代表野生牡丹组(Paeonia section Moutan DC.,Paeoniaceae)8个种,利用包括核基因(Adh1A、Adh2和GPAT)和叶绿体基因(trnS-trnG和rps16-trnQ)的DNA序列以及形态性状的多套数据来探讨野牛牡丹的种间关系.合并分析得到具高支持率的牡丹组物种间的系统发育关系.结果表明,芍药属牡丹组8个野生种分为两个亚组,即肉质花盘亚组subseet.Delavayanae和革质花盘亚组subsect.Vaginatae.肉质花盘亚组包括滇牡丹P delavayi和大花黄牡丹P.ludlowii;革质花盘亚组包括其余6个种.革质花盘亚组中,四川牡丹P.decomposita ssp.decomposita和紫斑牡丹P. rockii ssp. rockii关系密切;卵叶牡丹P.qiui和矮牡丹P. jishanenMs关系密切;银屏牡丹P. suffruticosa ssp.yinpingmudan与风丹P. ostii关系 密切,并且后两个分支为姊妹群.     

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.     

Phylogenetic analysis of Paeonia sect. Moutan (Paeoniaceae) based on multiple DNA fragments and morphological data

Tree peony, being crowned the title “King of Flowers” in China, is of great medicinal, ornamental, and economic values. In the present study, the phylogeny of the wild tree peony species (section Moutan, Paeonia, Paeoniaceae), represented by twelve accessions collected from all eight species in the section, was investigated based on the DNA sequence in five DNA fragments from both nuclear (Adh1A, Adh2 and GPAT) and chloroplast (trnS-trnG and rps16-trnQ) genomes, as well as morphological characters. Both maximum parsimony (MP) and Bayesian inference of phylogeny (BI) trees were reconstructed based on the combined data of the DNA sequences and morphological data, respectively. The MP and BI trees have the similar topology, and the sect. Moutan clearly branched into two clades. One clade consists of two species, P. delavayi and P. ludlowii, corresponding to the subsect. Delavayanae, and another clade is composed of other six species. Within the second clade, the six species can be divided into three subclades consisting of P. rockii and P. decomposita, P. jishanensis and P. qiui, P. suffruticosa and P. ostii, respectively. Among the three subclades, P. jishanensis/P. qiui is most closely related to P. suffruticosa/P. ostii. These results provide up to date the clearest picture of the phylogeny of wild tree peony species in the sect. Moutan.     

Testing four proposed barcoding markers for the identification of species within Ligustrum L. (Oleaceae)

DNA barcoding is a biological technique that uses short and standardized genes or DNA regions to facilitate species identification. DNA barcoding has been used successfully in several animal and plant groups. Ligustrum (Oleaceae) species occur widely throughout the world and are used as medicinal plants in China. Therefore, the accurate identification of species in this genus is necessary. Four potential DNA barcodes, namely the nuclear ribosomal internal transcribed spacer (ITS) and three chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA), were used to differentiate species within Ligustrum. BLAST, character-based method, tree-based methods and TAXONDNA analysis were used to investigate the molecular identification capabilities of the chosen markers for discriminating 92 samples representing 20 species of this genus. The results showed that the ITS sequences have the most variable information, followed by trnH–psbA, matK, and rbcL. All sequences of the four regions correctly identified the species at the genus level using BLAST alignment. At the species level, the discriminating power of rbcL, matK, trnH–psbA, and ITS based on neighbor-joining (NJ) trees was 36.8%, 38.9%, 77.8%, and 80%, respectively. Using character-based and maximum parsimony (MP) tree methods together, the discriminating ability of trnH–psbA increased to 88.9%. All species could be differentiated using ITS when combining the NJ tree method with character-based or MP tree methods. Overall, the results indicate that DNA barcoding is an effective molecular identification method for Ligustrum species. We propose the nuclear ribosomal ITS as a plant barcode for plant identification and trnH–psbA as a candidate barcode sequence.     

DNA barcoding of the recently evolved genus Holcoglossum (Orchidaceae: Aeridinae): a test of DNA barcode candidates

Orchidaceae is one of the largest families of flowering plants. Many species of orchid are endangered, and all species are included in Conventions on International Trade of Endangered Species of Fauna and Flora (CITES) I and II, but it is very difficult to identify orchid species, even those with fertile parts. The genus Holcoglossum (Orchidaceae: Aeridinae) has long been problematic in taxonomy. It consists of both long-evolved and radiated species and is an excellent case to use for testing DNA barcodes for Orchidaceae. We investigated the power of a subset of proposed plant barcoding loci [rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA and internal transcribed spacer (ITS)] to discriminate between species in this genus. Our results showed that all these DNA regions, except psbK-psbI and atpF-atpH, can be amplified easily from Holcoglossum and sequenced with established primers. The DNA regions matK and ITS had the highest variability. Among the six loci, matK resolved eight of the 12 Holcoglossum species and had the highest discriminatory ability. However, the combination of matK and ITS showed a greater ability to identify species than matK alone. Single or combined DNA markers discriminated between Holcoglossum species distributed in tropical areas effectively, but had less ability to identify radiated species from the temperate Hengduan Mountains of China. In the study, matK proved to be a useful DNA barcode for the genus Holcoglossum; however, complementary DNA regions are still required to accelerate the investigation and preservation of radiated species of orchid.     

DNA barcoding of the main cultivated yams and selected wild species in the genus Dioscorea

Distinguishing yam species based on morphological traits is extremely difficult and unreliable, posing a challenge to breeders and genebank curators. Development of a molecular assay based on DNA barcoding can facilitate rapid and accurate identification of important Dioscorea species. To develop a DNA barcoding system forDioscorea species identification, the rbcL and matK loci (in unison and in combination), the non-coding intergenic spacer trnH-psbA of the chloroplast genome, and the nuclear ITS regions were investigated using criteria for developing candidate DNA barcodes. All DNA barcoding sequences were assessed for ease of PCR amplification, sequence quality and species discriminatory power. Amongst the markers investigated, the matK locus performed well in terms of species identification (63.2%), in addition to detecting high interspecific variation with mean divergence of 0.0196 (SD=0.0209). The combination of the two coding regions (rbcL + matK) was determined to be the optimal (76.2%) DNA barcoding approach as 16 out of 21 species could be defined. While the rbcL exhibited good PCR amplification efficiency and sequence quality, its species discriminatory power was relatively poor with 47.6% identification. Similarly, the trnH-psbA region had a weak discrimination efficiency of only 36.8%. While the development of more robust DNA barcoding systems is an ongoing challenge, our results indicate that therbcL + matK combination can be utilized as multi-locus DNA barcode regions for Dioscorea species identification.     

DNA barcoding to analyse taxonomically complex groups in plants: the case of Thymus (Lamiaceae)

We evaluated the utility of the core barcode regions (matK and rbcL) and the plastid intergenic spacer trnH‐psbA to distinguish between Thymus spp. This is a taxonomically complex group that has been investigated so far mainly using morphological approaches. Thirty‐six samples representing nine different morphospecies were collected and used for molecular analysis. The three markers showed clear amplification and sequencing. However, the genetic variation and the resulting haplotype networks showed that only Thymus capitatus forms a well‐defined ‘barcoding gap’ compared with the other taxa. The identification problems observed in the other Thymus spp. may be related to reduced gene flow among populations, resulting in high intraspecific and low interspecific genetic variation. This situation does not permit the definition of species‐specific barcodes. A second hypothesis suggests that morphological traits used for the delimitation of Thymus spp. do not reflect real biological and molecular species boundaries. If this is the case, the taxonomy of Thymus should be revised through extensive sampling and analyses with different tools (i.e. molecular variability, morphology, geographical distribution, etc.) to define the natural units at the species level. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013 , 171 , 687–699.     

Molecular phylogenetics of Vanda and related genera (Orchidaceae)

The genus Vanda and its affiliated taxa are a diverse group of horticulturally important species of orchids occurring mainly in South‐East Asia, for which generic limits are poorly defined. Here, we present a molecular study using sequence data from three plastid DNA regions. It is shown that Vanda s.l. forms a clade containing approximately 73 species, including the previously accepted genera Ascocentrum, Euanthe, Christensonia, Neofinetia and Trudelia, and the species Aerides flabellata. Resolution of the phylogenetic relationships of species in Vanda s.l. is relatively poor, but existing morphological classifications for Vanda are incongruent with the results produced. Some novel species relationships are revealed, and a new morphological sectional classification is proposed based on support for these groupings and corresponding morphological characters shared by taxa and their geographical distributions. The putative occurrence of multiple pollination syndromes in this group of taxa, combined with complex biogeographical history of the South‐East Asian region, is discussed in the context of these results. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 549–572.     

Molecular phylogeny of Osmanthus (Oleaceae) based on non‐coding chloroplast and nuclear ribosomal internal transcribed spacer regions

Abstract The phylogenetic relationships of Osmanthus Lour. were investigated using the nuclear ribosomal internal transcribed spacer (ITS) regions and non‐coding chloroplast regions (psbA‐trnH, trnL‐F). The two datasets support the conclusion that Osmanthus is polyphyletic, with some species of the subtribe Oleinae nested within Osmanthus. Osmanthus didymopetalus P. S. Green is nested within the clade formed by species of section Osmanthus in two trees. Osmanthus attenuatus P. S. Green, O. yunnanensis P. S. Green, and O. gracilinervis R. L. Lu of traditional section Osmanthus are clearly divergent from other accessions, and do not form a monophyletic group with other Osmanthus accessions. Osmanthus marginatus Hemsl. is embedded in the clade formed by species of section Osmanthus in the ITS tree. In cpDNA trees all species of section Osmanthus are placed in the large clade and all species of section Leiolea formed a group. The taxonomic incongruence among trees for ITS and cpDNA indicate hybridization, as introgression may have occurred among some species of sections Osmanthus and Leiolea. Phylogeny of Osmanthus is discussed in light of molecular and morphological data, and a revised infrageneric classification with three sections (Leiolea, Siphosmanthu, and Osmanthus) is presented. The section Linocieroides is abandoned and united with section Osmanthus.     

Mitochondrial and chloroplast DNA-based phylogeny of Pelargonium (Geraniaceae)

Overall phylogenetic relationships within the genus Pelargonium (Geraniaceae) were inferred based on DNA sequences from mitochondrial(mt)-encoded nad1 b/c exons and from chloroplast(cp)-encoded trnL (UAA) 5' exon-trnF (GAA) exon regions using two species of Geranium and Sarcocaulon vanderetiae as outgroups. The group II intron between nad1 exons b and c was found to be absent from the Pelargonium, Geranium, and Sarcocaulon sequences presented here as well as from Erodium, which is the first recorded loss of this intron in angiosperms. Separate phylogenetic analyses of the mtDNA and cpDNA data sets produced largely congruent topologies, indicating linkage between mitochondrial and chloroplast genome inheritance. Simultaneous analysis of the combined data sets yielded a well-resolved topology with high clade support exhibiting a basic split into small and large chromosome species, the first group containing two lineages and the latter three. One large chromosome lineage (x = 11) comprises species from sections Myrrhidium and Chorisma and is sister to a lineage comprising P. mutans (x = 11) and species from section Jenkinsonia (x = 9). Sister to these two lineages is a lineage comprising species from sections Ciconium (x = 9) and Subsucculentia (x = 10). Cladistic evaluation of this pattern suggests that x = 11 is the ancestral basic chromosome number for the genus.     

Incongruence between chloroplast and species phylogenies in Eucalyptus subgenus Monocalyptus (Myrtaceae)

Seventy-eight polymorphic cpDNA (chloroplast DNA) characters were found in 13 closely related taxa from Eucalyptus series Amygdalinae (subgenus Monocalyptus) and seven potential outgroup taxa. The strict consensus of six cladograms generated from cpDNA data confirmed monophyly of Monocalyptus. However, cpDNA phylogeny within Monocalyptus was incongruent with taxonomic classification, being more related to geography, even when accessions were from divergent series. Monocalyptus cpDNA formed two major clades. On the island of Tasmania cpDNA was restricted to a single clade, exhibited very little variation, and was phylogenetically related to cpDNA found in central and western Victoria. In contrast, cpDNA of mainland monocalypt taxa was more variable, even within the Amygdalinae. Four out of six Tasmanian Amygdalinae species were polymorphic. The difference between cpDNA of replicates was often greater than differences between species from different series. The low level of cpDNA variation and extensive morphological intergradation between the Tasmanian endemics suggest recent speciation. However, the transfer of cpDNA through hybridization between lineages is the most likely explanation for the observed sharing of cpDNA across series. This study highlights that the geographical pattern to cpDNA variation in Eucalyptus may be an important source of information on past plant distributions in Australia.     

Phylogenetics of Annona cherimola (Annonaceae) and some of its closest relatives

Annona cherimola is a woody perennial species in the Annonaceae family that produces edible fruits and has economic importance in several regions of the world with subtropical climates. Together with other 10‐12 species, A. cherimola belongs to the section Atta of the Annona genus with a center of origin in Central America and the Caribbean. Species of the section Atta produce soft skin ripe fruits with raised areoles bounded by recessed furrows. Annona cherimola is the only species of the section naturally found in the Andean region of South America. Currently, no information is available at the molecular level on the phylogenetic relationships of most of the species of Atta and closely related sections in Annona. In order to fill this gap, in this work a phylogenetic approach was performed using five coding and non‐coding plastid DNA regions, to determine the phylogenetic relationships between A. cherimola and other related species included in Atta and other sections of the genus. The results obtained support recent studies that demonstrated the likely Mesoamerican origin of A. cherimola based on biogeographical analysis with SSR markers, rather than the previously considered South American origin hypothesis. In addition, the species belonging to the Atta section did not show monophyly. Finally, A. cherimola and A. pruinosa seem to be phylogenetically close species and additional studies are needed to discern the relations between them.     

Plastid DNA sequence data help to clarify phylogenetic relationships and reticulate evolution in Lycoris (Amaryllidaceae)

The temperate East Asian genus Lycoris is a well known lineage of ornamental geophytes consisting of at least 20 species, some of which are thought to be of natural hybrid origin. Previous genetic studies have supported this hypothesis, but these have relied solely on the use of karyology and/or nuclear ribosomal ITS sequences. No plastid DNA data have been available to address interspecific relationships within Lycoris until now. In this study, 500 individuals from 29 populations representing 16 of the 20 published Lycoris spp. were sampled, and DNA sequences were generated for two plastid markers (trnS‐trnfM and trnC‐ycf6). From these data we inferred phylogenetic relationships among the sampled taxa at the species and population levels using concatenated phylogenetic methods. A well resolved and strongly supported phylogenetic reconstruction for Lycoris was obtained. Although the plastid DNA topology differs from that derived previously using ITS, both genomes produce trees that cluster Lycoris spp. into three clades. One of these, containing polyploid taxa such as L. albiflora, L. caldwellii, L. straminea and L. houdyshelii, shows strong evidence of reticulation, and we discuss the identity of potential parents of these allopolyploids. In contrast, we offer evidence that challenges the hypothesis that triploid individuals of L. radiata are the result of hybridization. Instead, they appear to be autotriploids that have arisen in more than one location. By comparing the phylogenetic results obtained using nuclear genomic data to those from the plastid genome, a much clearer picture of the role that hybridization and reticulation have played in the evolution of Lycoris is emerging. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 115–126.     

Preliminary investigations of hybridization/reticulate evolution in Guaranidrilus (Enchytraeidae: Oligochaeta)

The identification of gut diverticula at 7/8 as a synapomorphy and recognition of all taxa of Guaranidrilus has been obscured by losses of gut diverticula within the lineage. The homoplastic occurrence of basally unpaired peptonephridia in some enchytraeid species has similarly obscured the limits of Hemienchytraeus. Taxa with unpredictable relationships, morphogenic irregularities in some reproductive structures, and, apparently, modified modes of reproduction, suggest the possibilities of hybridization between taxa with both close and distant relationships.     

Chloroplast DNA phylogeny and biogeography of Lepidium (Brassicaceae)

Two intergenic spacers, trnT-trnL and trnL-trnF, and the trnL intron of cpDNA were sequenced to study phylogenetic relationships and biogeography of 73 Lepidium taxa. Insertions/deletions of ≥3 bp (base pairs) provided reliable phylogenetic information whereas indels ≤2 bp, probably originating from slipped-strand mispairing, are prone to parallelism in the context of our phylogenetic framework. For the first time, an hypothesis of the genus Lepidium is proposed based on molecular phylogeny, in contrast to previous classification schemes into sections and greges (the latter category represents groups of related species within a given geographic region), which are based mainly on fruit characters. Only a few of the taxa as delimited in the traditional systems represent monophyletic lineages. The proposed phylogeny would suggest three main lineages, corresponding to (1) sections Lepia and Cardaria, (2) grex Monoplocoidea from Australia, and (3) remaining taxa, representing the bulk of Lepidium species with more or less resolved sublineages that sometimes represent geographical correspondence. The fossil data, easily dispersible mucilaginous seeds, widespread autogamous breeding systems, and low levels of sequence divergence between species from different continents or islands suggest a rapid radiation of Lepidium by long-distance dispersal in the Pliocene/Pleistocene. As a consequence of climatic changes in this geological epoch, arid/semiarid areas were established, providing favorable conditions for the radiation of Lepidium by which the genus attained its worldwide distribution.     

Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China

Ficus, with about 755 species, diverse habits and complicated co‐evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH‐psbA, psbK‐psbI, atpF‐atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra‐ and inter‐specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK‐psbI and trnH‐psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF‐atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH‐psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.