cpDNA Analysis Research Articles

Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives

In Japan, two Eutrema species, wasabi (Eutrema japonicum, the important traditional Japanese condiment) and yuriwasabi (E. tenue), have been recognized as endemic species. We sequenced complete chloroplast (cp) genomes of seven wasabi and yuriwasabi accessions from Japan to study their phylogeny and evolution, using molecular dating of species divergence. Phylogenetic analyses of the complete cp DNA of these two Japanese species and five other Eurasian Eutrema species revealed that wasabi and yuriwasabi did not form a monophyletic group. One yuriwasabi accession (Gifu) formed a clade with E. yunnanense from China, indicating that this accession should be considered as a different species from the other yuriwasabi accessions. We reveal that Japanese Eutrema species diverged from the ‘E. yunnanense–yuriwasabi (Gifu)’ clade approximately 1.3 million years ago (Mya), suggesting that the connection between Japan and the Eurasian continent has existed more recently than the Quaternary period. The abundance of cp sequence data in this study also allowed the detection of genetic differentiation among wasabi cultivars. The two polymorphic sites detected between ‘Fujidaruma’ and ‘Shimane No.3’ were used to develop genotyping markers. The cp genome information provided here will thus inform the evolutionary histories of Japanese Eutrema species and help in genotyping wasabi cultivars.

Landscape Features and Climatic Forces Shape the Genetic Structure and Evolutionary History of an Oak Species (Quercus chenii) in East China.

Major topographic features facilitate intraspecific divergence through geographic isolation. This process may be enhanced by environmental isolation along climatic gradients, but also may be reduced by range shifts under rapid climatic changes. In this study, we examined how topography and climate have interacted over time and space to influence the genetic structure and evolutionary history of Quercus chenii, a deciduous oak species representative of the East China flora. Based on the nuclear microsatellite variation at 14 loci, we identified multiple genetic boundaries that were well associated with persistent landscape barriers of East China. Redundancy analysis indicated that both geography and climate explained similar amounts of intraspecific variation. Ecological differences along altitudinal gradients may have driven the divergence between highlands and lowlands. However, range expansions during the Last Interglacial as inferred from approximate Bayesian computation (ABC) may have increased the genetic diversity and eliminated the differentiation of lowland populations via admixture. Chloroplast (cp) DNA analysis of four intergenic spacers (2,866 bp in length) identified a total of 18 haplotypes, 15 of which were private to a single population, probably a result of long-term isolation among multiple montane habitats. A time-calibrated phylogeny suggested that palaeoclimatic changes of the Miocene underlay the lineage divergence of three major clades. In combination with ecological niche modeling (ENM), we concluded that mountainous areas with higher climatic stability are more likely to be glacial refugia that preserved higher phylogenetic diversity, while plains and basins may have acted as dispersal corridors for the post-glacial south-to-north migration. Our findings provide compelling evidence that both topography and climate have shaped the pattern of genetic variation of Q. chenii. Mountains as barriers facilitated differentiation through both geographic and environmental isolation, whereas lowlands as corridors increased the population connectivity especially when the species experienced range expansions.

Misidentifications of Echinochloa crus‐galli var. formosensis as Echinochloa oryzicola as a result of similar cpDNA sequences

Echinochloa crus‐galli (L.) Beauv. var. formosensis Ohwi (2n = 6x = 54, AABBCC genomes) and Echinochloa oryzicola (Vasinger) Vasinger (2n = 4x = 36, AABB) are major paddy weeds in East and Southeast Asia. E. oryzicola has been generally considered to be a paternal genome donor of E. crus‐galli s. l., which includes E. crus‐galli var. formosensis based on cpDNA sequences. Thus, molecular characterization using polymerase chain reaction‐restriction fragment length polymorphism analysis of cpDNA has been proposed as a reliable method for discriminating between the two species. In this study, we report that four accessions of E. crus‐galli var. formosensis from Okinawa, Nagasaki, Shizuoka and Tokyo had similar cpDNA sequences to E. oryzicola and had been misidentified as E. oryzicola using molecular methods. In addition, our results demonstrated that these accessions likely inherited their chloroplast genomes from E. oryzicola and not from an anonymous diploid species during polyploidization. Our findings provide new insights into the evolution of E. crus‐galli s. l. and suggest that identification using the cpDNA molecular method alone is not an appropriate approach to differentiate E. crus‐galli var. formosensis and E. oryzicola.

Discovery of chloroplast capture in Juniperus excelsa complex by multi- locus phylogeny

Previous studies of nrDNA (nuclear DNA) of Juniperus seravschanica indicated its nuclear DNA (ITS) was from an ancestor of J. polycarpos. However, analysis of cpDNA (chloroplast DNA) suggested the taxon had derived its chloroplast from an ancestor of J. foetidissima. That study has been viewed as putative, because the ITS region is sometimes unreliable for the detection of ancestral hybrids due to concerted evolution and lineage sorting. The recent availability of several single copy nuclear genes (SCNGs) with primers specifically designed for Juniperus presented an opportunity to fully investigate this case of putative chloroplast capture. Three phylogenetic analyses using five SCNGs (LHCA4, maldehy, myb, CnAIP3 and 4CL), ITS region, and four cpDNAs (petN- psbM, trnD-trnT, trnL-trnF and trnS-trnG) were performed on J. seravschanica, as well as other members of the J. excelsa complex: J. excelsa, J. polycarpos, and J. p. var. turcomanica. Analyses revealed incongruence between SCNGs, ITS region and cpDNA showing that J. seravschanica contains an ancestral J. foetidissima/ J. thruifera cp genome. In addition, the phylogenies indicate that the J. excelsa complex is composed of three distinct clades at the species level: J. excelsa, J. polycarpos and J. seravschanica and two varieties of J. polycarpos: J. p. var. polycarpos and J. p. var. turcomanica.

Environmental variation shapes genetic variation in Bouteloua gracilis: Implications for restoration management of natural populations and cultivated varieties in the southwestern United States.

With the increasing frequency of large‐scale restoration efforts, the need to understand the adaptive genetic structure of natural plant populations and their relation to heavily utilized cultivars is critical. Bouteloua gracilis (blue grama) is a wind‐dispersed, perennial grass consisting of several cytotypes (2n = 2×–6×) with a widespread distribution in western North America. The species is locally dominant and used regularly in restoration treatments. Using amplified fragment length polymorphism (AFLP) and cpDNA analyses, we assessed the genetic variability and adaptive genetic structure of blue grama within and among 44 sampling sites that are representative of the species’ environmental and habitat diversity in the southwestern United States. Five cultivars were also included to investigate genetic diversity and differentiation in natural versus cultivated populations. Three main findings resulted from this study: (a) Ninety‐four polymorphic AFLP markers distinguished two population clusters defined largely by samples on and off the Colorado Plateau; (b) substructure of samples on the Colorado Plateau was indicated by genetic divergence between boundary and interior regions, and was supported by cytotype distribution and cpDNA analysis; and (c) six AFLP markers were identified as “outliers,” consistent with being under selection. These loci were significantly correlated to mean annual temperature, mean annual precipitation, precipitation of driest quarter, and precipitation of wettest quarter in natural populations, but not in cultivated samples. Marker × environment relationships were found to be largely influenced by cytotype and cultivar development. Our results demonstrate that blue grama is genetically variable, and exhibits genetic structure, which is shaped, in part, by environmental variability across the Colorado Plateau. Information from our study can be used to guide the selection of seed source populations for commercial development and long‐term conservation management of B. gracilis, which could include genetic assessments of diversity and the adaptive potential of both natural and cultivated populations for wildland restoration.

Different species or genetically divergent populations? Integrative species delimitation of the Primulina hochiensis complex from isolated karst habitats

To consistently and objectively delineate species-level divergence from population subdivision has been a challenge in systematics. This is particularly evident in naturally fragmented and allopatric systems in which small population size often leads to extreme population structuring. Here we evaluated the robustness of the species delimitation methods implemented in BEAST, BPP, and iBPP in the Primulina hochiensis complex comprising four described and one candidate species (five taxa in total) distributed in karst landscapes of southern China. We analyzed levels of molecular and morphological divergence among species using multilocus sequence data (nine chloroplast loci and 10 nuclear loci), and morphological data (16 quantitative and 12 qualitative traits), for 124 individuals from 25 populations of the complex. Independent analyses of cpDNA and nDNA sequence data revealed high levels of genetic differentiation among the five taxa. Both BPP and iBPP delimited five candidate species, which correspond to the five genetic clusters recovered with population structure analysis. In contrast, morphological differences among populations were more limited, so that results from principal component analysis (PCA) recovered only three distinct clusters. We ruled out the possibility of morphologically cryptic species because reciprocally monophyletic groups were not supported among the morphologically un-differentiated taxa. Our results represent a case where extreme population genetic structuring leads to oversplit of species diversity by molecular data using the multispecies coalescent (MSC) methods. The observed congruence across multiple analyses corroborates the recognition of a new species P. lianpingensis and indicates its sister species relationship with P. yingdeensis. This study highlights the dangers of violating model assumption and the importance of incorporating multiple evidence into species delimitation of a particular system.

Differentiation of the endemic Greek genus Hymenonema and its relatives of subtribe Scolyminae (Compositae, Cichorieae) based on a multilocus species tree reconstruction

Hymenonema (Compositae, tribe Cichorieae) together with the genera Catananche, Gundelia, and Scolymus forms the subtribe Scolyminae. It is endemic to Greece and consists of two species, Hymenonema laconicum and Hymenonema graecum, which occur in the south Peloponnisos and central Aegean area, respectively. The present contribution aims at a phylogenetic reconstruction of evolutionary relationships among the 12 species of the subtribe, focusing on the temporal and spatial framework for its evolution. The phylogenetic relationships among the members of Scolyminae were inferred from molecular data based on the multi-copy region of the nrDNA internal transcribed spacers ITS1 and ITS2, two intergenic spacers of the cpDNA (trnL-trnF, rpl32-trnL), and one single-copy nuclear region (D10). The gene trees were reconstructed using Bayesian phylogenetic methods. All gene trees support the monophyly of Hymenonema and the sister-group relationship with the genus Scolymus. The further sister-group relationship of this group (Hymenonema–Scolymus) with Catananche is also supported by nrDNA and cpDNA analyses. Finally, a species tree (inferred in a Bayesian coalescent framework) was reconstructed and dates the divergence time between the two Hymenonema species to the Pleistocene (around 1.3 Ma ago). Maximum likelihood-based biogeographical reconstructions suggest a Miocene (pre-Messinian) differentiation of the subtribe on the northern Tethyan platform, followed by Miocene/Pliocene dispersal events to the western Mediterranean and North-African platforms and final, small-scale vicariance events within the genera in the Pleistocene.

Untangling the generic boundaries in tribe Marrubieae (Lamiaceae: Lamioideae) using nuclear and plastid DNA sequences

AbstractTribe Marrubieae (Lamiaceae: Lamioideae) comprises about 91 species classified in four genera distributed from Europe to West and Central Asia and North Africa. Here, we present a phylogenetic analysis of cpDNA (rpl32–trnL, rps16, trnL intron, trnL–trnF spacer) and nuclear ribosomal (ITS) sequence data of 68 accessions representing all genera and most sections of Marrubieae. Our taxon sampling covers is dense for the genera Ballota and Marrubium, including a total of 60 of 85 currently recognized taxa (species and subspecies). Parsimony and Bayesian analyses of individual nrITS and cpDNA markers, as well as the combined plastid dataset, produced largely congruent trees. Our results confirm the monophyly of tribe Marrubieae. All analyses retrieved four well supported clades in the tribe. The phylogenetic position of the genera Acanthoprasium and Moluccella is consistent with previous studies and both are recovered as monophyletic. The most important result of the present study is the splitting of the genus Ballota, in its traditional concept, into two well–supported clades matching well with the genera Ballota and Pseudodictamnus. Moreover, Marrubium, in its current circumscription, is paraphyletic, but will become monophyletic by transferring Ballota deserti to this genus. In conclusion, we propose the recognition of five genera in tribe Marrubieae: Acanthoprasium, Ballota, Moluccella, Marrubium and the resurrected segregate Pseudodictamnus. The required new combinations are proposed.

Polyphyletic origin in Pimpinella (Apiaceae): evidence in Western Europe.

The genus Pimpinella L. comprises about 150 species, being one of the largest genera within the family Apiaceae (subfamily Apioideae). Previous molecular phylogenetic studies have shown that Pimpinella is a taxonomically complex group. In this study, evolutionary relationships among representatives from Western Europe have been inferred from phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer (ITS 1 and ITS 2) and plastid sequences (trnL intron and the trnL-F spacer), with a representative sampling included (168 accessions in the ITS analysis, representing 158 species; and 42 accessions in the cpDNA analysis representing 35 taxa of Pimpinella and closely related species). All analyses resolved that Pimpinella is a non-monophyletic group, and Pimpinella's taxa that grow in Western Europe are part of phylogenetically independent groups that correspond to three different tribes of the subfamily Apioideae: Pimpinelleae (core group), Pyramidoptereae and Smyrnieae.

Unveiling the origin of Quercus serrata subsp. mongolicoides found in Honshu, Japan, by using genetic and morphological analyses

AbstractQuercus mongolica is a tree found in temperate deciduous forests in east Asia. In Japan, Q. mongolica var. crispula is commonly found; moreover, an oak whose morphology is similar to that of Q. mongolica var. mongolica of the Asian continent has been found in certain areas of Honshu and Hokkaido. Recently, the oak found in Honshu was described as Q. serrata subsp. mongolicoides (QSM). However, genetic comparison between this oak and Q. mongolica var. mongolica of the Asian continent has not been performed; the origin of QSM is thus unknown. Therefore, we aimed to determine the origin of QSM by conducting nuclear microsatellite (nSSR), chloroplast DNA (cpDNA) and leaf morphology analyses for the three taxa, as well as other congeners. The cpDNA variation overlapped among the three taxa, suggesting low discrimination ability for these taxa. Although morphological congruency was found between QSM and Q. mongolica var. mongolica, results of nSSR analyses showed that QSM contained a genetic admixture of Q. mongolica var. mongolica of the Asian continent and Q. mongolica var. crispula of Japan, bolstering an admixture hypothesis. The nSSR and cpDNA analyses suggested that Q. mongolica var. crispula can be the progenitor of Q. mongolica var. mongolica and harbors the ancestral cpDNA haplotypes. Therefore, we concluded that QSM might have been created by an admixture that likely occurred within Japan between Q. mongolica var. crispula and putative relict Q. mongolica var. mongolica, which might have diverged in or around Japan from Q. mongolica var. crispula during the late Pleistocene.

Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae).

Previous molecular phylogenetic analyses have resolved the Australian bloodwood eucalypt genus Corymbia (~100 species) as either monophyletic or paraphyletic with respect to Angophora (9–10 species). Here we assess relationships of Corymbia and Angophora using a large dataset of chloroplast DNA sequences (121,016 base pairs; from 90 accessions representing 55 Corymbia and 8 Angophora species, plus 33 accessions of related genera), skimmed from high throughput sequencing of genomic DNA, and compare results with new analyses of nuclear ITS sequences (119 accessions) from previous studies. Maximum likelihood and maximum parsimony analyses of cpDNA resolve well supported trees with most nodes having >95% bootstrap support. These trees strongly reject monophyly of Corymbia, its two subgenera (Corymbia and Blakella), most taxonomic sections (Abbreviatae, Maculatae, Naviculares, Septentrionales), and several species. ITS trees weakly indicate paraphyly of Corymbia (bootstrap support <50% for maximum likelihood, and 71% for parsimony), but are highly incongruent with the cpDNA analyses, in that they support monophyly of both subgenera and some taxonomic sections of Corymbia. The striking incongruence between cpDNA trees and both morphological taxonomy and ITS trees is attributed largely to chloroplast introgression between taxa, because of geographic sharing of chloroplast clades across taxonomic groups. Such introgression has been widely inferred in studies of the related genus Eucalyptus. This is the first report of its likely prevalence in Corymbia and Angophora, but this is consistent with previous morphological inferences of hybridisation between species. Our findings (based on continent-wide sampling) highlight a need for more focussed studies to assess the extent of hybridisation and introgression in the evolutionary history of these genera, and that critical testing of the classification of Corymbia and Angophora requires additional sequence data from nuclear genomes.

Evidence for continual hybridization rather than hybrid speciation between Ligularia duciformis and L.paradoxa (Asteraceae).

BackgroundHybrids possess phenotypic traits that are often intermediate between their parental taxa, which commonly serves as evidence of hybridization in morphological analyses. Natural hybridization has been shown to occur frequently in Ligularia (Asteraceae). In a previous study, Ligularia ×maoniushanensis was demonstrated as a natural hybrid species between L. duciformis and L. paradoxa based on morphological and reproductive traits.MethodsWe used three chloroplast (cpDNA) fragments (psbA-trnH, trnL-rpl32 and trnQ-5′rps16), the nuclear ribosomal internal transcribed spacer (nrITS), and co-dominant SSR and dominant ISSR markers to study natural hybridization between L. duciformis and L. paradoxa growing sympatrically in two locations. Parental taxa were inferred using network analyses of cpDNA and nrITS haplotypes. Admixture among individuals was examined using the Bayesian clustering programs STRUCTURE and NewHybrids based on the SSR and ISSR data; and potential introgression in the SSR loci was assessed using the INTROGRESS package.ResultsThe putative parental species were clearly distinguished from other sympatric Ligularia species by nrITS data, and L. ×maoniushanensis individuals were confirmed to be the hybrid offspring of L. duciformis and L. paradoxa. Moreover, introgression was detected among several individuals morphologically identified as L. duciformis or L. paradoxa. Analyses of the cpDNA data revealed primarily unidirectional hybridization between L. duciformis and L. paradoxa, with L. paradoxa as the maternal parent in Mt. Maoniu, whereas bidirectional but asymmetrical hybridization was inferred to occur in Heihai Lake. The STRUCTURE analyses based on the SSR data detected two distinct clusters among the three taxa. The NewHybrids analyses showed that individuals circumscribed as L. ×maoniushanensis were dominated by early- and later-generation and backcrossing hybrids. The NewHybrids results based on the ISSR data were congruent with SSR results. In addition, introgression was detected in some SSR loci, and heterogeneity among loci was found in terms of detected patterns of introgression.ConclusionsOur data provide strong evidence for hybridization and introgression between L. duciformis and L. paradoxa. Ligularia ×maoniushanensis was demonstrated to be of hybrid origin. Since no evident reproductive isolation was found between the two parental species, detected hybrids appear to be part of hybrid swarms resulting from frequent and ongoing gene flow, which might impede the formation of a new hybrid species.

Do we have different varieties in Acer velutinum (Sapindaceae): Morphological and molecular studies

The genus Acer L. has about 156 species which are distributed in Asia, Europe, Northern Africa and North America. The species of Acer show extensive morphological and molecular diversity and the species that co-occur may hybridize; therefore, sometimes the species delimitation turns out to be difficult. Eight Acer (maple) species have been reported in Iran. The velvet maple—Acer velutinum Boiss (1846: 28)—is one of the largest maples in the world and the most frequent maple species in Iran. Morphologically speaking, two varieties have been cited for this species. Due to the extensive morphological variability, however, the researchers encounter difficulty delimiting these two varieties. Against this backdrop, the present study was performed with the aim to delimit two varieties of Acer velutinum by using both morphological and molecular approaches. It also aimed to proffer data on the genetic diversity of this species in the country. Nighty-four plant specimens of A. velutinum were randomly collected within 14 geographical populations at four provinces in Hyrcanian forests of Iran. The PsbA-trnHGUG intergenic spacer sequence of cp-DNA and the ISSR molecular markers along with the morphometric analysis and image analysis of the leaf shape were used. The numerical and phylogenetic analyses of the tree specimens from the presumed varieties of A. velutinum—based on morphology, image analysis of leaf shape, ISSR and cp-DNA—did not delimit these varieties; hence, A. velutinum was considered to have a high level of intergrading morphological variability. The population genetic study revealed significant molecular differences among the studied populations. The STRUCTURE plot and gene flow analysis (Nm), nevertheless, revealed some degree of shared alleles among the populations. The Mantel tests demonstrated significant correlation between the geographical and genetic distance of the studied populations; it further signaled the fact that the gene flow occurred mainly between the coterminous populations.

Asymmetrical natural hybridization varies among hybrid swarms between two diploid Rhododendron species.

The extent to which hybridization leads to gene flow between plant species depends on the structure of hybrid populations. However, if this varies between locations, species barriers might prove permeable in some locations but not in others. To assess possible variation in hybrid population structure, the magnitude and direction of natural hybridization between two Chinese endemic species, Rhododendron spiciferum and Rhododendron spinuliferum , were evaluated. Thirteen nuclear microsatellite markers were employed to characterize 566 individuals collected from 15 non-allopatric populations and nine allopatric parental populations. Chloroplast DNA (cpDNA) sequences were obtained from a subset of samples. Genetic structure and direction of gene flow was determined using a combination of STRUCTURE and NEWHYBRIDS analysis. Nuclear analysis revealed that parental taxa formed two genetically distinct clusters and hybrids shared the genetic background of both parents and did not form a separate genetic lineage. Overall, hybrid swarms were dominated by early- and later-generation hybrids, with a significantly higher proportion of hybrids (59·6%) possessing >50% R. spiciferum-like nuclear germplasm. The cpDNA analysis further indicated that a significantly greater proportion of hybrids (61·1%) possessed the R. spiciferum cpDNA haplotype. Gene flow between R. spiciferum and R. spinuliferum was found to be bidirectional in 14 of the 15 hybrid swarms and asymmetrical in six hybrid swarms. Asymmetrical gene flow was evident for only nuclear DNA (nDNA) in two populations, for only cpDNA in three populations, and for both nDNA and cpDNA in one population. Collectively, the variation in genetic structure found among the 15 hybrid swarms suggests that introgression rather than hybrid speciation is a more likely outcome of hybridization between these hybridizing taxa.

Spatial patterns and intraspecific diversity of the glacial relict legume species Vavilovia formosa (Stev.) Fed. in Eurasia

Vavilovia formosa is one of five genera in tribe Fabeae, (Fabaceae, Leguminosae) with close phylogenetic relationships to Pisum. It grows in subalpine and alpine levels in Armenia, Azerbaijan, Georgia, Iran, Iraq, Lebanon, Russia and Turkey and is recognized as an endangered and protected plant. This study was conducted to reveal its intraspecific variability, as well as to predict the past, extant and future species distribution range. We analysed 51 accessions with common phylogenetic markers (trnF-trnL, trnS-trnG, matK, rbcL, psbA-trnH and ITS). These represent in total up to 2551 bp of chloroplast and 664 bp of nuclear sequences per sample. Two populations from Turkey and Armenia were analysed for genetic diversity by AFLP. Leaf morphometry was conducted on 1457 leaflets from 43 specimens. Extracted bioclimatic parameters were used for niche-modelling approach. Analysis of cpDNA revealed two haplotypes, 12 samples from Armenia, Daghestan, Nakhichevan and Iran belonged to H1 group, while 39 samples of all Turkish and part of Armenian were in H2 group. The mean intrapopulation diversity based on AFLP was low (H E = 0.088) indicating limited outcrossing rate. A significantly positive correlation between geographical latitude and leaf area ( $$\rho$$ = 0.527, p < 0.05) was found. Niche modelling has shown temporal variation of predicted occurrence across the projected time periods. Vavilovia formosa has suffered a range reduction following climate warming after last glacial maximum, which classify this species as cold-adapted among the Fabeae species as well as a glacial relict.

Diversity analysis of Tomato leaf curl New Delhi virus-[potato], causing apical leaf curl disease of potato in India

The complete coat protein (cp) gene sequence of eighty Tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) isolates collected from eleven states were determined. Phylogenetic analysis based on cp gene grouped the isolates into two major clades (I & II) and they shared 95.9–100.0% identity. The DNA A and DNA B of eight representative isolates (six from clade I and two from clade II) were 2739–2740 and 2692–2694 nts long and shared 94.6–99.4% and 97.2–99.5% homology within the isolates, respectively. Among the eight isolates, the DNA A of two isolates (Clade II), GWA-5 and FAI-19 had 94.6–95.3% sequence identity to other six isolates and formed a sub-clade within the ToLCNDV-[potato] isolates. Similar grouping was also revealed with AC1 and AC4 genes of these eight isolates. The DNA A components shared more than 90.0% identity with the DNA A of ToLCNDV isolates from cucurbitaceous crops, tomato, bhendi, 89.0–90.0% with ToLCNDV-papaya isolates and 70.4–74.0% with other tomato leaf curl viruses. Hence, the begomovirus infecting potatoes are the ToLCNDV isolates, designated as ToLCNDV-[potato]. Whereas, the DNA B components shared 86.6–91.7% identity with ToLCNDV isolates from cucurbits, tomato and bhendi. Evidence for intra-species recombination was detected only in DNA A with a maximum of three events in GWA-5 and FAI-19 isolates. Analysis of cp gene, DNA A, iterons and recombination events clearly indicate that two groups of ToLCNDV-[potato] infects potato in India.

Taxonomic resolution of the Tetratheca hirsuta (Elaeocarpaceae) species complex using an integrative approach

The Tetratheca hirsuta Lindl. species complex from south-west Western Australia is one of the last unresolved complexes in this Australian endemic genus, and comprises the highly variable T. hirsuta, two rare, phrase-named taxa, and the closely allied T. hispidissima Steetz. An integrative approach, incorporating multivariate morphometric analysis and molecular phylogenetic and phenetic analyses of nrDNA (ETS) and cpDNA (ndhF–trnL, rpl16, trnS–trnG5ʹ2S), was used to investigate taxonomic boundaries within the complex. Morphological data showed clear divergence within the complex, and allowed several taxonomically uncertain individuals to be assigned. Phenetic and phylogenetic analyses of ETS showed substantial congruence with morphology, indicating that the groups recognised through morphometric analyses are also genetically divergent. By comparison, the chloroplast regions yielded incongruent gene trees, perhaps owing to incomplete lineage sorting, hybridisation or slow evolution of cpDNA. The present results support the recognition of the following four taxa: a morphologically and geographically expanded T. hispidissima, which is highly divergent from the remainder of the complex, and a closer grouping of T. hirsuta subsp. boonanarring Joyce &amp; R.Butcher subsp. nov., T. hirsuta subsp. viminea (Lindl.) Joyce comb. et stat. nov. and T. hirsuta subsp. hirsuta.

Allopolyploid Origin of Chenopodium album s. str. (Chenopodiaceae): A Molecular and Cytogenetic Insight.

Reticulate evolution is characterized by occasional hybridization between two species, creating a network of closely related taxa below and at the species level. In the present research, we aimed to verify the hypothesis of the allopolyploid origin of hexaploid C. album s. str., identify its putative parents and estimate the frequency of allopolyploidization events. We sampled 122 individuals of the C. album aggregate, covering most of its distribution range in Eurasia. Our samples included putative progenitors of C. album s. str. of both ploidy levels, i.e. diploids (C. ficifolium, C. suecicum) and tetraploids (C. striatiforme, C. strictum). To fulfil these objectives, we analysed sequence variation in the nrDNA ITS region and the rpl32-trnL intergenic spacer of cpDNA and performed genomic in-situ hybridization (GISH). Our study confirms the allohexaploid origin of C. album s. str. Analysis of cpDNA revealed tetraploids as the maternal species. In most accessions of hexaploid C. album s. str., ITS sequences were completely or nearly completely homogenized towards the tetraploid maternal ribotype; a tetraploid species therefore served as one genome donor. GISH revealed a strong hybridization signal on the same eighteen chromosomes of C. album s. str. with both diploid species C. ficifolium and C. suecicum. The second genome donor was therefore a diploid species. Moreover, some individuals with completely unhomogenized ITS sequences were found. Thus, hexaploid individuals of C. album s. str. with ITS sequences homogenized to different degrees may represent hybrids of different ages. This proves the existence of at least two different allopolyploid lineages, indicating a polyphyletic origin of C. album s. str.

Species delimitation, genetic diversity and structure of the European indigenous wild pear (Pyrus pyraster) in Saxony, Germany

The present study investigated genetic diversity, structure and hybridization in a collection of the endangered wild pear species Pyrus pyraster (L.) Burgd. A total of 278 putative ‘true type’ P. pyraster trees originating from seven populations in the federal state of Saxony in Germany were analyzed along with 35 pear cultivars commonly cultivated in Saxony. The genetic analysis was performed using nine nuclear microsatellite markers (ncSSR) and two paternally inherited chloroplast marker (cpDNA) amplifying in the intergenic spacer region trnQ–rps16 and the intron region rps16. On basis of the ncSSR dataset after STRUCTURE analysis 80 % of the wild pear individuals were assigned as ‘true type’ P. pyraster genotypes. The cpDNA analysis showed shared haplotypes in P. pyraster and P. communis but with an unequal frequency in both species. The analysis of molecular variance resulted in a moderate (ncSSR) and great (cpDNA) variation among ‘true type’ P. pyraster and the pear cultivars. The genetic diversity in the ‘true type’ P. pyraster populations was still high and the genetic structure between the populations low (ncSSR and cpDNA) indicating a genetic exchange between the populations by pollen and seeds. The clear discrimination between the P. pyraster and P. communis confirms our expectation of the existence of ‘true type’ P. pyraster individuals in the study area. The existing genetic integrity and the high genetic diversity argue for the implementation of preservation measures in P. pyraster.

Ecological distributions, phenological isolation, and genetic structure in sympatric and parapatric populations of the Larrea tridentata polyploid complex.

Polyploidy is widely recognized as a mechanism of diversification. Contributions of polyploidy to specific pre- and postzygotic barriers-and classifications of polyploid speciation as "ecological" vs. "non-ecological"-are more contentious. Evaluation of these issues requires comprehensive studies that test ecological characteristics of cytotypes as well as the coincidence of genetic structure with cytotype distributions. We investigated a classical example of autopolyploid speciation, Larrea tridentata, at multiple areas of cytotype co-occurrence. Habitat and phenological differences were compared between diploid, tetraploid, and hexaploid populations on the basis of edaphic, community composition, and flowering time surveys. Frequency of hybridization between diploids and tetraploids was investigated using a diploid-specific chloroplast DNA (cpDNA) marker; genetic structure for all cytotypes was assessed using amplified fragment length polymorphisms (AFLPs). Across contact zones, we found cytotypes in habitats distinguished by soil and vegetation. We observed modest differences in timing and production of flowers, indicating a degree of assortative mating that was asymmetric between cytotypes. Nonetheless, cpDNA analyses in diploid-tetraploid contact zones suggested that ∼5% of tetraploid plants had hybrid origins involving unilateral sexual polyploidization. Genetic structure of AFLPs largely coincided with cytotype distributions in diploid-tetraploid contact zones. In contrast, there was little structure in areas of contact between tetraploids and hexaploids, suggesting intercytotype gene flow or recurrent hexaploid formation. Diploid, tetraploid, and hexaploid cytotypes of L. tridentata are segregated by environmental distributions and flowering phenology in contact zones, with diploid and tetraploid populations having corresponding differences in genetic structure.