Hybrid status of Tibouchina urvilleana Cogn. revealed by ITS sequences of nuclear ribosomal DNA

Tibouchina urvilleana Cogn. is native to southern Brazil and currently cultivated as an important ornamental shrub in frost free areas around the world. Its rapid vegetative growth and sterility suggests that it might be of hybrid origin. In this study, Internal Transcribed Spacer (ITS) of nuclear ribosomal DNA and chloroplast trnL-trnF spacer from T. urvilleana and three other congeneric species were sequenced to test its hybrid status. Cloning sequencing revealed two distinct types of ITS sequences from T. urvilleana, with one type almost identical with Tibouchina aspera. Genetic distance between the two types was much larger than the average interspecific genetic distances calculated from other Tibouchina species. Sequencing of chloroplast trnL-trnF spacer showed that T. urvilleana has identical sequence with T. aspera, but differed from other congeneric species by one nucleotide substitution and two indels. Molecular data demonstrated clearly that T. urvilleana indeed was a hybrid, with T. aspera or closely related species acting as the maternal parent.     

Extensive length variation in the cpDNA trnT-trnF region of hemiparasitic Pedicularis and its phylogenetic implications

The cpDNA trnT-trnF region, a molecular marker widely used in the phylogenetic reconstruction at lower taxonomic levels, is relatively conserved in size and structure. In this region single length variation over 100 bp is much less common than small deletion for congeneric species of angiosperms. Here we examined evolutionary patterns of the trnT-trnF region in 43 species of Pedicularis, a species-rich genus with adaptive radiation. Four independent large deletions, varying from 203 to 297 bp in length, were detected from nine species of the genus, which might result from slipped-strand mispairing. These deletions occurred in different locations of the cpDNA region and in different clades of the phylogenetic tree, indicating that the deletion of large cpDNA fragments may be very frequent in the hemiparasitic lineage of the family Orobanchaceae. Parsimony analyses showed that section Cyathophora of Pedicularis, endemic to the Sino-Himalayan region, was a strongly supported monophyletic group. This section could have a recent origin followed by rapid radiation, considering that it is characterized by a large deletion in the trnT-trnF region and a relatively low interspecific sequence divergence.     

Aspidistra obtusata sp. nov. (Asparagaceae) from Vietnam

Aspidistra obtusata is described and illustrated as a new species from southern Vietnam. The new species resembles A. truongii, but differs in smaller diameter of perigone (12.6–14.9 mm vs 25–35 mm), shape of tepals (elliptic, obtuse vs triangular‐ovate) and pistil (widely obconic vs mushroom‐shaped). A molecular characterization of the new species (based on data of plastid psbA‐trnH and nuclear 5S‐NTS regions) is also given, and compared with the most similar congeners.     

The complete chloroplast genome of Myriophyllum spicatum reveals a 4‐kb inversion and new insights regarding plastome evolution in Haloragaceae

Myriophyllum, among the most species‐rich genera of aquatic angiosperms with ca. 68 species, is an extensively distributed hydrophyte lineage in the cosmopolitan family Haloragaceae. The chloroplast (cp) genome is useful in the study of genetic evolution, phylogenetic analysis, and molecular dating of controversial taxa. Here, we sequenced and assembled the whole chloroplast genome of Myriophyllum spicatum L. and compared it to other species in the order Saxifragales. The complete chloroplast genome sequence of M. spicatum is 158,858 bp long and displays a quadripartite structure with two inverted repeats (IR) separating the large single copy (LSC) region from the small single copy (SSC) region. Based on sequence identification and the phylogenetic analysis, a 4‐kb phylogenetically informative inversion between trnE‐trnC in Myriophyllum was determined, and we have placed this inversion on a lineage specific to Myriophyllum and its close relatives. The divergence time estimation suggested that the trnE‐trnC inversion possibly occurred between the upper Cretaceous (72.54 MYA) and middle Eocene (47.28 MYA) before the divergence of Myriophyllum from its most recent common ancestor. The unique 4‐kb inversion might be caused by an occurrence of nonrandom recombination associated with climate changes around the K‐Pg boundary, making it interesting for future evolutionary investigations.     

Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia

Aim The aim of this study was to test hypotheses regarding some of the main phylogeographical patterns proposed for European plants, in particular the locations of glacial refugia, the post‐glacial colonization routes, and genetic affinities between southern (alpine) and northern (boreal) populations. Location The mountains of Europe (Alps, Balkans, Carpathians, Central Massif, Pyrenees, Scandinavian chain, Sudetes), and central European/southern Scandinavian lowlands. Methods As our model system we used Pulsatilla vernalis, a widely distributed European herbaceous plant occurring both in the high‐mountain environments of the Alps and other European ranges and in lowlands north of these ranges up to Scandinavia. Based on a distribution‐wide sampling of 61 populations, we estimated chloroplast DNA (cpDNA) variation along six regions using polymerase chain reaction–restriction fragment‐length polymorphisms (PCR–RFLPs) (trnH–trnK, trnK–trnK, trnC–trnD, psbC–trnS, psaA–trnS, trnL–trnF) and further sequencing of trnL–trnF and trnH–psbA. In addition, 11 samples of other European species of Pulsatilla were sequenced to survey the genus‐scale cpDNA variation. Results Eleven PCR–RFLP polymorphisms were detected in P. vernalis, revealing seven haplotypes. They formed two distinct genetic groups. Three haplotypes representing both groups dominated and were widely distributed across Europe, whereas the others were restricted to localized regions (central Alps, Tatras/Sudetes mountains) or single populations. Sequencing analysis confirmed the reliability of PCR–RFLPs and homology of haplotypes across their distribution. The chloroplast DNA variation across the section Pulsatilla was low, but P. vernalis did not share haplotypes with other species. Main conclusions The genetic distinctiveness of P. vernalis populations from the south‐western Alps with respect to other Alpine populations, as well as the affinities between the former populations and those from the eastern Pyrenees, is demonstrated, thus providing support for the conclusions of previous studies. Glacial refugia in the Dolomites are also suggested. Isolation is inferred for the high‐mountain populations from the Tatras and Sudetes; this is in contrast to the case for the Balkans, which harboured the common haplotype. Specific microsatellite variation indicates the occurrence of periglacial lowland refugia north of the Alps, acting as a source for the post‐glacial colonization of Scandinavia. The presence of different fixed haplotypes in eastern and western Scandinavia, however, suggests independent post‐glacial colonization of these two areas, with possible founder effects.     

Allium ursinum L. in Germany – surprisingly low genetic variability

Allium ursinum s.l. is a widely spread species of the herb layer in beech forests throughout Europe. Little is known about its phylogenetic origin and its biogeographic history. Molecular genetic analyses of eleven populations from Germany were used to clarify the relationship between populations of A. ursinum s.l. and its relationship to several other species of the genus Allium. The study focused mainly on the Teutoburg Forest in Lower Saxony and the Franconian mountain area in Bavaria. Sequences of the nuclear internal transcribed spacer ITS, and the external transcribed spacer ETS, as well as the plastidic trn L‐rpl 32 and the trn L‐trn F spacer regions were compared. No variation was detected within the species. Even sequences of populations from Belfast, Ireland did not differ from populations of Germany. The closest relative to Allium ursinum s.l. turned out to be Allium moly or Allium scorzonerifolium from the section Molium. Random amplified polymorphic DNA fingerprinting was performed and revealed 29% polymorphic bands. Genetic distances of the populations within the Teutoburg Forest coincided with geographical distances. Three populations (Osnabrück Westerberg, Osnabrück Honeburg and Leer, East Frisia) out of eleven analysed populations were identified as garden escapes. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Morphological and Molecular Identification of the New Species,Trichodina pseudoheterodentata sp. n. (Ciliophora,Mobilida, Trichodinidae) from the Channel Catfish,Ictalurus punctatus,in Chongqing China

It is difficult to differentiate similar trichodinids solely based on morphological examination, thus other identification methods, such as molecular identification, are necessary for identification. One mobilid ciliate named Trichodina pseudoheterodentata sp. n. was isolated from the gills of channel catfish, Ictalurus punctatus, in Chongqing, China. In the present study, its SSU rDNA was sequenced for the first time. Based on the results from both morphological identification and SSU rDNA sequencing, the new species was identified and compared with similar species. The morphological analysis revealed that T. pseudoheterodentata is a large Trichodina species (cell diameter 73.0–82.5 μm) and possesses robust denticles with broad blades and well‐developed blade connections. Characterization of its primary and secondary SSU rDNA structures indicated that T. pseudoheterodentata was distinctly different from congeneric species in H12, H15, E10_1, and V4 regions. Phylogenetic analysis revealed that the genetic distances among the new species and similar species reached interspecific levels, furthermore, the phylogenetic study also validated the identification of T. pseudoheterodentata and its placement in the genus Trichodina.     

Phylogenetic relationships within Pappophoreae s.l. (Poaceae: Chloridoideae): Additional evidence based on ITS and trnL-F sequence data

Historically, Pappophoreae included the genera Cottea, Enneapogon, Kaokochloa, Pappophorum and Schmidtia. Some authors consider this tribe as a well-supported monophyletic group; while other evidences reveals Pappophoreae as polyphyletic, with Pappophorum separated from the rest of the tribe. When the latter happens, it can form a clade with Tridens flavus. Molecular phylogenetic analyses of the subfamily Chloridoideae have included few species of Pappophoreae; therefore, further research involving more representatives of this tribe is needed. With the aim of providing new evidence to help clarify the phylogenetic position of Pappophorum and its relationships with other genera of the tribe and the subfamily Chloridoideae, eight new sequences of ITS and trnL-F regions of Pappophoreae species were generated. These sequences were analyzed together with other available sequence data obtained from GenBank, using maximum parsimony and Bayesian inference, for individual (trnL-F or ITS) or combined trnL-F/ITS data sets. All analyses reveal that Pappophoreae is polyphyletic, with Pappophorum separated from the rest of the tribe forming a well-supported clade sister to Tridens flavus.     

Multiple autopolyploidizations and range expansion of Allium przewalskianum Regel. (Alliaceae) in the Qinghai‐Tibetan Plateau

We used the Allium przewalskianum diploid–tetraploid complex on the Qinghai‐Tibetan Plateau (QTP) as a model to examine how this complex responded to the Quaternary climatic oscillations, and whether multiple autopolyploidizations have occurred. We sequenced five chloroplast DNA (cpDNA) fragments (accD‐psaI, trnH‐psbA, trnL‐trnF, trnS‐trnG and rpl16‐intron) in 306 individuals (all of known ploidy level) from 48 populations across the distribution of this species complex. We identified a total of 32 haplotypes—11 in diploids only, 13 in tetraploids only, and 8 found in both cytotypes. This, plus network analyses, indicated that tetraploids have arisen independently from diploids at least eight times. Most populations in the eastern QTP contained multiple haplotypes, but only a single haplotype was found for 17 tetraploid populations on the western QTP, suggesting a recent colonization of the western QTP. We further found that this species complex underwent an earlier range expansion around 5–150 thousand years ago (kya), after the largest glacial period (800–170 kya) in the QTP. In addition, the high frequencies of tetraploids in the QTP suggested that the tetraploid A. przewalskianum cytotype has evolutionary advantages over diploids in colonizing and/or surviving the arid habitats of the QTP.     

Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants

Abstract Species of Prunus L. sect. Persica are not only important fruit trees, but also popular ornamental and medicinal plants. Correct identification of seedlings, barks, or fruit kernels is sometimes required, but no reliable morphological characters are available. Nowadays, the technique of DNA barcoding has the potential to meet such requirements. In this study, we evaluated the suitability of 11 DNA loci (atpB‐rbcL, trnH‐psbA, trnL‐F, trnS‐G, atpF‐H, rbcL, matK, rpoB, rpoC1, nad1, and internal transcribed spacer [ITS]) as candidate DNA barcodes for peaches, using samples from 38 populations, covering all the species in sect. Persica. On the whole, the primers worked well in this group and sequencing difficulties were met only in the case of ITS locus. Five loci (rbcL, matK, rpoB, rpoC, and nad1) have very low variation rates, whereas atpB‐rbcL, atpF‐H, trnH‐psbA, trnL‐F and trnS‐G show more variability. The most variable loci, atpB‐rbcL and trnH‐psbA, can distinguish three of the five species. Two two‐locus combinations, atpB‐rbcL+trnL‐F and atpB‐rbcL+atpF‐H, can resolve all five species. We also find that identification powers of the loci are method‐dependent. The NeighborNet method shows higher species identification power than maximum parsimony, neighbor joining, and unweighted pair group method with arithmetic mean methods.     

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

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

TrnL-trnF sequence data imply paraphyly of Aquilaria and Gyrinops (Thymelaeaceae) and provide new perspectives for agarwood identification

The genera Aquilaria and Gyrinops (Thymelaeaceae, Malvales) are well known for the production of agarwood which is a highly wanted forest product of substantial economic value. The taxonomic status of Aquilaria and Gyrinops as separate genera is doubted as they are only distinguished by the number of stamens. We investigated their status by conducting phylogenetic analyses of DNA sequences from the plastid trnL-trnF spacer. Control of international trade of agarwood is currently hampered by the failure of traditional methods such as microscopy to identify samples to species level. We therefore evaluated the potential of molecular identification of agarwood by searching for species- and region-specific plastid DNA polymorphisms. DNA sequences were obtained from 31 Thymelaeaceae accessions encompassing 20 different species in six genera. Aquilaria and Gyrinops appear to be paraphyletic. Success in sequencing wood samples demonstrates that molecular markers provide new perspectives for agarwood identification.     

How do similarities in spatial distributions and interspecific associations affect the coexistence of Quercus species in the Baotianman National Nature Reserve,Henan, China

Congeneric species often have similar ecological characteristics and use similar resources. These similarities may make it easier for them to co‐occur in a similar habitat but may also lead to strong competitions that limit their coexistence. Hence, how do similarities in congeneric species affect their coexistence exactly? This study mainly used spatial point pattern analysis in two 1 hm² plots in the Baotianman National Nature Reserve, Henan, China, to compare the similarities in spatial distributions and interspecific associations of Quercus species. Results revealed that Quercus species were all aggregated under the complete spatial randomness null model, and aggregations were weaker under the heterogeneous Poisson process null model in each plot. The interspecific associations of Quercus species to non‐Quercus species were very similar in Plot 1. However, they can be either positive or negative in different plots between the co‐occurring Quercus species. The spatial distributions of congeneric species, interspecific associations with non‐Quercus species, neighborhood richness around species, and species diversity were all different between the two plots. We found that congeneric species did have some similarities, and the closely related congeneric species can positive or negative associate with each other in different plots. The co‐occurring congeneric species may have different survival strategies in different habitats. On the one hand, competition among congenerics may lead to differentiation in resource utilization. On the other hand, their similar interspecific associations can strengthen their competitive ability and promote local exclusion to noncongeneric species to obtain more living space. Our results provide new knowledge for us to better understand the coexistence mechanisms of species.     

Mesoptychia chinensis Bakalin,Vilnet & Xiong sp. nov. (Jungermanniaceae,Marchantiophyta) and comments on the distribution of Mesoptychia south of the boreal zone in Asia

Abstract

Morphologically unique specimens of Mesoptychia (Lindb.) A.Evans recently collected in Guizhou Province, China were sequenced and analysed using a combined trnL-F+trnG-intron cpDNA dataset from 10 known taxa of Mesoptychia. Using a combination of morphological and molecular data, Mesoptychia chinensis Bakalin, Vilnet & Xiong sp. nov. is described as new to science. Mesoptychia polymorpha subsp. pakistanica Bakalin & Vilnet subsp. nov. is established based on morphological data only. Mesoptychia ussuriensis is newly recorded for China. New molecular data for species delimitation within Mesoptychia distributed in southern and south-east Asia are provided.     

DNA barcoding of tuberous Orchidoideae: a resource for identification of orchids used in Salep

Tubers of terrestrial orchids are harvested and traded from the eastern Mediterranean to the Caspian Sea for the traditional product Salep. Overexploitation of wild populations and increased middle‐class prosperity have escalated prices for Salep, causing overharvesting, depletion of native populations and providing an incentive to expand harvesting to untapped areas in Iran. Limited morphological distinctiveness among traded Salep tubers renders species identification impossible, making it difficult to establish which species are targeted and affected the most. In this study, a reference database of 490 nrITS, trnL‐F spacer and matK sequences of 133 taxa was used to identify 150 individual tubers from 31 batches purchased in 12 cities in Iran to assess species diversity in commerce. The sequence reference database consisted of 211 nrITS, 158 trnL‐F and 121 matK sequences, including 238 new sequences from collections made for this study. The markers enabled unambiguous species identification with tree‐based methods for nrITS in 67% of the tested tubers, 58% for trnL‐F and 59% for matK. Species in the genera Orchis (34%), Anacamptis (27%) and Dactylorhiza (19%) were the most common in Salep. Our study shows that all tuberous orchid species in this area are threatened by this trade, and further stresses the urgency of controlling illegal harvesting and cross‐border trade of Salep tubers.     

Petrocodon tongziensis (Gesneriaceae), a new species from limestone areas in Guizhou,China based on morphological and molecular evidence

Petrocodon tongziensis R.B.Zhang & F.Wen, a new species from northern Guizhou province, China, is described and illustrated based on molecular and morphological evidence. The new species was found growing in crevices and on tufa of moist surfaces of limestone hills in Tongzi County. A maximum parsimony (MP) analysis based on the combined ITS and trnL‐F DNA regions showed that the new species falls within a large polytomy within Petrocodon, but is resolved as most closely related to an unidentified species (WF2014) and these two taxa are in turn resolved as sister to Petrocodon hunanensis X.L.Yu & Ming Li. This is congruent with the fact that both P. hunanensis and P. tongziensis have four fertile stamens, a character state likely to be an ancestral state distinguishing this clade from the rest of Petrocodon. Petrocodon tongziensis differs from P. hunanensis by lacking a terrestrial stem, as well as by the number of bracts, presence of bracteoles, shape of the lobes of the upper lip, and reduced number and length of staminodes.     

Gagea bohemica (Liliaceae), a highly variable monotypic species within Gagea sect. Didymobulbos

Abstract

Gagea bohemica s.l. (Liliaceae) consists of two morphologically extreme forms (saxatilis and bohemica) which depending on the varying opinions of the authors are considered as separate species, subspecies or as a single variable species. In this study, morphological (form and length of perianth segments, pilosity of pedicles and peduncles) and molecular data (cpDNA trnL–trnF IGS) of 55 samples corresponding to 43 populations from Western Europe to the Mediterranean area and eastern Caucasus were investigated. G. bohemica (Zauschn.) Schult. & Schult. f. represents a highly variable monotypic species from both morphological and karyological (2n = 24, 36, 48, 60, 72) aspects. Based on the molecular and morphological data presented here, a differentiation between specific and infraspecific taxa is not possible. We hypothesize that the morphological variability of this species is largely influenced by different site conditions and ploidy level.     

Dissimilar molecular and morphological patterns in an introgressed peripheral population of a sand dune species (Armeria pungens,Plumbaginaceae)

• Introgression is a poorly understood evolutionary outcome of hybridisation because it may remain largely undetected whenever it involves the transfer of small parts of the genome from one species to another. Aiming to understand the early stages of this process, a putative case from the southernmost border of the Armeria pungens range from its congener A. macrophylla is revisited following the discovery of a subpopulation that does not show phenotypic signs of introgression and resembles typical A. pungens.
• We analysed morphometrics, nuclear ribosomal DNA ITS and plastid DNA (trnL‐trnF) sequences, genome size, 45S and 5S rDNA loci‐FISH data and nrDNA IGS sequences.
• Within the study site, most individuals match morphologies of either of the two hybridising species, particularly the new subpopulation, with intermediate phenotypes being scarce. This pattern does not fully fit molecular evidence revealing two ITS ribotypes co‐occurring intragenomically in most plants from the study site and one single plastid haplotype. Genome size and structural features of the IGS sequences both indicate that A. pungens from the study site is genetically more similar to its sympatric congener than to the remainder of its conspecifics.
• Introgression of A. macrophylla into A. pungens and plastid capture explain all the evidence analysed. However, important features to understand the origin and fate of the introgressed population, such as the degree and direction of introgression, which are important for understanding early stages of hybridisation in plants with low reproductive barriers, should be addressed with new data.