Long-distance colonization of the Western Mediterranean by Cistus ladanifer (Cistaceae) despite the absence of special dispersal mechanisms

Aim There are few biogeographical and evolutionary studies that address plant colonization and lineage origins in the Mediterranean. Cistus serves as an excellent model with which to study diaspore dispersal and distribution patterns of plants exhibiting no special long‐distance dispersal mechanisms. Here we analyse the pattern of genetic variation and divergence times to infer whether the African–European disjunction of C. ladanifer L. is the result of long‐distance dispersal or of vicariance events. Location Principally the Western Mediterranean region, with a focus on the Strait of Gibraltar. Methods We used DNA sequence phylogenetic approaches, based on plastid (rbcL/trnK‐matK) and nuclear (ITS) sequence data sets, and the penalized likelihood method, to date the diversification of the 21 species of Cistus. Phylogenetic relationships and phylogeographical patterns in 47 populations of C. ladanifer were also analysed using two plastid DNA regions (trnS‐trnG, trnK‐matK). These sequence data were analysed using maximum parsimony, Bayesian inference and statistical parsimony. Results Dating estimates indicated divergence dates of the C. ladanifer lineage in the Pleistocene. Eight nucleotide‐substitution haplotypes distributed on the European (four haplotypes) and African (five haplotypes) sides of the Strait of Gibraltar were revealed from C. ladanifer sequences. Both the haplotype network and the phylogenetic analyses depicted two main Cistus lineages distributed in both Europe and North Africa. An Iberian haplotype forms part of the North African lineage, and another haplotype distributed on both continents is related to the European lineage. Haplotype relationships with respect to outgroup sequences supported the hypothesis that the centre of genetic diversity is in northern Africa. Main conclusions Based on lineage divergence‐time estimates and disassociation between geographical and lineage haplotype distributions, we inferred at least two intercontinental colonization events of C. ladanifer post‐dating the opening of the Strait of Gibraltar (c. 5 Ma). This result supports a hypothesis of long‐distance dispersal rather than a hypothesis of vicariance. We argue that, despite limited dispersal abilities, preference for disturbed habitats was integral to historical colonization after the advent of the Mediterranean climate (c. 3.2 Ma), when Cistus species diverged and became established as a dominant element in the Mediterranean scrub.     

Phylogenetic analysis of eastern Asian and eastern North American disjunct Lespedeza (Fabaceae) inferred from nuclear ribosomal ITS and plastid region sequences

Lespedeza (tribe Desmodieae, Fabaceae) follows a disjunct distribution in eastern Asia and eastern North America. Phylogenetic relationships among its species and related taxa were inferred from nuclear ribosomal internal transcribed spacer (ITS) and plastid sequences (trnH‐psbA, psbK‐psbI, trnK‐matK and rpoC1). We examined 35 species of Lespedeza, two of Kummerowia and one of Campylotropis, the sole constituents of the Lespedeza group. An analysis of these data revealed that the genus Campylotropis is sister to the other two genera. However, we were unable to resolve the relationships between Kummerowia and Lespedeza in the strict consensus trees of parsimony analyses based on plastid and combined DNA data. In the genus Lespedeza, the Old World subgenus Macrolespedeza is monophyletic, whereas the transcontinental subgenus Lespedeza is paraphyletic. Monophyly of eastern Asian species and of North American species is strongly supported. Although inconsistent with the traditional classification, this phylogenetic finding is consistent with seedling morphology. Three subgroups recognized in subgenus Macrolespedeza were unresolved in our phylogenetic trees. An incongruence length difference (ILD) test indicated that the two partitions (nuclear ITS and plastid sequences) were significantly incongruent, perhaps because of hybridization between species in Lespedeza. Most of the primary clades of tribe Desmodieae are Asian, implying that the relatively few New World ones, such as those in Lespedeza, are more recently derived from Asia. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 221–235.     

Sequence diversification of 45S rRNA ITS, trnH-psbA spacer, and matK genic regions in several Allium species

Allium is a very diverse genus with over 600 species distributed worldwide. Haplotype analyses of 45S rRNA ITS, trnH-psbA spacer, and matK gene sequences in 9 Allium species were carried out, subsequent to which phylogenetic relations of the nine species were also analyzed. Of the three genes, the nuclear 45S rRNA ITS sequences showed the highest variation with one haplotype in each species. The other two chloroplast genes revealed that more than one haplotype was present in each species, and each haplotype was present in several of the species. In the matK gene, EcoRI restriction revealed heteroplasmy in which the functional gene retains the EcoRI recognition site while the nonfunctional, pseudogene does not. Phylogenetic patterns were not consistent among the haplotypes of the 45 rRNA ITS, trnH-psbA spacer, and matK genic regions. This phylogenetic incongruency might be due to the presence of multiple haplotypes in each of the chloroplast genes. However, the inconsistency of the phylogenetic relationships, based on the 45S rRNA ITS sequences makes a strong case for further analysis.     

Genetic diversity,genetic structure and phylogeography of the Iberian endemic Gypsophila struthium (Caryophyllaceae) as revealed by AFLP and plastid DNA sequences: connecting habitat fragmentation and diversification

Iberian gypsum outcrops are highly fragmented and ecologically challenging environments for plant colonization. As gypsophytes occur exclusively in such habitats, they are ideal models for the study of both the effects of habitat fragmentation and selection on population genetic diversity and structure. In this study, we used amplified fragment length polymorphism (AFLP) and plastid DNA sequences to investigate the phylogeographical history of the Iberian plant Gypsophila struthium (Caryophyllaceae), a widespread endemic restricted to Iberian gypsum outcrops. Gypsophila struthium consists of two subspecies that differ in the architecture of their inflorescence and have mostly allopatric ranges. Gypsophila struthium subsp. struthium occurs in central, eastern and south‐eastern Iberia, whereas G. struthium subsp. hispanica occurs in northern and eastern areas. AFLPs revealed low but significant genetic differentiation between the subspecies, probably as a result of a recent diversification during the Pliocene–Pleistocene. In the geographical contact zone between the taxa, the Bayesian analyses revealed populations with mixed ancestries and genetic clusters predominantly of one or the other subspecies, indicating incomplete reproductive barriers between them. Plastid DNA haplotypes revealed strong geographical structure and testified to processes of isolation by distance and continuous range expansion for some haplotype clades. The Bayesian analyses of the population structure of AFLP data and nested clade phylogeographical analysis (NCPA) of plastid haplotypes revealed that the putative ancestral range corresponded to central and eastern populations of G. struthium subsp. struthium, with those lineages contributing through more recent expansion to increased genetic diversity and structure of the south‐eastern and eastern ranges of this subspecies and to the diversification of G. struthium subsp. hispanica in northern and eastern gypsum outcrops. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 654–675.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Genetic study reveals close link between Irish and Northern Spanish specimens of the protected Lusitanian slug Geomalacus maculosus

The slug Geomalacus maculosus is a prominent member of the Lusitanian fauna. As its global distribution is restricted to western Ireland and northern Iberia, it is protected under EU legislation. Nothing is known about the genetic variability and population structure of this species, so, with a special view to shedding light on the origin of the Irish G. maculosus, tissue samples from 78 specimens were collected from 13 locations within Ireland and ten locations within Iberia and partial sequences of the mitochondrial 16S rRNA and cytochrome oxidase subunit 1 (COI) and from the nuclear internal transcribed spacer 1 region (ITS‐1) were compared. The genetic diversity of the Irish G. maculosus was found to be greatly reduced compared with the Iberian populations, with only one (16S rRNA) and two (COI) mitochondrial haplotypes identified respectively. No private Irish ITS‐1 haplotype was found. Based on the COI sequences, the Irish specimens clustered closest to Spanish specimens from Northern Asturias and Cantabria, and the bGMYC analysis identified five further Iberian clades that were highly genetically differentiated suggesting long‐term allopatric divergence.     

Identification of species in the angiosperm family Apiaceae using DNA barcodes

Apiaceae (Umbelliferae) is a large angiosperm family that includes many medicinally important species. The ability to identify these species and their adulterants is important, yet difficult to do so because of their subtle fruit morphological differences and often lack of diagnostic features in preserved specimens. Moreover, dried roots are often the official medical organs, making visual identification to species almost impossible. DNA barcoding has been proposed as a powerful taxonomic tool for species identification. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL+matK as the core plant barcode. Recently, the China Plant BOL Group proposed that the nuclear ribosomal DNA internal transcribed spacer (ITS), as well as a subset of this marker (ITS2), be incorporated alongside rbcL+matK into the core barcode for seed plants, particularly angiosperms. In this study, we assess the effectiveness of these four markers plus psbA‐trnH as Apiaceae barcodes. A total of 6032 sequences representing 1957 species in 385 diverse genera were sampled, of which 211 sequences from 50 individuals (representing seven species) were newly obtained. Of these five markers, ITS and ITS2 showed superior results in intra‐ and interspecific divergence and DNA barcoding gap assessments. For the matched data set (173 samples representing 45 species in five genera), the ITS locus had the highest identification efficiency (73.3%), yet ITS2 also performed relatively well with 66.7% identification efficiency. The identification efficiency increased to 82.2% when using an ITS+psbA‐trnH marker combination (ITS2+psbA‐trnH was 80%), which was significantly higher than that of rbcL+matK (40%). For the full sample data set (3052 ITS sequences, 3732 ITS2 sequences, 1011 psbA‐trnH sequences, 567 matK sequences and 566 rbcL sequences), ITS, ITS2, psbA‐trnH, matK and rbcL had 70.0%, 64.3%, 49.5%, 38.6% and 32.1% discrimination abilities, respectively. These results confirm that ITS or its subset ITS2 be incorporated into the core barcode for Apiaceae and that the combination of ITS/ITS2+psbA‐trnH has much potential value as a powerful, standard DNA barcode for Apiaceae identification.     

Late Neogene history of the laurel tree (Laurus L., Lauraceae) based on phylogeographical analyses of Mediterranean and Macaronesian populations

Aim The post‐glacial range dynamics of many European plant species have been widely investigated, but information rapidly diminishes as one moves further back in time. Here we infer the historical range shifts of Laurus, a paradigmatic tree of the Tethyan flora that has covered southern Eurasia since the Oligo‐Miocene, by means of phylogenetic and phylogeographical analyses. Location Mediterranean Basin, Black Sea and Macaronesian archipelagos (Azores, Madeira, Canary Islands). Methods We analysed plastid DNA (cpDNA) sequence (trnK–matK, trnD–trnT) variation in 57 populations of Laurus and three Lauraceae genera. Phylogenetic methods (maximum parsimony and Bayesian inference) and statistical parsimony networks were used to reconstruct relationships among haplotypes. These results were contrasted with the fossil record and bioclimatic niche‐based model predictions of past distributions to infer the migration routes and location of refugia. Results The phylogenetic tree revealed monophyly for Laurus. Overall sequence variability was low within Laurus, but six different haplotypes were distinguished and a single network retrieved, portraying three lineages primarily related to geography. A strongly divergent eastern lineage occupied Turkey and the Near East, a second clade was located in the Aegean region and, lastly, a western clade grouped all Macaronesian and central and western Mediterranean populations. A close relationship was observed between the Macaronesian populations of L. azorica and the western populations of L. nobilis. Main conclusions The phylogeographical structure of Laurus preserves the imprints of an ancient contraction and break‐up of the range that resulted in the evolution of separate cpDNA lineages in its western‐ and easternmost extremes. Intense range dynamics in the western Mediterranean and multiple glacial refugia contributed to the generation and long‐term conservation of this phylogeographical pattern, as shown by the fit between the haplotype ranges and past suitable areas inferred from bioclimatic models. Finally, our results challenge the taxonomic separation of Laurus into two distinct species.     

Molecular systematics of the Cactaceae

Bayesian, maximum‐likelihood, and maximum‐parsimony phylogenies, constructed using nucleotide sequences from the plastid gene region trnK‐matK, are employed to investigate relationships within the Cactaceae. These phylogenies sample 666 plants representing 532 of the 1438 species recognized in the family. All four subfamilies, all nine tribes, and 69% of currently recognized genera of Cactaceae are sampled. We found strong support for three of the four currently recognized subfamilies, although relationships between subfamilies were not well defined. Major clades recovered within the largest subfamilies, Opuntioideae and Cactoideae, are reviewed; only three of the nine currently accepted tribes delimited within these subfamilies, the Cacteae, Rhipsalideae, and Opuntieae, are monophyletic, although the Opuntieae were recovered in only the Bayesian and maximum‐likelihood analyses, not in the maximum‐parsimony analysis, and more data are needed to reveal the status of the Cylindropuntieae, which may yet be monophyletic. Of the 42 genera with more than one exemplar in our study, only 17 were monophyletic; 14 of these genera were from subfamily Cactoideae and three from subfamily Opuntioideae. We present a synopsis of the status of the currently recognized genera.
© The Willi Hennig Society 2011.     

Testing four candidate barcoding markers in temperate woody bamboos (Poaceae: Bambusoideae)

Abstract Bambusoideae is an important subfamily of the grass family Poaceae that has considerable economic, ecologic and cultural value. In addition, Bambusoideae species are important constituents of the forest vegetation in China. Because of the paucity of flower‐bearing specimens and homoplasies of morphological characters, it is difficult to identify species of Bambusoideae using morphology alone, especially in the case of temperate woody bamboos (i.e. Arundinarieae). To this end, DNA barcoding has shown great potential in identifying species. The present study is the first attempt to test the feasibility of four proposed DNA barcoding markers (matK, rbcL, trnH–psbA, and internal transcribed spacer [ITS]) in identifying 27 species of the temperate woody bamboos. Three plastid markers showed high levels of universality, whereas the universality of ITS was comparatively low. A single plastid marker provided low levels of discrimination success at both the genus and species levels (<12%). Among the combinations of plastid markers, the highest discriminatory power was obtained using the combination of rbcL+matK (14.8%). Using a combination of three markers did not increase species discrimination. The nuclear region ITS alone could identify 66.7% of species, although fewer taxa were included in the ITS analyses than in the plastid analyses. When ITS was integrated with a single or combination of plastid markers, the species discriminatory power was significantly improved. We suggest that a combination of rbcL+ ITS, which exhibited the highest species identification power of all combinations in the present study, could be used as a potential DNA barcode for temperate woody bamboos.     

Global phylogeography in Sanionia uncinata (Amblystegiaceae: Bryophyta)

Global phylogeographic patterns in Sanionia uncinata are addressed based on information in internal transcribed spacer (ITS) (214 specimens) and the plastid markers trnL‐trnF (221) and rpl16 (217). ITS suggests a monophyletic Sanionia and a paraphyletic S. uncinata; this was neither supported nor rejected by plastid data. Northern or Eastern Eurasia and Alaska appear important in the early evolution of Sanionia and some populations dispersed into the Southern Hemisphere relatively early. Some haplotypes or groups of haplotypes are morphologically and ecologically distinct, biologically meaningful units that correspond with S. orthothecioides, S. symmetrica and S. georgicouncinata s.l. The latter includes two species that are indistinguishable by morphology, S. georgicouncinata s.s. (Southern Hemisphere) and S. nivalis (Northern Hemisphere). Tropical African and South American S. uncinata populations have separate origins and the Southern Hemisphere was colonized at least twice. In the northern circum‐Arctic region, the haplotype composition differs between the North Atlantic and Beringian areas. Eastern Eurasia has a higher S. uncinata haplotype diversity than other Holarctic regions, implying less devastating effects of recurrent glacial periods. For Eastern and Western Eurasia, North America and the Southern Hemisphere, most of the haplotype variation was found within the regions, but 14–18% can be referred to among region variation. Plastid haplotype diversity was lower in the Southern Hemisphere than in the Arctic to subarctic, possibly attributable to founder effects. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 19–42.     

Testing genome skimming for species discrimination in the large and taxonomically difficult genus Rhododendron

Standard plant DNA barcodes based on 2–3 plastid regions, and nrDNA ITS show variable levels of resolution, and fail to discriminate among species in many plant groups. Genome skimming to recover complete plastid genome sequences and nrDNA arrays has been proposed as a solution to address these resolution limitations. However, few studies have empirically tested what gains are achieved in practice. Of particular interest is whether adding substantially more plastid and nrDNA characters will lead to an increase in discriminatory power, or whether the resolution limitations of standard plant barcodes are fundamentally due to plastid genomes and nrDNA not tracking species boundaries. To address this, we used genome skimming to recover near-complete plastid genomes and nuclear ribosomal DNA from Rhododendron species and compared discrimination success with standard plant barcodes. We sampled 218 individuals representing 145 species of this species-rich and taxonomically difficult genus, focusing on the global biodiversity hotspots of the Himalaya-Hengduan Mountains. Only 33% of species were distinguished using ITS+matK+rbcL+trnH-psbA. In contrast, 55% of species were distinguished using plastid genome and nrDNA sequences. The vast majority of this increase is due to the additional plastid characters. Thus, despite previous studies showing an asymptote in discrimination success beyond 3–4 plastid regions, these results show that a demonstrable increase in discriminatory power is possible with extensive plastid genome data. However, despite these gains, many species remain unresolved, and these results also reinforce the need to access multiple unlinked nuclear loci to obtain transformative gains in species discrimination in plants.     

Molecular phylogenetic analysis of Tulipa (Liliaceae) based on noncoding plastid and nuclear DNA sequences with an emphasis on Turkey

We investigated the phylogenetic relationships in Tulipa in Turkey using DNA sequences from the plastid trnL‐trnF region and the internal transcribed spacer (ITS) of nuclear ribosomal DNA. We generated trnL‐trnF and nuclear ITS sequences for 11 Tulipa spp. from Turkey and compared the utility of trnL‐trnF and ITS sequences for phylogenetic analysis. Neighbor‐joining, Bayesian and maximum parsimony methods were implemented using the same matrices. Our study of Tulipa based on molecular data revealed congruent results with previous studies. Despite the relatively lower resolution of trnL‐trnF than that of ITS, both sequence matrices generated similar results. Three clades were clearly distinguished, corresponding to subgenera Tulipa, Eriostemones and Orithyia. It is not fully resolved whether Clusianae should be recognized as a separate section of subgenus Tulipa or a distinct subgenus. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 270–279.     

Molecular phylogeny and biogeography of Astilbe (Saxifragaceae) in Asia and eastern North America

Phylogenetic analyses were conducted for Astilbe (Saxifragaceae), an Asian/eastern North American disjunct genus, using sequences of nuclear ribosomal internal transcribed spacer (ITS) and plastid matK, trnL‐trnF and psbA‐trnH regions. The monophyly of Astilbe is well supported by both ITS and plastid sequences. Topological incongruence was detected between the plastid and the ITS trees, particularly concerning the placement of the single North American species, A. biternata, which may be most probably explained by its origin involving hybridization and/or allopolyploidy with plastid capture. In Astilbe, all species with hermaphroditic flowers constitute a well‐supported clade; dioecious species form a basal grade to the hermaphroditic clade. Astilbe was estimated to have split with Saxifragopsis from western North America at 20.69 Ma (95% HPD: 12.14–30.22 Ma) in the early Miocene. This intercontinental disjunction between Astilbe and Saxifragopsis most likely occurred via the Bering land bridge. The major clade of Astilbe (all species of the genus excluding A. platyphylla) was inferred to have a continental Asian origin. At least three subsequent migrations or dispersals were hypothesized to explain the expansion of Astilbe into North America, Japan and tropical Asian islands. The intercontinental disjunct lineage in Astilbe invokes a hybridization event either in eastern Asia or in North America. This disjunction in Astilbe may be explained by a Beringian migration around 3.54 Ma (95% high posterior density: 1.29–6.18 Ma) in the late Tertiary, although long‐distance dispersal from eastern Asia to North America is also likely. The biogeographical connection between continental Asia, Taiwan, the Philippines and other tropical Asian islands in Astilbe provides evidence for the close floristic affinity between temperate or alpine south‐western China and tropical Asia. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ●● , ●●–●●.     

Impact of mountain chains, sea straits and peripheral populations on genetic and taxonomic structure of a freshwater turtle, Mauremys leprosa (Reptilia, Testudines, Geoemydidae)

Mauremys leprosa, distributed in Iberia and North‐west Africa, contains two major clades of mtDNA haplotypes. Clade A occurs in Portugal, Spain and Morocco north of the Atlas Mountains. Clade B occurs south of the Atlas Mountains in Morocco and north of the Atlas Mountains in eastern Algeria and Tunisia. However, we recorded a single individual containing a clade B haplotype in Morocco from north of the Atlas Mountains. This could indicate gene flow between both clades. The phylogenetically most distinct clade A haplotypes are confined to Morocco, suggesting both clades originated in North Africa. Extensive diversity within clade A in south‐western Iberia argues for a glacial refuge located there. Other regions of the Iberian Peninsula, displaying distinctly lower haplotype diversities, were recolonized from within south‐western Iberia. Most populations in Portugal, Spain and northern Morocco contain the most common clade A haplotype, indicating dispersal from the south‐western Iberian refuge, gene flow across the Strait of Gibraltar, and reinvasion of Morocco by terrapins originating in south‐western Iberia. This hypothesis is consistent with demographic analyses, suggesting rapid clade A population increase while clade B is represented by stationary, fragmented populations. We recommend the eight, morphologically weakly diagnosable, subspecies of M. leprosa be reduced to two, reflecting major mtDNA clades: Mauremys l. leprosa (Iberian Peninsula and northern Morocco) and M. l. saharica (southern Morocco, eastern Algeria and Tunisia). Peripheral populations could play an important role in evolution of M. leprosa because we found endemic haplotypes in populations along the northern and southern range borders. Previous investigations in another western Palearctic freshwater turtle (Emys orbicularis) discovered similar differentiation of peripheral populations, and phylogeographies of Emys orbicularis and Mauremys rivulata underline the barrier status of mountain chains, in contrast to sea straits, suggesting common patterns for western Palearctic freshwater turtles.     

Intraspecific divergences of Rhodiola alsia (Crassulaceae) based on plastid DNA and internal transcribed spacer fragments

In the present study, we used two maternally inherited plastid DNA intergenic spacers, rpl20‐rps12 and trnS‐trnG, and the biparentally inherited nuclear ribosomal internal transcribed spacer (ITS) region to explore genetic variation and phylogeographical history of Rhodiola alsia, a herb endemic to the Qinghai‐Tibetan Plateau (QTP). Based on range‐wide sampling (18 populations and 227 individuals), we detected 45 plastid DNA haplotypes and 19 ITS sequence types. Only three plastid DNA haplotypes were widespread; most haplotypes were restricted to single sites or to neighbouring populations. Analysis of molecular variance revealed that most of the genetic variance was found within populations (51.24%) but that populations were also distinct (F_ST = 0.48759). We found three areas with relatively high plastid DNA diversity and these could further be recognized as potentially isolated divergence centres based on the ITS sequence type distribution. These represent three potentially isolated glacial refugia for R. alsia: one of them has long been recognized as an important refugium on the south‐eastern edge of the QTP, whereas the others are new and located in the north and south of the Tanggula Mountains on the plateau platform. Divergence time estimates based on ITS suggest that the main lineages of R. alsia diverged from each other 0.35–0.87 Mya, indicating that climatic oscillations during the Pleistocene may have been an important driver of intraspecific divergence in R. alsia. Rhodiola alsia probably experienced a phylogeographical history of retreat to isolated glacial refugia during Quaternary glaciations that led to different degrees of allopatric intraspecific divergence. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 204–215.     

Application of plastid and nuclear markers to DNA barcoding of Euro‐Mediterranean oaks (Quercus,Fagaceae): problems,prospects and phylogenetic implications

DNA barcoding, a species identification system based on sequences from a short, standardized DNA region, has emerged recently as a new tool for taxonomists. We investigated the discriminatory power of a subset of highly variable proposed plant barcoding loci (matK, trnH‐psbA, ITS2) in Quercus, a taxonomically complex tree genus of global importance. The research included all currently recognized species and some major variants of the Mediterranean region and Europe (32 taxa) and 17 East Asian and North American species used for comparison. Based on sequence character state, we assigned unique plastid haplotypes to 40.8% of the investigated species; ITS2 increased the resolution up to 87.8% of total taxa. Nevertheless, unsuccessful genetic distance‐based discrimination questioned the potential efficiency of correct species identification for future studies. Most species appeared to be nonmonophyletic in parallel phylogenetic tests. Three subgeneric groups were outlined, with different rates of within‐group variability and geographical differentiation. Members of one of these groups (corresponding to the Eurasian Group Ilex) were paraphyletic to Group Quercus from the New and Old World and the Eurasian Group Cerris. The data gathered indicate that barcoding markers may help to identify closely related species clusters and contribute to the inference of major diversification and evolutionary patterns in oaks, but the methodology per se appears to be of limited efficacy in defining species limits, unless we make a profound revision of traditional Quercus taxonomic categories. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 478–499.     

MOLECULAR PHYLOGEOGRAPHY, RETICULATION, AND LINEAGE SORTING IN MEDITERRANEAN SENECIO SECT. SENECIO (ASTERACEAE)

Abstract The Mediterranean species complex of Senecio serves to illustrate evolutionary processes that are likely to confound phylogenetic inference, including rapid diversification, gene tree‐species tree discordance, reticulation, interlocus concerted evolution, and lack of complete lineage sorting. Phylogeographic patterns of chloroplast DNA (cpDNA) haplotype variation were studied by sampling 156 populations (502 individuals) across 18 species of the complex, and a species phylogeny was reconstructed based on sequences from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. For a subset of species, randomly amplified polymorphic DNAs (RAPDs) provided reference points for comparison with the cpDNA and ITS datasets. Two classes of cpDNA haplotypes were identified, with each predominating in certain parts of the Mediterranean region. However, with the exception of S. gallicus, intraspecific phylogeographic structure is limited, and only a few haplotypes detected were species‐specific. Nuclear sequence divergence is low, and several unresolved phylogenetic groupings are suggestive of near simultaneous diversification. Two well‐supported ITS clades contain the majority of species, amongst which there is a pronounced sharing of cpDNA haplotypes. Our data are not capable of diagnosing the relative impact of reticulation versus insufficient lineage sorting for the entire complex. However, there is firm evidence that S. flavus subsp. breviflorus and S. rupestris have acquired cpDNA haplotypes and ITS sequences from co‐occurring species by reticulation. In contrast, insufficient lineage sorting is a viable hypothesis for cpDNA haplotypes shared between S. gallicus and its close relatives. We estimated the minimum coalescent times for these haplotypes by utilizing the inferred species phylogeny and associated divergence times. Our data suggest that ancestral cpDNA polymorphisms may have survived for ca. 0.4–1.0 million years, depending on molecular clock calibrations.     

Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae)

Although two plastid regions have been adopted as the standard markers for plant DNA barcoding, their limited resolution has provoked the consideration of other gene regions, especially in taxonomically diverse genera. The genus Gossypium (cotton) includes eight diploid genome groups (A–G, and K) and five allotetraploid species which are difficult to discriminate morphologically. In this study, we tested the effectiveness of three widely used markers (matK, rbcL, and ITS2) in the discrimination of 20 diploid and five tetraploid species of cotton. Sequences were analysed locus‐wise and in combinations to determine the most effective strategy for species identification. Sequence recovery was high, ranging from 92% to 100% with mean pairwise interspecific distance highest for ITS2 (3.68%) and lowest for rbcL (0.43%). At a 0.5% threshold, the combination of matK+ITS2 produced the greatest number of species clusters. Based on ‘best match’ analysis, the combination of matK+ITS2 was best, while based on ‘all species barcodes’ analysis, ITS2 gave the highest percentage of correct species identifications (98.93%). The combination of sequences for all three markers produced the best resolved tree. The disparity index test based on matK+rbcL+ITS2 was significant (P < 0.05) for a higher number of species pairs than the individual gene sequences. Although all three barcodes separated the species with respect to their genome type, no single combination of barcodes could differentiate all the Gossypium species, and tetraploid species were particularly difficult.