Effects of Volatiles of Essential Oils on Behavior of the Western Flower Thrips <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> Perg. (Thysanoptera,Thripidae)

The results of evaluating the effects of essential oils on the behavior and reproduction potential of the western flower thrips Frankliniella occidentalis Perg. are presented. Essential oils of Acorus calamus, Juniperus virginiana, and Melissa officinalis possessed a repellent effect on the larvae. A sample from Lutsea cubeba was characterized by an attractive property. Oil solutions of J. virginiana, Mentha spicata, Nepeta cataria, and Litsea cubeba repelled the thrips females off the leaves of the host plant. Reduced concentrations of oils from J. virginiana, M. officinalis, and M. spicata did not affect the distribution of thrips females between the experimental and control leaves, but the number of offspring was significantly lower on the treated leaves.     

Multiplex real-time PCR assay for detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and screening for non-O157 Shiga toxin-producing <Emphasis Type="Italic">E. coli</Emphasis>

Background

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency.

Methods

The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s.

Results

The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested.

Conclusions

A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

     

mtDNA-based identification of two widespread roach species (<Emphasis Type="Italic">Rutilus</Emphasis>, Cyprinidae) characterized by sympatric zone

Test systems using multiplex PCR and restriction analysis have been designed for the simple and rapid identification of two widespread species of roach, Rutilus rutilus and R. lacustris, characterized by the sympatric zone. This method is based on the variation of nucleotide sequences of the mitochondrial markers (cytochrome oxidase I subunit and cytochrome b); it allows identifying the species omitting the DNA sequencing.     

How salinity stress influences the thermal time requirements of seed germination in <Emphasis Type="Italic">Silybum marianum</Emphasis> and <Emphasis Type="Italic">Calendula officinalis</Emphasis>

Salinity is a major problem of many agricultural lands that is usually associated with drought stress in arid and semi-arid regions. In this study we examine the role of salinity stress on temperature requirements of two herbaceous species and how it could be modeled to quantify alterations. We applied four non-linear regression models (segmented, beta, beta modified, and dent-like) to describe the germination rate–temperature relationships of Silybum marinum L. and Calendua officinalis L. over six constant temperatures exposed to different levels of salinity stress. Our results revealed that salinity could affect the cardinal temperatures in both plants and, as a result, it is not possible to suggest one model for all levels of salinity stress. The best model to fit data to predict cardinal temperatures of Silybum marianum and Calendula officinalis at the no-salinity condition were dent-like (AICc?=?4.03) and beta (AICc = ??2.30), respectively. Knowing the thermal time constant (f_o) value helps us predict the minimum number of hours required for completion of germination at the optimal temperature. All models in this study were estimated higher f_o due to higher salinity stress in both Silybum marianum and Calendula officinalis seeds. The highest estimated f_o for Silybum marianum (91.5?±?59.6) and Calendula officinalis (178.9?±?26.5) was obtained from the results of germination rate prediction using a dent-like model at 200 mM salinity.     

Selection and Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Berliner) (Eubacteriales: Bacillaceae) Strains for <Emphasis Type="Italic">Ecdytolopha aurantiana</Emphasis> (Lima) (Lepidoptera: Tortricidae) Control

The citrus fruit borer, Ecdytolopha aurantiana (Lima, 1927) (Lepidoptera: Tortricidae), is responsible for major losses to the citrus industry because it causes rot and drop of fruits. The current study aimed to select and characterize Bacillus thuringiensis (Berliner, 1911) strains toxic to E. aurantiana. For this purpose, 47 B. thuringiensis strains were evaluated in selective bioassays using first instar larvae of E. aurantiana. The lethal concentration (LC₅₀) of the most toxic strains was estimated, and the strains were characterized by morphological, biochemical, and molecular methods. Of the 47 strains tested, 10 caused mortality above 85% and showed mean lethal concentrations between 1.05E+7 and 1.54E+8 spores mL^?1. The lowest LC₅₀ values were obtained for the HD-1 standard strain and the BR145, BR83, BR52, and BR09 strains. The protein profile showed the presence of Cry proteins of 60, 65, 70, 80, and 130 kDa. The molecular characterization showed the presence of cry1, cry2, cry3, and cry11 genes. The morphological analysis identified three different crystalline inclusions: bipyramidal, round, and cuboidal. The cry1 and cry2 genes were the most frequent among the B. thuringiensis strains evaluated and encode Cry proteins toxic to insects of the order Lepidoptera, which agree with the toxicity results obtained by the selective bioassays against E. aurantiana. The results showed four different B. thuringiensis strains toxic to E. aurantiana at the same level as the HD-1 standard strain, and these strains have biotechnological potential for E. aurantiana control through the production of transgenic plants or the formulation of biopesticides.     

Tracking the impact of drought on functionally different woody plants in a Mediterranean scrubland ecosystem

Climate warming is predicted to amplify drought stress. Thus, it is important to understand how coexisting plant species respond to severe droughts. Here we study how seven Mediterranean woody plant species with different evolutionary history and functional characteristics (Pinus halepensis Mill., Juniperus phoenicea L., Pistacia lentiscus L., Rhamnus lycioides L., Rosmarinus officinalis L., Genista scorpius (L.) DC., and Globularia alypum L.) responded to a severe winter drought during 2011–2012 in Spain. The study site is located in the Valcuerna valley, Monegros desert, northeastern Spain. We evaluated how the drought affected the annual growth-ring formation of the species by using dendrochronology and quantified the intensity of drought-induced defoliation and mortality and compared it between species and groups of species with different evolutionary history. Radial growth of all species was strongly reduced by the 2012 drought. The pre-Mediterranean species (P. halepensis, J. phoenicea, P. lentiscus and R. lycioides) reduced growth more than the Mediterranean species (R. officinalis, G. scorpius and G. alypum). Defoliation was significantly higher in pre-Mediterranean than in Mediterranean species. When species were analyzed separately we found that P. halepensis was the species with the highest growth reduction but J. phoenicea was defoliated more severely and showed higher mortality rates as a consequence of drought. In the case of the Mediterranean shrubs, drought-induced mortality was only noticeable in R. officinalis. Drought impacted growth of all species but this did not induce mortality in all of them. Growth reduction was dependent on evolutionary history. However, functional characteristics of the species such as leaf stomatal regulation and root architecture may be more important than evolutionary history on explaining drought-induced mortality. Indeed, species with shallow root systems such as J. phoenicea and R. officinalis were the most adversely affected by the drought.     

Morphological and molecular investigations of <Emphasis Type="Italic">Gagea</Emphasis> (Liliaceae) in southeastern Kazakhstan with special reference to putative altitudinal hybrid zones

The most important center of speciation in the genus Gagea is thought to be in Central Asia. Here, we focus on species diversity in southeastern Kazakhstan (around Almaty, Ili-Alatau range of the Western Tian-Shan mountains). We studied an elevational transect, reaching from lowland steppes to the alpine zone (500–2750 m a. s. l.), and carried out detailed morphological and molecular investigations for populations of Gagea spp. Nine species were identified in different altitudinal zones; one of these (Gagea almaatensis) is described as new to science. We could detect two altitudinal contact zones between closely related species: G. filiformis and G. granulosa (sect. Minimae), and G. almaatensis and G. kuraminica (sect. Gagea). Morphological and molecular investigations (ITS data and cpDNA networks) indicate ongoing hybridization of co-occurring G. filiformis into G. granulosa and putative bidirectional hybridization events between G. almaatensis and G. kuraminica.     

<Emphasis Type="Italic">Pseudodelphis eleginopsis</Emphasis> n. sp. (Nematoda: Guyanemidae), a new tissue-dwelling parasite of the Patagonian blennie <Emphasis Type="Italic">Eleginops maclovinus</Emphasis> (Cuvier) (Perciformes: Eleginopsidae) in Argentina,with notes on related forms

Based on light and scanning electron microscopical studies, a new nematode parasite, Pseudodelphis eleginopsis n. sp. (Dracunculoidea: Guyanemidae), is described from tissues behind the gills of the Patagonian blennie Eleginops maclovinus (Cuvier) (Perciformes: Eleginopsidae) off the Atlantic coast (San Matías and San José Gulfs) of Patagonia, Argentina. The new species is mainly characterised by the length of the body (males 10–13 mm, larvigerous females 31–59 mm), the number (14) and arrangement of cephalic papillae, the absence of a buccal capsule, the muscular to glandular oesophagus length ratio (1:3–4) of larvigerous females, the length of the spicules (48–63 µm) and the number (7 pairs) and arrangement of the caudal papillae in the male. Pseudodelphis eleginopsis n. sp. is the first species of this genus described from a marine fish in the Atlantic Ocean and the first known dracunculoid parasitising the fish host belonging to the family Eleginopsidae. As revealed by the examination of very young females of the new species, the female genital tract of Pseudodelphis spp. is monodelphic. The genus Syngnathinema Moravec, Spangenberg & Frasca, 2001 is considered a junior synonym of Pseudodelphis Adamson & Roth, 1990 and, consequently, S. californiense and S. chitwoodi are transferred to Pseudodelphis as P. californiensis (Moravec, Spangenberg & Frasca, 2001) n. comb. and P. chitwoodi (Moravec & Kuchta, 2013) n. comb., respectively. Two dracunculoid species, Pseudodelphis limnicola Brugni & Viozzi, 2006 and the previously established Philonema percichthydis Moravec, Urawa & Coria, 1997, both described from the same freshwater host species, Percichthys trucha (Valenciennes), in the same region (Patagonia), are considered to be identical; therefore, the valid name of this species is Pseudodelphis percichthydis n. comb. and P. limnicola becomes its junior synonym. A key to the species of Pseudodelphis is provided.     

Phylogenetic insights on Mediterranean and Afrotropical <Emphasis Type="Italic">Rhipicephalus</Emphasis> species (Acari: Ixodida) based on mitochondrial DNA

This study was performed to determine the tick species that infest cattle and humans throughout an altitudinal gradient in the Yungas Biogeographic Province of Argentina. The presence of tick-borne bacteria of the genera Rickettsia, Ehrlichia and Borrelia in the collected ticks was also evaluated. Samples of ticks parasitizing cattle and humans were carried out in different seasons. Questing ticks (adults and nymphs) were collected from vegetation and analyzed to detect the presence of Rickettsia, Ehrlichia and Borrelia by a battery of different PCRs. Five species of hard ticks were found parasitizing cattle: Amblyomma sculptum, Amblyomma tonelliae, Amblyomma hadanii, Haemaphysalis juxtakochi and Ixodes pararicinus. Amblyomma sculptum (immature and adults), A. tonelliae (immature and adults), A. hadanii (larvae) and one nymph of I. pararicinus were found attached to humans. Rickettsia amblyommatis was detected in one nymph of A. hadanii. DNA of a Borrelia genospecies belonging to the B. burgdorferi s.l. complex (phylogenetically related to haplotypes previously reported in Ixodes aragaoi from Uruguay and I. pararicinus from Argentina) was detected in adults of I. pararicinus. Amblyomma sculptum and I. pararicinus appear to be the tick species more frequent on cattle in the YBP from Argentina, and A. sculptum and A. tonelliae, were the main ticks found attached to humans. The medical importance of the bacteria of the genus Rickettsia and Borrelia detected in this work remains unknown.     

A re-evaluation of diversity of the Aporocotylidae Odhner, 1912 in <Emphasis Type="Italic">Siganus fuscescens</Emphasis> (Houttuyn) (Perciformes: Siganidae) and associated species

The aporocotylid fauna of the mottled spinefoot, Siganus fuscescens (Houttuyn), from Moreton Bay, Queensland, Australia, was characterised using a combined morphological and molecular approach. Four aporocotylid species were identified, three belonging to the genus Ankistromeces Nolan & Cribb, 2006 and one to Cardicola Short, 1953. Specimens of Cardicola matched an undescribed species from the same host and locality; this species is described as Cardicola mogilae n. sp. Phylogenetic analyses of ITS2 and 28S data showed that C. mogilae n. sp. forms a strongly supported clade with other Cardicola species from siganid fishes. We record Ankistromeces olsoni Nolan & Cribb, 2006 in Moreton Bay for the first time, redescribe A. dunwichensis Nolan & Cribb, 2006 on the basis of new specimens and sequence data and re-report Ankistromeces sp. X from Moreton Bay based on molecular data. We review the status of the ten putative species of aporocotylids reported from siganids. Small variation in ITS2 rDNA sequences, in association with different geographic localities, was previously used to separate Cardicola lafii Nolan & Cribb, 2006 from C. parilus Nolan & Cribb, 2006, C. bartolii Nolan & Cribb, 2006 from C. watsonensis Nolan & Cribb, 2006, C. tantabiddii Nolan & Cribb, 2006 from Cardicola sp. 2, Ankistromeces sp. Y from A. olsoni and Ankistromeces sp. X from Ankistromeces sp. Z. These five combinations are reinterpreted as each representing a single species; Cardicola lafii is recognised as the senior synonym of C. parilus and C. bartolii as the senior synonym of C. watsonensis. This study thus suggests that six, rather than ten, species should be recognised as infecting S. fuscescens. This richness remains greater than is known for any other fish species and siganids are, so far, unique among fishes in harbouring two strongly radiated lineages of aporocotylids.     

Descriptions of eight new species of <Emphasis Type="Italic">Ligophorus</Emphasis> Euzet &; Suriano, 1977 (Monogenea: Ancyrocephalidae) from Red Sea mullets

Eight new species of Ligophorus Euzet &; Suriano, 1977 (Monogenea: Ancyrocephalidae) are described from two species of mullets from the Red Sea. Ligophorus bykhowskyi n. sp. and L. zhangi n. sp. from Crenimugil crenilabris (Forsskål) differ from other species of the genus in the structure of the male copulatory organ, which has a simple accessory piece and a wide copulatory tube that arises from a large, single-chambered, expanded base. Ligophorus simpliciformis n. sp., L. bipartitus n. sp., L. campanulatus n. sp., L. mamaevi n. sp., L. lebedevi n. sp. and L. surianoae n. sp. from Liza carinata (Valenciennes) are differentiated on the basis of the morphometrics of the hard parts of the haptor and male copulatory organ. The eight species represent the first records of species directly attributed to Ligophorus from the Red Sea. Measurements of the haptoral hard-parts and the male copulatory organ of the new species are analysed with the aid of Principal Component Analysis. Three morphological types of male copulatory organ, five types of anchor, and two types of ventral and three types of dorsal bars were distinguished among these species. L. bykhowskyi and L. zhangi from C. crenilabris have the same type of male copulatory organ and anchors. Those species from Liza carinata have only one common morphological character, a thick copulatory tube, but have two types of accessory piece, four types of anchors and three types of bars. All species of Ligophorus found on mullets in the Red Sea have an accessory piece without a distal bifurcation and thus differ from most species of this genus from other regions of the world’s oceans.     

<Emphasis Type="BoldItalic">Barrmaelia</Emphasis> and <Emphasis Type="BoldItalic">Entosordaria</Emphasis> in Barrmaeliaceae (fam. nov., Xylariales) and critical notes on <Emphasis Type="BoldItalic">Anthostomella</Emphasis>-like genera based on multigene phylogenies

Phylogenetic analyses of a combined DNA data matrix containing ITS, LSU, rpb2 and tub2 sequences of representative Xylariales revealed that the genus Barrmaelia is a well-defined monophylum, as based on four of its described species (B. macrospora, B. moravica, B. oxyacanthae, B. rhamnicola) and the new species B. rappazii. The generic type of Entosordaria, E. perfidiosa, is revealed as the closest relative of Barrmaelia, being phylogenetically distant from the generic type of Clypeosphaeria, C. mamillana, which belongs to Xylariaceae sensu stricto. Entosordaria and Barrmaelia are highly supported and form a distinct lineage, which is recognised as the new family Barrmaeliaceae. The new species E. quercina is described. Barrmaelia macrospora, B. moravica and B. rhamnicola are epitypified and E. perfidiosa is lecto- and epitypified. Published sequences of Anthostomella and several Anthostomella-like species from the genera Alloanthostomella, Anthostomelloides, Neoanthostomella, Pseudoanthostomella and Pyriformiascoma are evaluated, demonstrating the necessity of critical inspection of published sequence data before inclusion in phylogenies. Verified isolates of several species from these genera should be re-sequenced to affirm their phylogenetic affinities. In addition, the generic type of Anthostomella should be sequenced before additional generic re-arrangements are proposed.     

Genomic clues to the parental origin of the wild flowering cherry <Emphasis Type="Italic">Prunus yedoensis</Emphasis> var. <Emphasis Type="Italic">nudiflora</Emphasis> (Rosaceae)

Prunus yedoensis Matsumura is one of the popular ornamental flowering cherry trees native to northeastern Asia, and its wild populations have only been found on Jeju Island, Korea. Previous studies suggested that wild P. yedoensis (P. yedoensis var. nudiflora) is a hybrid species; however, there is no solid evidence on its exact parental origin and genomic organization. In this study, we developed a total of 38 nuclear gene-based DNA markers that can be universally amplifiable in the Prunus species using 586 Prunus Conserved Orthologous Gene Set (Prunus COS). Using the Prunus COS markers, we investigated the genetic structure of wild P. yedoensis populations and evaluated the putative parental species of wild P. yedoensis. Population structure and phylogenetic analysis of 73 wild P. yedoensis accessions and 54 accessions of other Prunus species revealed that the wild P. yedoensis on Jeju Island is a natural homoploid hybrid. Sequence-level comparison of Prunus COS markers between species suggested that wild P. yedoensis might originate from a cross between maternal P. pendula f. ascendens and paternal P. jamasakura. Moreover, approximately 81% of the wild P. yedoensis accessions examined were likely F1 hybrids, whereas the remaining 19% were backcross hybrids resulting from additional asymmetric introgression of parental genotypes. These findings suggest that complex hybridization of the Prunus species on Jeju Island can produce a range of variable hybrid offspring. Overall, this study makes a significant contribution to address issues of the origin, nomenclature, and genetic relationship of ornamental P. yedoensis.     

Mealybugs of the genus <Emphasis Type="Italic">Fonscolombia</Emphasis> Licht. (Homoptera,Pseudococcidae) of the fauna of Russia and adjacent countries

The genus name Euripersia Borchs. is considered a junior synonym of Fonscolombia Licht. based on the absence of essential differences between type species of these genera. The key to Fonscolombia includes, in addition to the type species, nine species from Russia and adjacent countries. Eight of these species are redescribed and illustrated, F. butorinae Danzig et Gavrilov, 2005 has been recently described as new. Two species originally described in Euripersia do not belong to this genus. One is transferred to Trionymus Berg, and thus a new combination is established: Trionymus mongolicus (Danzig), comb. n. Synonymy of Euripersia caulicola with Phenacoccus incertus Kir. has been established recently (Danzig, 2005). Other species presently placed in Euripersia (Ben-Dov, 1994) need a revision. Lectotypes are designated for 4 nominal species. Fonscolombia is related to Phenacoccus Sign., the separation of these two genera is not clear. Fonscolombia is characterized by broad-oval and convex body, poorly developed anal lobes, short, 6-8-segmented (F. edentata and F. herbacea with 7–9-segmented) antennae, and cerarii present on the last abdominal segments only (C ₃ sometimes present). Most species live on roots of grasses (F. butorinae between leaves near soil, F. herbacea, on dicotyledonous herbs) and are characterized by ovoviviparity.     

<Emphasis Type="Italic">Cacopsylla biwa</Emphasis> sp. nov. (Hemiptera: Psyllidae): a new pest of loquat <Emphasis Type="Italic">Eriobotrya japonica</Emphasis> (Rosaceae) in Japan

A new psyllid species, Cacopsylla biwa Inoue, sp. nov., is described from Tokushima Prefecture, Shikoku, Japan. This new species develops only on Eriobotrya japonica (Thunb.) Lindl. (Rosaceae), causing severe damage to its fruits and flowers. It is strongly suspected that C. biwa is an alien species. Morphological similarities and host-plant relationships indicate that C. biwa is most closely related to Cacopsylla eriobotryae (Yang) comb. nov. (transferred from Edentatipsylla Li), which occurs in Taiwan and feeds on Eriobotrya deflexa (Hemsl.) Nakai. Morphological diagnostic characteristics of C. biwa and differences from the other congeners are discussed. Information is provided on the biology and life cycle of the new species.     

Fingerprinting by amplified fragment length polymorphism (AFLP) and barcoding by three plastidic markers in the genus <Emphasis Type="Italic">Wolffiella</Emphasis> Hegelm

Amplified fragment length polymorphism (AFLP) fingerprinting and three different plastidic DNA regions (rpl16, rps16, atpF-atpH) were used to investigate species identity in the genus Wolffiella. For this purpose, clones (67 in total) belonging to all ten species were selected. Almost all the species were represented by more than one clone. The fingerprinting technique, AFLP, clearly distinguished the species, W. caudata, W. gladiata, W. neotropica, W. rotunda, and W. welwitschii. Apart from confirming the molecular identity of these five species, the plastidic markers could delineate two additional species, W. hyalina and W. denticulata, although the conclusion concerning the latter is restricted by the availability of only one clone. The efficiency of the plastid-derived markers in identifying the number of species-specific clusters followed the sequence rps16 > rpl16 > atpF-atpH. The species W. lingulata, W. oblonga, and W. repanda could not be identified by any of the molecular methods presented here, but could be strictly defined on a morphological basis. In several clones, high amounts of genetic admixtures between different species were detected. Further, simulation studies demonstrated that these clones are genetic hybrids. This might be one of the obstacles in molecular identification of species in the genus Wolffiella.     

Fen meadows on the move for the conservation of <Emphasis Type="Italic">Maculinea (Phengaris) teleius</Emphasis> butterflies

In the Netherlands, a single population of the obligate myrmecophilic butterfly Maculinea (Phengaris) teleius has survived on only 3 ha of habitat for more than 25 years, whereas at least 40 ha of habitat are thought to be required for a sustainable metapopulation. Therefore, 170 ha of farmland is being restored to wet meadows within a LIFE?+?project by large-scale soil excavation and hay inoculation. For successful restoration, the habitat requirements of the butterfly, with Sanguisorba officinalis as host plant and its particular life cycle as parasite of the ant species Myrmica scabrinodis, have to be taken into account. We tested whether colonization of nests of this ant species in the restoration areas is facilitated by translocation of sods collected from fen meadows. We divided 54 sods, each sized 1 m², randomly over six patches and measured vegetation development and ant presence in the sods and surrounding control plots for 2 years. In the first summer, significantly more Myrmica ants were found in the transplanted sods in comparison to the surrounding area. Herb cover had a significant positive effect on Myrmica ant presence while it did not affect the presence of the pioneer ant species Lasius niger. In the second year, Myrmica ants were found in the surrounding control plots as well. This study contributes to the knowledge-base required for the design of restoration projects aimed at expanding the habitat of the critically endangered butterfly Maculinea (Phengaris) teleius.     

Polymorphic sites in transcribed spacers of 35S rRNA genes as an indicator of origin of the <Emphasis Type="Italic">Paeonia</Emphasis> cultivars

Region ITS1–5.8S rDNA–ITS2 is sequenced in 27 varieties of cultivated ornamental peonies, ten of which presumably originate from Paeonia lactiflora, one from P. officinalis, 13 from hybridization of P. lactiflora and P. peregrina, or P. officinalis, and three are Itoh hybrids. Comparative analysis of distribution patterns of polymorphic sites (PS) for the obtained DNA sequences and data from GenBank is carried out. Hypotheses of origin of the studied varieties, except for two, which, as previously assumed, originate from hybridization of P. lactiflora and P. peregrina, are confirmed. It is shown that the sequence ITS1–5.8S rDNA–ITS2 is a good genetic marker for cultivars of the P. lactiflora group and Itoh hybrids, and that the PS distribution patterns in these sequences can provide valuable information on the kinship and origin of individual varieties. However, insufficient knowledge of wild species from the P. officinalis kinship group limits the use of this marker in the study of varieties obtained through interspecific hybridization within the Paeonia section.     

Phylogeography of the Macaronesian Lettuce Species <Emphasis Type="Italic">Lactuca watsoniana</Emphasis> and <Emphasis Type="Italic">L. palmensis</Emphasis> (Asteraceae)

The phylogenetic relationships and phylogeography of two relatively rare Macaronesian Lactuca species, Lactuca watsoniana (Azores) and L. palmensis (Canary Islands), were, until this date, unclear. Karyological information of the Azorean species was also unknown. For this study, a chromosome count was performed and L. watsoniana showed 2n = 34. A phylogenetic approach was used to clarify the relationships of the Azorean endemic L. watsoniana and the La Palma endemic L. palmensis within the subtribe Lactucinae. Maximum parsimony, Maximum likelihood and Bayesian analysis of a combined molecular dataset (ITS and four chloroplast DNA regions) and molecular clock analyses were performed with the Macaronesian Lactuca species, as well as a TCS haplotype network. The analyses revealed that L. watsoniana and L. palmensis belong to different subclades of the Lactuca clade. Lactuca watsoniana showed a strongly supported phylogenetic relationship with North American species, while L. palmensis was closely related to L. tenerrima and L. inermis, from Europe and Africa. Lactuca watsoniana showed four single-island haplotypes. A divergence time estimation of the Macaronesian lineages was used to examine island colonization pathways. Results obtained with BEAST suggest a divergence of L. palmensis and L. watsoniana clades c. 11 million years ago, L. watsoniana diverged from its North American sister species c. 3.8 million years ago and L. palmensis diverged from its sister L. tenerrima, c. 1.3 million years ago, probably originating from an African ancestral lineage which colonized the Canary Islands. Divergence analyses with *BEAST indicate a more recent divergence of the L. watsoniana crown, c. 0.9 million years ago. In the Azores colonization, in a stepping stone, east-to-west dispersal pattern, associated with geological events might explain the current distribution range of L. watsoniana.