Cryopreservation of the critically endangered golden paintbrush (<Emphasis Type="Italic">Castilleja levisecta</Emphasis> Greenm.): from nature to cryobank to nature

In this study conservation of Castilleja levisecta Greenm., a globally endangered species was addressed through in vitro cryopreservation of shoot tips. In vitro cultures were successfully established using seedlings received from British Columbia, Canada. Shoot tips excised from in vitro propagated plants were cryopreserved using a droplet-vitrification method following optimization of individual protocol steps such as pre-culture, treatment with vitrification solutions, and unloading. The highest plant regrowth after cryopreservation (66%) was achieved when shoot tips were pre-cultured in 0.3 M sucrose for 17 h followed by 0.5 M sucrose for 4 h, incubated in an osmo-protectant solution (17.5% [v/v] glycerol and 17.5% [w/v] sucrose) for 20 min, exposed to vitrification solution A3 (37.5% [v/v] glycerol plus 15% [v/v] dimethylsulfoxide (DMSO) plus 15% [v/v] ethylene glycol (EG) plus 22.5% [w/v] sucrose) on ice for 40 min, and unloaded in 0.8 M sucrose solution for 30 min. Healthy plants were developed from cryopreserved shoot tips and propagated in vitro using nodal segments. Plants derived from in vitro culture and from cryopreserved tissues were successfully rooted and acclimated in a greenhouse with 100% survival rate. Acclimatized plants were reintroduced in a naturalized propagation area at the Conservation Nursery at Fort Rodd Hill, Canada. Twenty of 94 reintroduced plants (21%) survived the transit from lab to field and some had started to flower. This is the first report for cryopreservation of C. levisecta, an important step in conserving and re-introducing this critically imperiled species in nature.     

Water quality effects following establishment of the invasive <Emphasis Type="Italic">Dreissena polymorpha</Emphasis> (Pallas) in a shallow eutrophic lake: implications for pollution mitigation measures

This study investigates whether ecosystem alteration occurred in a shallow, lowland lake following establishment of the alien zebra mussel, Dreissena polymorpha (Pallas). Measurements of total phosphorus (TP) loads, lake TP concentrations, phytoplankton (chlorophyll a) and transparency levels for the period 1990–2008 were examined to determine the water quality effects of D. polymorpha. The period of time also included the implementation of catchment measures aimed at reducing phosphorus (P) loading to the lake. A range of loading-response models was tested to explore changes in the net sedimentation rate for P. Results show that while high TP loads from the catchment were reduced, TP concentrations in the lake remained high after D. polymorpha invasion. Decoupling of the previous chlorophyll a–TP relationship also occurred. Results from a TP loading-response model that closely simulated observed concentrations in the lake prior to establishment of D. polymorpha indicated that measured TP post establishment was statistically higher than predicted for the same conditions but without the presence of D. polymorpha. Data presented in the paper highlight the need to consider the potential impacts of invasive species in evaluations of the effectiveness of measures aimed at mitigating aquatic pollution.     

Cryopreservation of <Emphasis Type="Italic">Citrus</Emphasis> anthers in the National Crop Genebank of China

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L^?1 sucrose, 0.1-g L^?1 boric acid, 1.0-g L^?1 calcium nitrate, 0.1-g L^?1 potassium nitrate, 0.3-g L^?1 magnesium sulfate, and 10-g L^?1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.     

A conserved repetitive DNA element located in the centromeres of chromosomes in <Emphasis Type="Italic">Medicago</Emphasis> genus

In most eukaryotic species, centromeres harbor large arrays of tandem repeated satellite DNA sequences. In this study, we report on the genomic distribution of a centromere satellite repeat “MtR3” in Medicago genus and three distantly related genera. Fluorescence in situ hybridization (FISH) results showed MtR3 repeats were detected in the centromere regions in M. truncatula, M. minima, M. edgeworthii, M. ruthenica, M. caerulea, M. sativa, and M. falcata (4×), but no signals were discovered in M. lupulina, M. polymorpha, and M. falcata (2×), Melilotus officinalis, Crotalaria medicaginea, and Trifolium repens. However, sequence analysis showed this MtR3 DNA had genomic distribution in all species and was highly conserved across the entire Medicago genus and three other genera. The conservation and widespread presence suggested MtR3 repeats may play important roles in centromeric function.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Cryopreservation of an endangered <Emphasis Type="Italic">Hladnikia pastinacifolia</Emphasis> Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.     

Genetic diversity of the house mouse <Emphasis Type="Italic">Mus musculus</Emphasis> and geographic distribution of its subspecies-specific RAPD markers on the territory of Russia

Genetic diversity and geographic distribution of taxon-specific RAPD markers was examined in ten local populations of the house mouse Mus musculus (n = 42). The house mice were generally characterized by moderate genetic variation: polymorphism P ₉₉ = 60%, P ₉₅ = 32.57%; heterozygosity H = 0.12; the observed allele number n _a = 1.6; the effective allele number n _e = 1.18; the within-population differentiation ?_s = 0.388; and Shannon index I = 0.19. The degree of genetic isolation of individual local populations was greatly variable. The genetic subdivision index G _st varied from 0.162 to 0.770 at the gene flow of Nm = 2.58?0.149, while the among-population distances D _N varied from 0.026 to 0.178. The largest part of the genetic diversity was found among the populations (H _T = 0.125), while the within-population diversity was twice lower (H _S = 0.06). The samples examined were well discriminated relative to the sets of RAPD markers. The character distribution pattern provided conditional subdivision of the mice into the “western” and the “eastern” groups with the putative boarder along the Baikal Lake. The first group was characterized by the prevalence of the markers typical of M. m. musculus and M. m. domesticus. The second group was characterized by the prevalence of the markers typical of M. m. musculus, M. m. gansuensis, M. m. castaneus, M. m. domesticus, and M. m. wagneri. The genotype of the nominative subspecies M. m. musculus was background for all populations. In the populations examined some of earlier described subspecies-specific molecular markers were found at different frequencies, pointing to the involvement of several subspecies of M. musculus in the process of hybridization.     

Droplet-vitrification cryopreservation of <Emphasis Type="Italic">in vitro</Emphasis>-grown shoot tips of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.     

A first report on coexistence and hybridization of <Emphasis Type="Italic">Mytilus trossulus</Emphasis> and <Emphasis Type="Italic">M. edulis</Emphasis> mussels in Greenland

Semi-sessile Mytilus mussels are used as indicators of climate changes, but their geographic distribution is not sufficiently known in the Arctic. The aim of this study was to investigate the taxonomic status and genetic differentiation of Mytilus populations in a Northwest Greenlandic fjord at Maarmorilik, impacted by contaminations from a former mine. In this study, mussels were collected at three sites differing in exposure to environmental factors. A total of 54 polymorphic SNPs found in the Mytilus EST and DNA sequences analyzed were successfully applied to 256 individuals. The results provided the first evidence for the existence of M. trossulus in Greenland. The mussel from M. trossulus and M. edulis taxa are shown to coexist and hybridize in the fjord. The three studied sites were found to differ significantly in the distribution of taxa with a higher prevalence of M. trossulus in the inner fjord. The identified M. edulis × M. trossulus hybrids mostly had a hybrid index score of about 0.5, indicating a similar number of alleles characteristic for M. trossulus and M. edulis. There was a low number of backcrosses between ‘pure’ taxa and hybrids. This newly discovered hybrid zone between the two taxa is unique in comparison with the Canadian populations. As Mytilus mussels in Greenland hitherto have been regarded as the one taxon M. edulis, the results have importance for biogeography and future monitoring and environmental studies.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Taxonomic position of Afghan vole (Subgenus <Emphasis Type="Italic">Blanfordimys</Emphasis>) by the sequence of the mitochondrial <Emphasis Type="Italic">cytb</Emphasis> gene

It is rather difficult to construct a system of gray voles of the tribe Microtini by a set of morphological and karyological characters because form generation is mosaic at these organization levels. The sequence of the mitochondrial cytochrome b gene was used to study the phylogenetic relationships and taxonomic position of the Central Asian subgenus Blanfordimys. Afghan vole Microtus (Blanfordimys) afghanus and Bucharian vole M. (Blanfordimys) bucharensis clustered with Pamir vole M. (Neodon) juldaschi, which is conventionally assigned to another subgenus. The last two species proved to be significantly closer to each other than either of them was to M. (Blanfordimys) afghanus, which disagrees with the monophyletic origin accepted for Blanfordimys. The genetic distances between the species of the subgenus Blanfordmys and M. juldaschi were comparable with the distances between the sister subgenera Microtus s. str. and Sumeriomys or Pallasiinus and Alexandromys and with the basal divergence of supraspecific clades in the subgenus Terricola. It was assumed that a special Central Asian group of species exists within the tribe Microtini and includes species of the subgenus Blanfordimys and M. juldaschi and that the subgenera Neodon and Blanfordimys should be revised.     

Phylogenetic relationship and time of divergence of <Emphasis Type="Italic">Mus terricolor</Emphasis> with reference to other <Emphasis Type="Italic">Mus</Emphasis> species

Mitochondrial DNA control region of Mus terricolor, three aboriginal species M. spretus, M. macedonicus, M. spicilegus; the Asian lineage M. caroli, M. cervicolor, M. cookii; and the two house mice, M. musculus domesticus and M. m. castaneus were analysed to estimate the substitution rate, phylogenetic relationship and the probable time of divergence. Results showed that M. spretus, M. caroli and M. terricolor are highly diverged from each other (caroli/terricolor = 0.146, caroli/spretus = 0.147 and terricolor/spretus = 0.122), whereas M. spretus showed less divergence with two house mice species (0.070 and 0.071). Sequence divergence between M. terricolor and the Palearctic group were found to be ranging from 0.121 to 0.134. Phylogenetic analysis by minimum evolution, neighbour-joining, unweighed pair group method with arithmetic mean and maximum parsimony showed almost similar topology. Two major clusters were found, one included the Asian lineage, M. caroli, M. cookii and M. cervicolor and the other included the house mice M. m. domesticus, M. m. castaneus and the aboriginal mice M. macedonicus and M. spicilegus along with M. spretus, forming the Palearctic clade. M. terricolor was positioned between the Palearctic and Asian clades. Results showed that Palearctic-terricolor and the Asian lineages diverged 5.47 million years ago (Mya), while M. terricolor had split around 4.63 Mya from their ancestor. M. cervicolor, M. cookii and M. caroli diverged between 4.70 and 3.36 Mya, which indicates that M. terricolor and the Asian lineages evolved simultaneously. M. spretus is expected to have diverged nearly 2.9 Mya from their most recent common ancestor.     

Clavicipitaceous entomopathogens: new species in <Emphasis Type="Italic">Metarhizium</Emphasis> and a new genus <Emphasis Type="Italic">Nigelia</Emphasis>

In several surveys in the tropical forests in Thailand, specimens that looked morphologically similar to Metarhizium martiale and Cordyceps variegata, as well as other Metarhizium species were collected and cultured in vitro. A combined phylogeny of several genes including the small (18S) and large (28S) subunits of the ribosomal DNA, elongation factor 1-α (TEF), RNA polymerase II subunits 1 and 2 (RPB1, RPB2) genes has shown these to be new taxa in the Clavicipitaceae. Nigelia is described as a new genus closely related to Metarhizium, to the scale insect pathogens Aschersonia (Hypocrella), Samuelsia and Moelleriella, and to plant pathogens in Claviceps and Balansia, and other relatives. Nigelia comprises M. martiale and a new species Nigelia aurantiaca, which has been found infecting lepidopteran larvae and which produces pseudoimmersed, obliquely arranged, obpyriform perithecia with curved or bent ostioles and with whole (non-separating) cylindric ascospores. Metarhizium chaiyaphumense, M. kalasinense, M. prachinense, M. samlanense, and M. takense are described as new species of Metarhizium. Metarhizium martiale is transferred to Nigelia, and Paecilomyces reniformis is transferred to Metarhizium.     

Exploring virulence of new and less studied species of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. from Brazil for two-spotted spider mite control

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 10⁷ conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT₅₀). The lowest LT₅₀ (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.     

Survival and genetic stability of shoot tips of <Emphasis Type="Italic">Hedeoma todsenii</Emphasis> R.S.Irving after long-term cryostorage

Endangered and rare species for which seed banking is not possible require alternative methods of ex situ conservation for long-term preservation. These methods depend primarily on cryopreservation methods, such as shoot tip cryopreservation, but there are few datasets with information on the long-term survival of shoot tips stored in liquid nitrogen. In this study, survival and genetic stability of shoot tips of the endangered species, Hedeoma todsenii, banked over multiple years were examined. In vitro cultures cryopreserved with both the encapsulation dehydration and the encapsulation vitrification methods showed good average survival after up to 13 yr of storage in liquid nitrogen. The application of droplet vitrification to this species increased survival significantly, with an average of 72%, compared with 24–45% survival obtained with other methods. As measured with microsatellite and sequence-related amplified polymorphism (SRAP) markers, the genetic stability of the same genotypes stored over different periods of time typically did not change. However, there was an average of 10.4% band loss between replicate samples that did indicate a potential change in DNA composition. These results demonstrate the use of shoot tip cryopreservation as an effective ex situ conservation tool for this species, but genetic stability of the cryopreserved tissues should be closely monitored.     

In vitro multiplication and genetic stability of two species of <Emphasis Type="Italic">Micranthocereus</Emphasis> Backeb. (Cactaceae) endemic to Bahia,Brazil

The two targeted species of this study, Micranthocereus flaviflorus subsp. densiflorus and M. polyanthus subsp. alvinii, are endemic to the state of Bahia and have ornamental value. The main goal of this work was to micropropagate these species and to evaluate the genetic stability of the regenerated plants. To do so, shoots originated from in vitro germinated plants were inoculated in MS/2 (Murashige, Skoog, Physiol Plant 15:473–497, 1962) media containing 1.34 μmol L^?1 of α-naphthaleneacetic acid (NAA) for morphogenesis induction. This was repeated for three consecutive subcultures, and the subcultured shoots were designated by order of production as S1, S2 and S3. Retention of morphogenic potential and acceleration of organogenic response was observed after the three subsequent propagation events, so that if shoots were used as explant source the in vitro propagation of M. flaviflorus could be achieved in 90 days and that of M. polyanthus could be optimized to 60 days of duration. In order to perform genetic stability analysis along subcultures, ISSR markers were used and genetic variation between shoots of each subculture and their donor plant was measured with Jaccard’s similarity coefficient. This analysis revealed high genetic stability in the in vitro propagation of all the donor plants of M. flaviflorus and M. polyanthus in regards to three consecutive shoot subcultures, in which similarities were 100% for both species. The study of a greater number of subcultures is suggested to assess morphogenesis potential and genetic fidelity in long term.     

Phylogenetic assessment and taxonomic revision of <Emphasis Type="Italic">Mariannaea</Emphasis>

Four new species of Mariannaea were described in this paper, namely M. chlamydospora, M. cinerea, M. fusiformis, and M. lignicola. Mariannaea chlamydospora is characterized by its cream-colored, zonate colonies on PDA, smooth conidiophores, fusiform conidia, and abundant chlamydospores. Mariannaea cinerea forms grey colonies and ellipsoidal to subglobose conidia. Mariannaea fusiformis forms purple colonies and fusiform to subglobose conidia. Mariannaea lignicola has brown conidiophores and broad hyphae. The molecular phylogeny was inferred using ITS, LSU, and TUB-2 loci. The type species of Mariannaea (M. elegans) is epitypified. The variety M. elegans var. punicea is raised to species rank. Mariannaea clavispora is excluded from Mariannaea because of its cylindrical phialides, straight conidial chains and deviating phylogenetic affinity. Mariannaea nipponica did not fit well the generic concept of Mariannaea based on their morphological characters, and its generic placement remains uncertain. A key to the currently accepted 15 species of Mariannaea is provided.