Host‐specific differentiation in the anther smut fungus Microbotryum violaceum as revealed by microsatellites

The anther smut fungus Microbotryum violaceum ( = Ustilago violacea) is a parasite of many species of the Caryophyllaceae. Its host specificity has been debated since early this century, when cross‐inoculation experiments indicated the existence of host‐specific lineages. Recently, on the basis of spore ultrastructure, all presumed host races were lumped within M. violaceum. To measure gene flow among natural populations of anther smuts from different host species, we used microsatellite variation at 5 loci among samples from 8 Silene, 2 Saponaria, 2 Dianthus and 1 Gypsophila species. Most of the 326 M. violaceum samples investigated originated from the Swiss Alps and close surroundings. Microsatellite variation revealed almost perfect isolation among anther smut fungi from different host species. In addition, differentiation was supported by the nonrandom distribution of null alleles among samples from different host species and host genera. Null alleles were most abundant in anther smut samples from non‐Silene hosts. The resolution of genetic differentiation among anther smuts from different host species was highest in those from Silene species. Genetic relationships among samples as indicated by Neighbour‐Joining analysis based on genetic distances are discussed with respect to host phylogeny and host ecology. One sample was identified as Ustilago gausseni because it had verrucose instead of reticulate spores and was collected from Silene italica. Neighbour‐Joining analysis revealed that this sample was similar to the M. violaceum samples from other Silene host species. Therefore, our data question spore morphology as a reliable character for anther smut systematics.     

Isolation and characterization of microsatellite loci in Cylindrocladium pauciramosum

Ten polymorphic microsatellite markers were developed for Cylindrocladium pauciramosum, a plant pathogen with a wide host range, which poses a serious problem in South African Eucalyptus nurseries. Polymorphism was evaluated on 43 isolates collected from Colombia and South Africa. Each locus had between three and six alleles. Testing for random mating showed multilocus equilibrium for a population of 40 isolates from a South African forestry nursery. Cross‐species transferability tested for 19 other Cylindrocladium species found amplification only in C. spathulatum, which is phylogenetically closely related to C. pauciramosum.     

Structural refuges in two stem-boring weevils on Rumex crispus

Abstract.

• 1 Differences in feeding positions, parasitoid complexes and parasitization rates of the two dominant insect species in stems of Rumex crispus were investigated. Apion miniatum (inhabiting the lower 10% of the stem) and Apion violaceum (occurring all along the stem) exploited distinctly different feeding positions, partly due to differences in time of oviposition.
• 2 A.miniatum suffered less parasitization thanA.violaceum, measured both by number of parasitoid species and parasitization rates. Its feeding position constitutes a refuge.
• 3 Parasitization of A.violaceum was least in individuals feeding in basal, thick parts of the stem. There is a trend that the developed refuge increases with increasing stem diameter.
• 4 The refuge of A.violaceum in basal positions only affected idiobiont ectoparasitoids, because koinobiont endoparasitoids attack the weevils before they enter the stem. Moreover, the koinobiont did benefit from the refuge itself by avoiding hyperparasitism.
• 5 Densities of A.violaceum increased with plant size, whereas percentage parasitism did not differ. Its three most abundant parasitoid species showed small-scale density-independent patterns of attack over all plants. Therefore a constant proportion of weevil larvae mine within the refuge independent of its potential size.
• 6 Synchronization between weevils and plants, as well as between parasitoids and hosts, appears to be the dominant factor for the spatial community structure of the stem borers of Rumex crispus and their enemies.

     

Isolation and characterization of microsatellite markers in the solitary hunting wasp Ancistrocerus adiabatus (Vespidae: Eumeninae) and their cross utility in other species of Ancistrocerus

We have developed seven microsatellite primers for the solitary wasp Ancistrocerus adiabatus. The number of alleles per locus ranged from three to 14. The markers were developed to study population structure, inbreeding and sex determination of A. adiabatus. For each locus, we genotyped 30 female wasps collected from Southwest Michigan. The observed heterozygosity ranges from 0.067 to 0.933. We have also determined that primers for these microsatellite loci amplify in three other species in the genus: Ancistrocerus antilope, Ancistrocerus catskill, and Ancistrocerus campesterus.     

Microsatellite markers in a complex of Horismenus sp. (Hymenoptera: Eulophidae), parasitoids of bruchid beetles

Parasitoids of the genus Horismenus (Hymenoptera: Eulophidae) are the main natural enemies of bruchid beetles that feed on several species of Phaseolus beans. Samples of Horismenus depressus, H. missouriensis and H. butcheri were collected from seeds of three Phaseolus species throughout Mexico to examine the impact of plant variability on the genetic structure of parasitoid populations. For this purpose, six microsatellite loci were isolated and characterized. These loci are of great interest in understanding the taxonomy of the genus Horismenus, the most important Eulophid genus in the Neotropics.     

Isolation and characterization of polymorphic microsatellite markers in Zabrotes subfasciatus Boheman (Coleoptera: Bruchidae)

Bruchid beetles of the genus Zabrotes (Coleoptera: Bruchidae) are important worldwide pests of legume seeds. Samples of Zabrotes subfasciatus and Z. sylvestris were collected from seeds of two Phaseolus species throughout Mexico to assess the role of host plant variation and plant domestication on the evolution of host use in this species. For this purpose six polymorphic microsatellite loci were isolated and characterized for the bruchid Z. subfasciatus. Cross‐species amplification tests were performed on Z. sylvestris and revealed that three loci amplified successfully and were polymorphic in this closely related species.     

Comparison of genetic variation and differentiation using microsatellite markers among three rare threatened and one widespread toad lily species of Tricyrtis section Flavae (Convallariaceae) in Japan

Genetic diversities were examined using six microsatellite markers amplifiable in three rare and one widespread species of Tricyrtis section Flavae, which are endemic to Japan. Contrary to a general expectation, the three rare species, Tricyrtis flava, Tricyrtis ohsumiensis and Tricyrtis perfoliata, have comparable genetic variation at the species level to that of the widespread Tricyrtis nana. This is probably because T. nana has not sufficiently recovered genetic diversity from the bottleneck at speciation or because recent range contractions have occurred in the three rare species. Genetic diversity at the population level was smaller in the putative selfing species T. nana than in the other three outcrossing species. Compared with a preceding study using allozyme markers, the genetic diversity in microsatellite loci was considerably larger, probably resulting from higher mutation rates at the microsatellite loci. Owing to the high genetic diversity of the microsatellite markers, genetic differentiation among populations could be estimated even in T. nana with little allozyme polymorphism.     

Dinucleotide microsatellite markers from the Antarctic seals and their use in other Pinnipeds

Twenty‐four microsatellite loci were isolated from three species of Antarctic seals (Subfamily Monachinae, Tribe Lobodontini). Eleven loci were cloned from Weddell seal, Leptonychotes weddellii, seven from leopard seal, Hydrurga leptonyx, and six from crabeater seal, Lobodon carcinophagus. Variability was assessed in Weddell seals collected in McMurdo Sound, leopard seals from Bird Island, South Georgia, and crabeater seals sampled in the eastern Ross Sea. All loci were variable in the three species used for cloning and 22 of these loci amplified variable products in the Ross seal, Ommatophoca rossii. Cross‐species amplification was largely successful, with an average of 19 loci amplifying products in other phocids.     

Mucilage secretion: an adaptive mechanism to reduce seed removal by soil erosion?

Diaspores of many plant species inhabiting open vegetation in semi‐arid environments secrete mucilage after wetting (myxospermy) that glues the diaspores to the ground and prevents movement when the mucilage dries. In the present study, we test whether mucilage secretion can be considered as a selective response to soil erosion in plant species inhabiting semi‐arid environments. We relate the amount and type of mucilage secretion by seeds of Helianthemum violaceum and Fumana ericifolia (Cistaceae) to the number of raindrop impacts needed to remove these seeds after gluing them with their own mucilage to the ground and also the time that these seeds resist water run‐off without detaching. We also compare the amount of seed mucilage production by plants growing in habitats without erosion and plants affected by severe erosion by fitting mixed effect models. Our results show an important phenotypic variation in the amount of mucilage secretion in both species, although it is suggested that the effect of mucilage secretion in the rate of seed removal by erosion is species‐ and mechanism‐dependent. For F. ericifolia, the amount of mucilage secreted by the seeds is directly proportional to their resistance to raindrop impacts and is positively related to the intensity of the erosive processes that the plants experience. Nevertheless, all the seeds resist the force of run‐off during 60 min, irrespective of the amount of mucilage they produce. In H. violaceum, mucilage secretion per se, and not the amount of mucilage produced by the seeds, has an effect on the rate of seed removal by erosive processes. Furthermore, cellulosic fibrils were found only in the mucilage of F. ericifolia but not in H. violaceum. Overall, our results only partially support the hypothesis that a selective response to soil erosion exists. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 241–251.     

Characterization of microsatellite loci in White‐chinned Petrel (Procellaria aequinoctialis) and cross‐amplification in six other procellariiform species

Six polymorphic dinucleotide microsatellite loci were isolated and characterized from the White‐chinned Petrel Procellaria aequinoctialis, using a degenerate primer and PCR‐based technique to construct and screen an enriched genomic library. Preliminary data on three populations show heterozygosity levels ranging from 0.22 to 0.67 and allele numbers from three to nine. Preliminary data also suggest genetic distance between these three populations (F_ST 0.088). Cross‐species amplification of these six microsatellite loci and one further locus were tested in six other procellariiform species of the genus Procellaria, Macronectes, Thalassarche and Diomedea.     

Microsatellite loci to assess genetic variation in Tor putitora

Seven polymorphic microsatellite DNA loci were identified in golden mahseer, Tor putitora, through cross‐species amplification. Thirty‐two primers developed for three cyprinid fishes were tested in the study. The genetic variation detected at each microsatellite locus in T. putitora specimens (n = 107), collected from three different rivers and one lake was assessed. The allele frequencies deviated significantly from that expected under Hardy–Weinberg equilibrium. The mean observed heterozygosity values ranged from 0.29 to 0.40. Significant genotype heterogeneity indicated that the samples were not drawn from the same gene pool. The results indicate that the identified microsatellite loci exhibit promise for use in fine scale population structure analyses of T. putitora.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

Porphyridium violaceum,eine marine neue Art

Zusammenfassung 1. Die neubeschriebene marinePorphyridium-Art unterscheidet sich von dem terrestrisch lebenden, aber auch im Meerwasser gefundenenPorphyridium purpureum durch ihre größeren, violett gefärbten Zellen.2. Teilung und Wachstum der Zellen vonPorphyridium violaceum passen sich dem Belichtungsrhythmus an. Die während der 10stündigen Dunkelheit geteilten Zellen sind 9 bis 11µ dick, sie vergrößern sich während der Lichtperiode auf 11 bis 14µ.

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Porphyridium violaceum, a marine new species

The new species is marine, whereasPorphyridium purpureum (= P. cruentum), first known only from terrestrial habitats, is halophilic and has been found several times in seawater.Porphyridium violaceum differs from the above mentioned species by a larger diameter and the colour of its cells.

     

The impact of genome defense on mobile elements in <Emphasis Type="Italic">Microbotryum</Emphasis>

Repeat induced point mutation (RIP), a mechanism causing hypermutation of repetitive DNA sequences in fungi, has been described as a ‘genome defense’ which functions to inactivate mobile elements and inhibit their deleterious effects on genome stability. Here we address the interactions between RIP and transposable elements in the Microbotryum violaceum species complex. Ten strains of M. violaceum, most of which belong to different species of the fungus, were all found to contain intragenomic populations of copia-like retrotransposons. Intragenomic DNA sequence variation among the copia-like elements was analyzed for evidence of RIP. Among species with RIP, there was no significant correlation between the frequency of RIP-induced mutations and inferred transposition rate based on diversity. Two strains of M. violaceum, from two different plant species but belonging to the same fungal lineage, contained copia-like elements with very low diversity, as would result from a high transposition rate, and these were also unique in showing no evidence of the hypermutation patterns indicative of the RIP genome defense. In this species, evidence of RIP was also absent from a Class II helitron-like transposable element. However, unexpectedly the absolute repetitive element load was lower than in other strains.     

Genetic diversity in the blackberry rust pathogen, Phragmidium violaceum, in Europe and Australasia as revealed by analysis of SAMPL

Indigenous to Europe, the blackberry rust fungus Phragmidium violaceum was introduced to Australia and subsequently appeared in New Zealand, with the most recent authorised introductions to Australia specifically for the biological control of European blackberry. Markers for ‘selective amplification of microsatellite polymorphic loci’ (SAMPL) were developed for studying the population genetics of P. violaceum. Modification of one of the two SAMPL primers with a HaeIII adapter (H) revealed significantly greater levels of genetic variation than primers used to generate AFLPs, the latter revealing little or no variation among 25 Australasian and 19 European isolates of P. violaceum. SAMPL was used to describe genetic variation among these 44 isolates of P. violaceum from 51 loci generated using primer pairs (GACA)₄ + H–G and R1 + H–G. The European isolates were more diverse than Australasian isolates, with 37 and 22 % polymorphic loci, respectively. Cluster analysis revealed geographic clades, with Australasian isolates forming one cluster separated from two clusters comprising the European isolates. However, low bootstrap support at these clades suggested that Australian isolates had not differentiated significantly from European isolates since the first record of P. violaceum in Australia in 1984. In general, the results support two hypotheses. First, that the population of P. violaceum in Australia was founded from a subset of individuals originating from Europe. Second, that P. violaceum in New Zealand originated from the Australian population of P. violaceum, probably by wind dispersal of urediniospores across the Tasman Sea. The application of SAMPL markers to the current biological control programme for European blackberry is discussed.     

Chloroplast microsatellite markers for the mangrove tree species Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa, and cross-amplification in other mangrove species

Chloroplast microsatellite (cpSSR) markers were developed for three ecologically and economically important tree species in the mangrove family, Rhizophoraceae: Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa. Noncoding regions of chloroplast DNA (cpDNA) from each species were separately amplified using universal chloroplast primers. Six, two, and three polymorphic cpSSR loci in B. gymnorrhiza, K. candel, and R. stylosa, respectively, were developed from amplified noncoding cpDNA regions. Characterization of 216, 156, and 253 individuals of B. gymnorrhiza, K. candel, and R. stylosa, respectively, collected from different natural mangrove populations (B. gymnorrhiza, 9; K. candel, 7; R. stylosa, 9) on Iriomote Island in Japan showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.027 and 0.480. These cpSSR markers will be useful for analyzing the maternal lineage distributions and population genetic structures of the three species. Several of these markers may also be useful in similar studies of other mangrove species.     

Development of microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

Isolation and characterization of microsatellite markers for Pinellia ternata and cross-species amplification

In this study, we isolated and characterized 12 microsatellite loci for Pinellia ternata. Polymorphism of these 12 loci was assessed in 46 individuals collected from two wild populations. All the loci were polymorphic with four to 13 alleles per locus and the observed and expected heterozygosities ranged from 0.312 to 0.680 and from 0.506 to 0.734, respectively. None of the loci showed significant deviation from Hardy–Weinberg equilibrium (P < 0.05). No significant linkage disequilibrium was observed between pairs of studied loci. In addition, most markers amplified successfully in three closely related taxa that are Pinellia cordata, P. peltata and P. pedatisecta. These microsatellite markers could provide a useful tool for genetic structure studies of the Pinellia species.     

Microsatellite markers for the invasive plant species white sweetclover (Melilotus alba) and yellow sweetclover (Melilotus officinalis)

We describe specific primers that amplify nine microsatellite DNA loci from Melilotus alba and Melilotus officinalis, both invasive plant species (Fabaceae) throughout North America. Allelic diversity was slightly lower for M. alba than for M. officinalis, as was expected heterozygosity. For both species, heterozygote deficit was observed at several loci. Genotypic diversity was very high for both species; the 29 plant samples of each species all had different multilocus genotypes. These markers will be used to determine the origins of the sweetclover invasion in Alaska and to compare patterns of diversity between subarctic and lower latitude populations.     

Isolation of polymorphic microsatellite loci in three phantom midge species of the genus Chaoborus (Diptera: Chaoboridae)

Because of its widespread distribution in lakes and ponds Chaoborus is of great interest to many freshwater ecologists. Interestingly some species are restricted to small fish‐less water bodies, whereas other species live mostly in large lakes. To eventually test the genetic and evolutionary implications of these different lifestyles we identified microsatellite loci in three species in this preliminary study: C. obscuripes, C. crystallinus and C. flavicans. Using a biotin/streptavidin capture technique of repetitive sequences in a 96 well format, we obtained microsatellite‐enriched genomic libraries for all three species and identified six polymorphic microsatellite markers for each species.