Unveiling biases in soft‐tissue phosphatization: extensive preservation of musculature in the Cretaceous (Cenomanian) polychaete Rollinschaeta myoplena (Annelida: Amphinomidae)

The process of soft‐tissue phosphatization (the replication of labile tissues by calcium phosphate) is responsible for many instances of high‐resolution soft tissue preservation, often revealing anatomical insights into the animals that so preserved. However, while much work has gone into exploring key issues such as biases and micro‐controls, phosphatization remains poorly understood as a taphonomic process. Here, using camera lucida, plain‐light microscopy and SEM imagery, we address this issue by describing the taphonomy and fidelity of the musculature of Rollinschaeta myoplena Parry et al., a phosphatized annelid from the Cretaceous Konservat‐Lagerstätten of Hakel and Hjoula, Lebanon, with an unprecedented quantity of three‐dimensional soft‐tissue preservation. Analysis highlights two strong, previously recognized biases affecting the process of phosphatization: (1) a taxonomic bias restricted to R. myoplena that triggers unusually extensive phosphatization; and (2) a tissue bias whereby longitudinal and parapodial musculature show markedly higher fidelity in comparison to the musculature of the intestine and body wall circular muscles. Potential explanations for these biases include internal phosphate‐enrichment by relative muscle density, the relative rate of decay and the physiology of musculature. Incongruence between experimental decay series for polychaetes and the prevalence of labile tissue preservation over recalcitrant tissues in R. myoplena exposes the limits of decay experiments for understanding exceptional preservation.     

Molecular Characterization of Phytoplasmas Related to Apple Proliferation and Aster Yellows Groups Associated with Pear Decline Disease in Iran

Pear trees showing pear decline disease symptoms were observed in pear orchards in the centre and north of Iran. Detection of phytoplasmas using universal primer pair P1A/P7A followed by primer pair R16F2n/R16R2 in nested PCR confirmed association of phytoplasmas with diseased pear trees. However, PCR using group‐specific primer pairs R16(X)F1/R16(X)R1 and rp(I)F1A/rp(I)R1A showed that Iranian pear phytoplasmas are related to apple proliferation and aster yellows groups. Moreover, PCR results using primer pair ESFYf/ESFYr specific to 16SrX‐B subgroup indicated that ‘Ca. Phytoplasma prunorum’ is associated with pear decline disease in the north of Iran. RFLP analyses using HaeIII, HhaI, HinfI, HpaII and RsaI restriction enzymes confirmed the PCR results. Partial 16S rRNA, imp, rp and secY genes sequence analyses approved that ‘Ca. Phytoplasma pyri’ and ‘Ca. Phytoplasma asteris’ cause pear decline disease in the centre of Iran, whereas ‘Ca. Phytoplasma prunorum’ causes disease in the north of Iran. This is the first report of the association of ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma prunorum’ with pear decline disease worldwide.     

The control of red colour by a family of MYB transcription factors in octoploid strawberry (Fragaria × ananassa) fruits

Octoploid strawberry (Fragaria × ananassa Duch.) is a model plant for research and one of the most important non‐climacteric fruit crops throughout the world. The associations between regulatory networks and metabolite composition were explored for one of the most critical agricultural properties in octoploid strawberry, fruit colour. Differences in the levels of flavonoids are due to the differences in the expression of structural and regulatory genes involved in flavonoid biosynthesis. The molecular mechanisms underlying differences in fruit colour were compared between red and white octoploid strawberry varieties. FaMYB genes had combinatorial effects in determining the red colour of fruit through the regulation of flavonoid biosynthesis in response to the increase in endogenous ABA at the final stage of fruit development. Analysis of alleles of FaMYB10 and FaMYB1 in red and white strawberry varieties led to the discovery of a white‐specific variant allele of FaMYB10, FaMYB10‐2. Its coding sequence possessed an ACTTATAC insertion in the genomic region encoding the C‐terminus of the protein. This insertion introduced a predicted premature termination codon, which suggested the loss of intact FaMYB10 protein playing a critical role in the loss of red colour in white octoploid strawberry.     

EST‐derived polymorphic microsatellites from cultivated strawberry (Fragaria×ananassa) are useful for diversity studies and varietal identification among Fragaria species

Microsatellite or simple sequence repeat markers derived from expressed sequence tags (ESTs) provide genetic markers within potentially functional genes, which could be very useful for breeding programs. To date, the development of microsatellite markers in the genus Fragaria has focused mainly on Fragaria vesca. However, most of the interests of breeding programs relate to specific characteristics of cultivated strawberry. Here, we describe a set of 10 EST‐derived microsatellites from Fragaria × ananassa. These markers showed high levels of polymorphism within strawberry cultivars and among different Fragaria species, indicating their potential for genetic studies not only on strawberry but also in other species within the genus.     

Stability Studies on Random Folded RNAs (‘Never Born RNAs’), Implications for the RNA World

In the last years, we have developed a method for the study of random RNA sequences with the aim of investigating their capacity to assume folded and possibly functionally active (ribozymes) structures. The RNA Foster assay developed in our laboratory is a powerful, simple, and fast method to investigate the structural properties of RNA by assessing the presence of secondary domains and their thermal stability through the combination of S1 nuclease activity at different temperatures. In this work, we investigated the structural properties of totally de novo random RNAs, 97‐nucleotides‐long, of which 60 nucleotides were completely random. The rationale behind this was to assess whether and to what extent random RNAs would acquire a stable fold as a prerequisite for catalytic activity. A library of random sequences was created, and 18 sequences were randomly selected and analyzed. Surprisingly, most of the RNAs were resistant to S1 nuclease digestion at up to 50°, and two sequences were resistant even at 70°, suggesting that these totally random RNA molecules could posses a stable secondary structure over a broad range of temperatures under the conditions used. We discuss the possible relevance of these data for the general scenario of the RNA world.     

Potential effects of the invasive ‘killer shrimp’ (Dikerogammarus villosus) on macroinvertebrate assemblages and biomonitoring indices

1. Water quality monitoring data from 10 watercourses and laboratory mesocosm studies were used to assess the potential impacts of the crustacean amphipod invader Dikerogammarus villosus on resident macroinvertebrate assemblage structure in Central European fresh waters. 2. The presence of D. villosus was associated with a decline in the prevalence of many native species, pollution sensitive as well as pollution tolerant, and changes in biotic indices, despite the trends of improved water quality coinciding with the invasion period. A general increase in the prevalence of other invaders was also noted. The potential impacts of D. villosus were substratum dependent, differing between stone, concrete and sand‐dominated sites. 3. Mean Multimetric Macroinvertebrate Index Flanders (MMIF) values were marginally lower when D. villosus was present (P < 0.06), as opposed to when other amphipod species or no amphipods were present, despite the improved water quality. Mesocosm studies showed that several macroinvertebrate taxa were completely eliminated in treatments with D. villosus, oligochaete worms, Caenidae mayfly, chironomids and tipulids being particularly vulnerable to D. villosus predation. Biological Monitoring Working Party (BMWP) scores were lower in mesocosms with D. villosus as opposed to the native Gammarus pulex or no amphipods at all. 4. We predict that resident macroinvertebrate assemblages in both Central Europe and Britain will come under increasing pressure as D. villosus invasions progress. Consequently, macroinvertebrate biotic indices, such as the MMIF or BMWP, may need to be revised to account for changes in taxa sensitivities to water quality as well as increased predation and competition.     

Accounting for female space sharing in St. Kilda Soay sheep (Ovis aries) results in little change in heritability estimates

When estimating heritability in free‐living populations, it is common practice to account for common environment effects, because of their potential to generate phenotypic covariance among relatives thereby biasing heritability estimates. In quantitative genetic studies of natural populations, however, philopatry, which results in relatives being clustered in space, is rarely accounted for. The two studies that have been carried out so far suggest absolute declines in heritability estimates of up to 43% when accounting for space sharing by relatives. However, due to methodological limitations these estimates may not be representative. We used data from the St. Kilda Soay sheep population to estimate heritabilities with and without accounting for space sharing for five traits for which there is evidence for additive genetic variance (birthweight, birth date, lamb August weight, and female post‐mortem jaw and metacarpal length). We accounted for space sharing by related females by separately incorporating spatial autocorrelation, and a home range similarity matrix. Although these terms accounted for up to 18% of the variance in these traits, heritability estimates were only reduced by up to 7%. Our results suggest that the bias caused by not accounting for space sharing may be lower than previously thought. This suggests that philopatry does not inevitably lead to a large bias if space sharing by relatives is not accounted for. We hope our work stimulates researchers to model shared space when relatives in their study population share space, as doing so will enable us to better understand when bias may be of particular concern.     

Identification of a Novel 1033‐Nucleotide Deletion Polymorphism in the Prophage Region of ‘Candidatus Liberibacter asiaticus’: Potential Applications for Bacterial Epidemiology

The prophage/phage region in the genome of ‘Candidatus Liberibacter asiaticus’, an alpha‐proteobacterium associated with citrus Huanglongbing, included many valuable loci for genetic diversity studies. Previously, a mosaic genomic region (CLIBASIA_05640 to CLIBASIA_05650) was characterized, and this revealed inter‐ and intracontinental variations of ‘Ca. L. asiaticus’. In this study, 267 ‘Ca. L. asiaticus’ isolates collected from eight provinces in China were analysed with a primer set flanking the same mosaic region plus downstream sequence. While most amplicon sizes ranged from 1400 to 2000 bp, an amplicon of 550 bp (S550) was found in 14 samples collected from south‐western China. Sequence analyses showed that S550 was the result of a 1033 bp deletion which included the previously known mosaic region. The genetic nature of the deletion event remains unknown. The regional restriction of S550 suggests that the ‘Ca. L. asiaticus’ population from south‐western China is different from those in eastern China. The small and easy‐to‐detect S550 amplicon could serve as a molecular marker for ‘Ca. L. asiaticus’ epidemiology.     

Female reproductive success in gynodioecious Thymus vulgaris: pollen versus nutrient limitation and pollinator foraging behaviour

Gynodioecy is a dimorphic breeding system in which female individuals coexist with hermaphroditic individuals in the same population. Females only contribute to the next generation via ovules, and many studies have shown that they are usually less attractive than hermaphrodites to pollinators. Several mechanisms have been proposed to explain how females manage to persist in populations despite these disadvantages. The ‘resource reallocation hypothesis’ (RRH) states that females channel resources not invested in pollen production and floral advertisement towards the production of more and/or larger seeds. We investigated pollination patterns and tested the RRH in a population of Thymus vulgaris. We measured flower display, flower size, nectar production, visitation rates, pollinator constancy and flower lifespan in the two morphs. In addition, we measured experimentally the effects of pollen and resource addition on female reproductive success (fruit set, seed set, seed weight) of the two morphs. Despite lower investment in floral advertisement, female individuals were no less attractive to pollinators than hermaphrodites on a per flower basis. Other measures of pollinator behaviour (number of flowers visited per plant, morph preference and morph constancy) also showed that pollinators did not discriminate against female flowers. In addition, stigma receptivity was longer in female flowers. Accordingly, and contrary to most studies on gynodioecious species, reproductive success of females was not pollen limited. Instead, seed production was pollen limited in hermaphrodites, suggesting low levels of cross‐pollination in hermaphrodites. Seed production was resource limited in hermaphrodites, but not in females, thus providing support for the RRH. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 395–408.     

Niger Seed (Guizotia abyssinica), a New Host of ‘Candidatus Phytoplasma asteris’ in Iran

Shrubs of niger seed with phyllody and internode elongation symptoms suggestive of phytoplasma infections occurred in the central regions of Iran. Phytoplasma was detected by polymerase chain reaction (PCR) and nested PCR amplifications using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2. Using aster yellows group–specific primer pair rp(I)F1A/rp(I)R1A, a fragment of 1212 bp of the rp genes was amplified from DNA samples of infected plants. Random fragment length polymorphism (RFLP) analyses of R16F2n/R16R2‐amplified products using the CfoI restriction enzyme confirmed that Iranian niger seed phyllody phytoplasma is associated with aster yellows group phytoplasmas. Sequence analyses of the partial rp genes fragment indicated that the Iranian niger seed phyllody phytoplasma, which was collected from central regions of Iran, is related to ‘Candidatus Phytoplasma asteris’. This is the first report of a phytoplasma infecting the niger seed plant.     

A UDP‐glucosyltransferase functions in both acylphloroglucinol glucoside and anthocyanin biosynthesis in strawberry (Fragaria × ananassa)

Physiologically active acylphloroglucinol (APG) glucosides were recently found in strawberry (Fragaria sp.) fruit. Although the formation of the APG aglycones has been clarified, little is known about APG glycosylation in plants. In this study we functionally characterized ripening‐related glucosyltransferase genes in Fragaria by comprehensive biochemical analyses of the encoded proteins and by a RNA interference (RNAi) approach in vivo. The allelic proteins UGT71K3a/b catalyzed the glucosylation of diverse hydroxycoumarins, naphthols and flavonoids as well as phloroglucinols, enzymatically synthesized APG aglycones and pelargonidin. Total enzymatic synthesis of APG glucosides was achieved by co‐incubation of recombinant dual functional chalcone/valerophenone synthase and UGT71K3 proteins with essential coenzyme A esters and UDP‐glucose. An APG glucoside was identified in strawberry fruit which has not yet been reported in other plants. Suppression of UGT71K3 activity in transient RNAi‐silenced fruits led to a loss of pigmentation and a substantial decrease of the levels of various APG glucosides and an anthocyanin. Metabolite analyses of transgenic fruits confirmed UGT71K3 as a UDP‐glucose:APG glucosyltransferase in planta. These results provide the foundation for the breeding of fruits with improved health benefits and for the biotechnological production of bioactive natural products.     

Multiple nuclear and mitochondrial DNA sequences provide new insights into the phylogeny of South African Lacertids (Lacertidae,Eremiadinae)

Eremiadinae, one of three subfamilies of Lacertidae, are distributed throughout Asia and Africa. Previous phylogenetic studies suggested that one of the main groups of Eremiadinae (the Ethiopian clade) consist of two clades with predominately East‐African and South‐African distribution. Yet, especially the latter one, which includes the genera Pedioplanis, Meroles, Ichnotropis, Tropidosaura and Australolacerta, was not well supported in the molecular phylogenetic analysis. In this study, we analysed the phylogenetic relationships among the genera of the ‘South African clade’ to assess whether this group actually forms a highly supported clade and to address questions concerning the monophyly of the genera. We sequenced sections of the widely used mitochondrial genes coding for 16S rRNA, 12S rRNA and cytochrome b (altogether 2045 bp) as well as the nuclear genes c‐mos, RAG‐1, PRLR, KIF24, EXPH5 and RAG‐2 (altogether 4473 bp). The combined data set increased the support values for several nodes considerably. Yet, the relationships among five major lineages within the ‘South African clade’ are not clearly resolved even with this large data set. We interpret this as a ‘hard polytomy’ due to fast radiation within the South African lacertids. The combined tree based on nine marker genes provides strong support for the ‘South African Clade’ and its sister group relationship with the ‘East African Clade’. Our results confirm the genus Tropidosaura as a monophylum, while Ichnotropis is paraphyletic in our trees: Ichnotropis squamulosa appears more closely related to Meroles than to Ichnotropis capensis. Furthermore, the monophyly of Meroles is questionable as well. Based on our results, I. squamulosa should be transferred from Ichnotropis into the genus Meroles. Also, the two species of Australolacerta (A. australis and A. rupicola) are very distantly related and the genus is perhaps paraphyletic, too. Finally we propose a phylogeographical scenario in the context of palaeoclimatic data and compare it with a previously postulated hypothesis.     

A new genus of pliosaur (Reptilia: Sauropterygia) from the Lower Jurassic of Holzmaden,Germany

Abstract: The exquisitely preserved holotype of the pliosaur ‘Rhomaleosaurus’victor (SMNS 12478) is described from the Toarcian Posidonien‐Schiefer (Upper Lias, Lower Jurassic) of Holzmaden (Baden‐Württemberg), Germany. The specimen presents a novel combination of synapomorphies and unique morphometric proportions separating it from Rhomaleosaurus sensu stricto and warranting the erection of a new genus, Meyerasaurus gen. nov. Historically, the name ‘Thaumatosaurus’ has been interchangeable with Rhomaleosaurus and is frequently associated with SMNS 12478 in the literature. However, this is an invalid taxon and cannot be reinstated. The anatomy of Meyerasaurus victor is compared in detail with other pliosaurs, and its taxonomic affinity is reviewed. M. victor belongs to the family Rhomaleosauridae and shares several anatomical characters with Rhomaleosaurus including a short and robust premaxillary rostrum (length‐to‐width ratio c. 1.0), parallel premaxilla–maxilla sutures anterior to the nares, vomers contacting the maxillae posterior to the internal nares, and c. 28 cervical vertebrae minus the atlas–axis. The known geographical distribution of Rhomaleosaurus, which previously extended across the German and English palaeobiogeographical zones, is reduced to the English zone as a consequence of the referral of SMNS 12478 to a new genus. This is significant because it contributes to an ongoing trend of increasing generic separation between the German and English zones, while increasing the generic diversity within the German zone itself.     

Baring it all: undressing Cambrian ‘Orsten’ phosphatocopine crustaceans using synchrotron radiation X‐ray tomographic microscopy

Synchrotron radiation X‐ray tomographic microscopy (SRXTM) was used to virtually dissect and peel the shields off of the microscopic, bivalved phosphatocopine crustaceans in the Cambrian ‘Orsten’ type of preservation of Sweden. Doing so opened up for an array of concealed internal structures to be observed in a fully enclosed specimen of Hesslandona ventrospinata and a semi‐enclosed specimen of Hesslandona angustata. For comparison, also a head‐larva stage specimen of H. angustata, with shields in ‘butterfly position’, was analysed. The X‐ray tomographic data sets revealed excellently preserved structures, such as labrum, sternum, antennae, mandibular and post‐mandibular limbs with their minute setae, all of which were more or less disguised by the enclosing shields. This, moreover, allowed assignment to growth stages of the specimens, which is impossible based solely on external morphology and size. Micro‐spherules observed inside the shields of the semi‐enclosed H. angustata specimen may represent remains of food particles, and the feeding biology of phosphatocopines is discussed in detail. Our analyses suggest that phosphatocopines were particle feeders. The SRXTM technique offers the ability to three‐dimensionally reconstruct the morphology in high resolution, construct virtual serial sections and study concealed structures. The resulting data allow for new structures to be revealed for previously known taxa and for new taxa to be identified, with the added benefit of not destroying the specimens in the process. Hence, we do not longer have to rely on serendipitous finds of broken and/or open phosphatocopine specimens to elucidate their diagnostic ventral morphology.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.