The Leptynia hispanica species complex (Insecta Phasmida): polyploidy, parthenogenesis, hybridization and more

The Leptynia hispanica stick insect species complex includes bisexuals, triploid and tetraploid parthenogenetic populations, suggesting that polyploidy has played a central role in the evolution of this complex. An analysis of karyotype, mitochondrial DNA (cox2) and nuclear DNA (ef1-alpha) markers was carried out to clarify phylogenetic relationships and microevolutionary/phylogeographical patterns of the L. hispanica complex. Our analyses suggested a subdivision of bisexual populations into four groups, tentatively proposed as incipient species. Moreover, triploids and tetraploids showed two independent origins, the latter being more ancient than the former. From ef1-alpha analysis, triploids showed hybrid constitution, while the hybrid constitution of tetraploids is likely, but more data are needed. We suggest that L. hispanica is a case of 'geographical parthenogenesis' with parthenogenetic strains colonizing large peripheral ranges, and bisexuals confined to glacial refuge areas. Moreover, the age, wide distribution and competitive advantage of polyploids over diploids, demonstrate their significance in the evolution of the L. hispanica species complex.     

A taxonomic revision of Crambe, sect. Leptocrambe (Brassicaceae)

As part of a revision of the genus Crambe based on the morphological study of herbaria and cultivated material, the systematics of sect. Leptocrombe DC. is presented here. Section Leptocrambe is considered to comprise five species: C. kilimandscharica O. E. Schulz, C. sinuatodentata Hochst. ex Petri, C. hispanica L., C. filiformis Jacq. and C. kralikii Coss. C. hispanica includes three subspecies, subsp. hispanica , subsp. glabrata (DC.) Cout. and subsp. abyssnica (Hochst. ex R. E. Fr.) stat. nov. which includes var. dyssinica and var. meyeri (O. E. Schulz) comb. nov. C. kralikii includes two subspecies, subsp. kralikii and subsp. garamas (Maire) Podlech.     

Molecular phylogenetics of Iberian wall lizards (Podarcis): is Podarcis hispanica a species complex?

Phylogenetic relationships between species and morphotypes of Podarcis wall lizards from the Iberian Peninsula and north Africa were estimated using partial 12S rRNA and cytochrome b mitochondrial DNA sequences. All species except Podarcis hispanica form monophyletic units. P. hispanica is paraphyletic, although all identified morphotypes are monophyletic. These morphotypes represent highly divergent lineages showing 10-15% pairwise sequence divergence with the cytochrome b gene. The data suggest that P. hispanica is a species complex. We recommend using P. hispanica* until additional sampling delimits the number and ranges of species currently referred to P. hispanica. P. carbonelli, which has recently been raised to species status, is confirmed as a genetically distinct form. P. atrata is genetically distinct, but much more closely related to some populations of P. hispanica than previously thought.     

Essential oils in the ranunculaceae family: chemical composition of hydrodistilled oils from Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum seeds

In this study, we analyzed the chemical composition of volatile oils hydrodistilled from seeds of Consolida regalis, Delphinium elatum, Nigella hispanica, and N. nigellastrum using GC and GC/MS. In C. regalis, octadecenoic (77.79%) and hexadecanoic acid (8.34%) were the main constituents. Similarly, the oils from D. elatum and N. hispanica seeds consisted chiefly of octadecadienoic (42.83 and 35.58%, resp.), hexadecanoic (23.87 and 28.59%, resp.), and octadecenoic acid (21.67 and 19.76%, resp.). Contrastingly, the monoterpene hydrocarbons α-pinene (34.67%) and β-pinene (36.42%) were the main components of N. nigellastrum essential oil. Our results confirm the presence of essential oils in the family Ranunculaceae and suggest chemotaxonomical relationships within the representatives of the genera Consolida, Delphinium, and Nigella. In addition, the presence of various bioactive constituents such as linoleic acid, (-)-β-pinene, squalene, or carotol in seeds of D. elatum, N. hispanica, and N. nigellastrum indicates a possible industrial use of these plants.     

Interaction between the introduced eastern mosquitofish and two autochthonous Spanish toothcarps

In aquaria, adults and young-of-the-year (YOY) of both Spanish toothcarp Aphanius iberus and Valencia toothcarp Valencia hispanica received more aggression in the presence of eastern mosquitofish Gambusia holbrooki . YOY of both species also showed decreased feeding rates, as did V. hispanica adults. Adult V. hispanica also were more aggressive themselves and had their reproductive behaviour disrupted. Cases of eastern mosquitofish predation on small A. iberus were also observed. The occurrence and intensity of these potentially harmful effects, however, seemed to be modulated by water temperature, reproductive condition, relative size of the interacting fishes and species-specific habitat preferences. Thus, aggression almost disappeared at low water temperatures. Valencia hispanica and eastern mosquitofish used similar vertical positions in the water column and this apparently increased the frequency of their interactions. In contrast, A. iberus preferred positions closer to the bottom than the other two species and this vertical segregation appeared to lead to less direct interaction with eastern mosquitofish. In an outdoor mesocosm experiment, under semi-natural conditions, the presence of eastern mosquitofish resulted in an almost 70% reduction in the number of o.spring produced by A. iberus . The addition of extra, non-breeding males of A. iberus had no significant effect. YOY A. iberus from cages with eastern mosquitofish or extra males were significantly smaller (c. 1 mm, or 8% shorter) than those from cages without additional fish. Results for V. hispanica were not conclusive, as very few YOY were recovered. In contrast, eastern mosquitofish did not affect the survivorship of breeding adults of both native species.     

CTP synthase forms cytoophidia in archaea

CTP synthase(CTPS) is an important metabolic enzyme that catalyzes the rate-Iimiting reaction of nucleotide CrP de novo synthesis.Since 2010,a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes,which are termed cytoophidia.However,it is unknown whether cytoophidia exist in the third domain of life,archaea.Using Haloarcula hispanica as a model system,here we demonstrate that CTPS forms distinct intracellular compartments in archaea.Under stimulated emission depletion microscopy,we find that the structures of H.hispanica CTPS are elongated,similar to cytoophidia in bacteria and eukaryotes.When Haloarcula cells are cultured in lowsalt medium,the occurrence of cytoophidia increases dramatically.In addition,treatment of H.hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly.Our study reveals that CTPS can fo rm cytoophidia in all three domains of life,suggesting that forming cytoophidia is an ancient property of CTPS.     

Soil ascomycetes from Spain. XIII. Two new species of Apiosordaria

Apiosordaria hispanica sp. nov. and Apiosordaria globosa sp. nov. isolated from soil samples collected in Tarragona, Catalonia, Spain, are described and illustrated. Both species are morphologically close to A. otanii. The ascospores of A. hispanica have tuberculate walls, while those of A. otanii have small warts. Apiosordaria globosa differs from those species by the globose upper cell of the ascospores, which has a small apical protrusion with sub-apical germ pore when young. In A. hispanica and A. globosa the lower cells of the ascospores are slightly warted, while in A. otanii the lower cell of the ascospores is smooth-walled.     

Allozymic variation and relationships of the endangered cyprinodontid genus Valencia and its implications for conservation

Allozyme analysis was used to determine patterns of genetic variation and relationships within the genus Valencia , a group with two allopatric species, V. letourneuxi inhabiting the Balkan Peninsula, and V. hispanica which is endemic to the Iberian Peninsula. Products of 25 gene loci were analysed with AK *, FH *, LDH -1* and LDH -2* monomorphic when Fundulus heteroclitus macrolepidotus was not taken into account. Nine loci were diagnostic for both species of the genus Valencia . Levels of genetic variation of V. hispanica ( P =0·416, H ^e=0·118) and V. letourneuxi ( P =0·160, H _e=0·040) were higher than values reported previously. High population subdivision ( F _st=0·321) among V. hispanica populations indicates a clear interruption of genetic exchange among populations. High genetic variation and differentiation exhibited by populations of V. hispanica suggest that a recovery program should be carried out with natural stocks from the same localities.     

Cytogenetic analysis of Anaecypris hispanica and its relationship with the paternal ancestor of the diploid-polyploid Squalius alburnoides complex.

The karyotype of the endangered fish Anaecypris hispanica was revisited using advanced cytogenetic techniques to elucidate its putative relationship with the paternal ancestor of the hybrid complex Squalius alburnoides and to clarify some of the recently described cytogenetic patterns of the complex. The results of chromomycin A3 and Ag staining, as well as fluorescent in situ hybridization with 28S and 5S rDNA and the (TTAGGG)n telomeric probes, were compared with the patterns observed in specimens of the all-male nonhybrid lineage of S. alburnoides complex, which is considered to reconstitute the nuclear genome of the probably extinct paternal ancestor. Several cytogenetic features observed in A. hispanica specimens were indeed shared by S. alburnoides nuclear nonhybrid males, supporting the hypothesis of a close evolutionary link between A. hispanica and the paternal ancestor of the complex. The genomic rearrangements involving 28S rDNA sites previously described in the S. alburnoides complex and in its maternal ancestor (S. pyrenaicus) were not detected in A. hispanica; they are, therefore, probably due to mechanisms related to hybridization and polyploidy.     

Genetic structure of the diploid–polyploid fish Squalius alburnoides in southern Iberian basins Tejo and Guadiana, based on microsatellites

Several studies have demonstrated the hybrid origin of Squalius alburnoides , a complex of diploid, triploid and tetraploid fish, with Squalius pyrenaicus as the maternal ancestor and an Anaecypris hispanica -like species as the paternal ancestor. The aim of this study was to assess patterns of genetic diversity and similarity between sympatric forms of the different ploidy levels of S. alburnoides and related parental species in the two largest basins that are part of its southern Iberian distribution, the Tejo and Guadiana, using microsatellites. High levels of genetic diversity were found in populations of S. alburnoides from both basins, reflecting the hybrid origin, the high diversity of forms in these river basins and the different modes of reproduction of the complex. Microsatellites indicated low values of genetic differentiation between the non-hybrid all-male form of S. alburnoides and A. hispanica , supporting the view that these forms share a common ancestor. In some instances, diploid and triploid hybrids from the same river basin were more closely related to each other than to their counterparts in the other basins, suggesting an ancient origin for the complex in these Iberian southern basins.     

No trade-off between sprinting and climbing in two populations of the Lizard Podarcis hispanica (Reptilia: Lacertidae).

Biomechanical reflections suggest that lizards that have specialized in running should differ in parts of their morphological design from lizards that have specialized in climbing. Moreover, adaptation in one direction may conflict with performance in the other. We tested this assumed trade-off by measuring climbing and running performance in two subspecies of the Mediterranean lizard Poaarcis hispanica. One subspecies, P. h. atrata , inhabits a number of small islets near the Spanish coast. It is mainly ground dwelling. The other subspecies, P. h. hispanica , was sampled on the mainland of Spain. It frequendy occupies vertical elements within its habitat. Our data do not support the notion of a trade-off between both types of locomotion. Individuals of P. h. hispanica both run and climb faster than those of P. h. atrata. There is no difference between subspecies in ability to cling onto tilted substrates. Predictions on the morphology of both subspecies, inspired by biomechanical arguments, are not supported by our measurements.     

Variation in morphology, gait characteristics and speed of locomotion in two populations of lizards

Locomotor behaviour varies between two subspecies of the Spanish wall lizard Podarcis hispanica. One subspecies inhabits the Columbretes islands, the other lives on the Spanish mainland. Size standardized voluntary speeds (as measured in unrestrained laboratory conditions) are lower in the island population (P. h. atrata) than in the mainland population (P. h. hispanica). Maximal running performance (when chased) is much higher in the mainland population than in the island population. High speed video recordings show that subspecies differ in gait characteristics: individuals from the mainland modulate running velocity primarily by modifying stride length, individuals from the island primarily by altering stride frequency. P. h. hispanica's strategy for modulating speed probably allows this mainland subspecies to attain higher maximal speeds than the island subspecies P. h. atrata. Theoretical considerations suggest that at high speeds, P. h. hispanica's running style is energetically more favourable, but this hypothesis awaits experimental verification. We suggest that the differences in locomotion efficiency between the subspecies result from differences in predation pressure between the mainland and the island. The mainland study site has a higher predator diversity and offers less hiding opportunities to the lizards.     

Zinc accumulation in the telencephalon of lizards

The zinc concentration in the brains of two species of lizard was determined by atomic-absorption spectrophotometry. The zinc concentration was found to be highest in the telencephalon of Lacerta galloti (21.1 micrograms/g fresh weight) and Podarcis hispanica (16.77 +/- 0.8 micrograms/g) while the mesencephalon and brain stem exhibited lower zinc concentrations, i.e., 7.0 micrograms/g in Lacerta galloti and 6.08 +/- 0.4 micrograms/g in Podarcis hispanica. This high telencephalic concentration of zinc is paralleled by intense and well-defined Timm reactivity used for demonstrating the presence of zinc-containing boutons at the light-microscope level. Volumetric-densitometric studies of these Timm-reactive zones were performed using serial transverse sections of the same lizard brains.     

Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons.

We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea.     

Cross-species testing of 27 pre-existing microsatellites in Podarcis gaigeae and Podarcis hispanica (Squamata: Lacertidae)

We tested 27 microsatellite loci for cross-species amplification in the lacertids Podarcis gaigeae and Podarcis hispanica. We detected 11 and 15 polymorphic loci in the former and the latter species, respectively. In a larger sample of individuals from a single population of each species, the number of alleles ranged from five to 23 in 10 of the polymorphic loci in P. gaigeae, and between four and 13 in nine of polymorphic loci in P. hispanica. Two locus deviated from Hardy-Weinberg equilibrium in P. hispanica. Between 11 and 16 of the 27 loci also amplified successfully in three other Podarcis species.     

Colopha hispanica sp.n. (Homoptera: Aphidoidea: Pemphigidae): an unusual endophytic aphid living inside the stems of Scirpus holoschoenus (Cyperaceae)

Abstract. A new aphid species of the genus Colopha (Pemphigidae: Eriosomatinae), Colopha hispanica is described from Spain. The apterae differ from those of other species of Colopha in the shortness of their antennae and legs, in the greater number of their wax glands and in the underdevelopment of the setal socket on the forelegs. These aphids live inside the stems of Scirpus holoschoenus (Cyperaceae); this is the first record of a truly endophytic aphid. The adults are immobile. C. hispanica is compared with the other known species of Colopha , with Kaltenbachiella pallida , a similar species also found in Spain and with Gharesia polunini , a primitive species of Eriosomatinae. The distribution and host plants of Colopha species are reviewed and discussed.     

Anaerobic metabolism and activity of European lacertid lizards

1. Lactate production and physical performance during intense activity were measured in Lacerta vivipara, Podarcis muralis and Podarcis hispanica, temperate lizards from progressively lower latitudes. 2. Rates of lactate production during the initial 30 sec of bursts of activity (means = 1.75 mg g-1) and their Q10S (means = 1.36) are similar to previously obtained values for lizards. 3. Maximal anaerobic scope at activity body temperatures is lowest in Lacerta vivipara, which correlates with a tendency to less strenuous behaviour than that of the Podarcis species and may reflect the need for energy economy. 4. Q10S of maximal performance are lower in Lacerta vivipara and Podarcis muralis than Podarcis hispanica, a difference having no demonstrated metabolic basis but which is thermally adaptive for these cooler climate species.     

Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences.

Twenty-two extremely halophilic aerobic archaeal strains were isolated from enrichments prepared from Dead Sea water samples collected 57 years ago. The isolates were phenotypically clustered into five different groups, and a representative from each group was chosen for further study. Almost the entire sequences of the 16S rRNA genes of these representatives, and of Haloarcula hispanica ATCC 33960, were determined to establish their phylogenetic positions. The sequences of these strains were compared to previously published sequences of 27 reference halophilic archaea (members of the family Halobacteriaceae) and two other archaea, Methanobacterium formicicum DSM 1312 and Methanospirillum hungatei DSM 864. Phylogenetic analysis using approximately 1,400 base comparisons of 16S rRNA-encoding gene sequences demonstrated that the five isolates clustered closely to species belonging to three different genera--Haloferax, Halobacterium, and Haloarcula. Strains E1 and E8 were closely related and identified as members of the species Haloferax volcanii, and strain E12 was closely related and identified as a member of the species Halobacterium salinarum. However, strains E2 and E11 clustered in the Haloarcula branch with Haloarcula hispanica as the closest relative at 98.9 and 98.8% similarity, respectively. Strains E2 and E11 could represent two new species of the genus Haloarcula. However, because strains of these two new species were isolated from a single source, they will not be named until additional strains are isolated from other sources and fully characterized.     

Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae

Aims:  To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers.
Methods and Results:  EPSs synthesized by Idiomarina fontislapidosi F32^T, Idiomarina ramblicola R22^T and Alteromonas hispanica F23^T were collected and analysed under optimum conditions: MY medium supplemented with 7·5% (w/v) salts; 32°C; and 1% (w/v) glucose. Polymers were synthesized mainly during the early stationary growth phase with yields ranging from 1 to 1·5 g l⁻¹. The Idiomarina species each produced an anionic EPS composed mainly of glucose, mannose and galactose. A. hispanica synthesized an anionic EPS composed mainly of glucose, mannose and xylose. Solutions of all the polymers were low in viscosity and pseudoplastic in their behaviour. They showed emulsifying activity and the capacity to bind some metals.
Conclusions:  The Alteromonadaceae species studied in this work produced EPSs with physical and chemical properties different from those produced by other halophilic and nonhalophilic bacteria, suggesting that the wide diversity of micro-organisms being encountered nowadays in hypersaline environments offers enormous potential resources for biotechnological applications.
Significance and Impact of the Study:  We have optimized the EPS production and analysed new biopolymers produced by some recently described, moderately halophilic bacteria. These biopolymers are chemically and physically different from others already in use in biotechnology and offer hopes for new applications, especially in the case of A. hispanica , which may prove to be a viable source of xylo-oligosaccharides.     

An improved transposon for the halophilic archaeon Haloarcula hispanica

An improved transposon (ThD73) for Haloarcula hispanica is described. Based on the halobacterial insertion sequence ISH28, it showed little target sequence specificity but was biased toward a lower G+C content. Twenty randomly selected ThD73 mutants were analyzed, and the DNA flanking their insertions revealed several recognizable sequences, including two (unrelated) ISH elements.