[Antibiotic stewardship programs in the Spanish emergency services: PROA-URG Study]. / Programas de optimización del uso de antimicrobianos en los servicios de urgencias españoles: Estudio PROA-URG.

OBJECTIVE: Antimicrobial stewardship programs (ASP) have become a key tool in the adaptation of these drugs to the health system. The information available on the application and indicators used in these programs in emergency departments is scarce. The objective of this study is to know the extent of ASP implementation in the emergency departments, as well as the use of antimicrobials in these units. METHODS: Multicenter retrospective study. An invitation was sent to all participants of the REDFASTER-SEFH emergency pharmacist working group. A questionnaire was used consisting of 21 items, answered by a team made up of a pharmacist, emergency room specialist, infectious disease specialist and microbiologist. RESULTS: Eighteen hospitals completed the survey. Fourteen (77.8%) had an ASP manager. The DDD value per 1000 admissions ranged between 36.5 and 400.5 (median: 100.4 [IQR:57.2-157.3]). Both carbapenem and macrolide group presented wide variability in use. Six (33.3%) hospitals had an annual report on the specific resistance profile for urine and blood cultures. The percentage of multi-drug resistant strains in urine cultures was 12.5% and in blood cultures 12.2%. The percentage of adequacy in the bacteremia treatment was 81.0% (IQR:74.6-85.0%), while in urinary tract infections was 78.0% (IQR:71.5-88.0). CONCLUSIONS: Despite the existence of ASP members in emergency services, as well as the training activity and local guidelines is common. knowledge of the use of antimicrobials and resistances is limited. Future activities must be aimed at improving information about the ASP results in these units.

Biologically synthesized silver nanoparticles as potent antibacterial effective against multidrug-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most worrisome infectious bacteria due to its intrinsic and acquired resistance against several antibiotics and the recalcitrance of its infections; hence, the development of novel antimicrobials effective against multidrug-resistant P. aeruginosa is mandatory. In this work, silver nanoparticles obtained by green synthesis using a leaf extract and fungi were tested against a battery of clinical strains from cystic fibrosis, pneumonia and burnt patients, some of them with multidrug resistance. Both nanoparticles showed a potent antibacterial effect, causing severe damage to the cell wall, membrane and DNA, and inducing the production of reactive oxygen species. Moreover, the nanoparticles derived from fungi showed synergistic antibacterial effects with the antibiotics meropenem and levofloxacin for some clinical strains and both kinds of nanoparticles were nontoxic for larvae of the moth Galleria mellonella, encouraging further research for their implementation in the treatment of P. aeruginosa infections.

Antibacterial properties of phenothiazine derivatives against multidrug-resistant Acinetobacter baumannii strains.

AIM: As options to treat recalcitrant bacterial infections which are increasingly limited due to multidrug-resistant strains, searching for new, effective antibacterial compounds is necessary. One strategy is to generate treatment alternatives by drug repurposing. METHODS AND RESULTS: In this work, phenotypic microarrays were used for the screening of miscellaneous compounds against the growth and biofilm formation of Acinetobacter baumannii, an important emergent multidrug-resistant opportunistic pathogen. The results showed that the phenothiazine derivatives, such as promethazine, trifluoperazine, thioridazine, and chlorpromazine, inhibited the growth of antibiotic-sensitive and multidrug-resistant strains (showing minimal inhibitory concentrations ranging from 0·05 to 0·6 g l-1 and minimal bactericidal concentrations ranging from 0·1 to 2·5 g l-1 ). All phenothiazine derivatives were active against biofilm cells (with minimal biofilm eradication concentrations ranging from 0·5 to >3 g l-1 ). Chlorpromazine promoted reactive oxigen species (ROS) production, and cell membrane and DNA damage. Chlorpromazine showed synergy with antibiotics such as ceftazidime, meropenem, and colistin and was an effective treatment for experimentally infected Galleria mellonella when combined with ceftazidime. CONCLUSIONS: It was demonstrated that phenothiazine derivatives, especially chlorpromazine, are drugs with attractive antibacterial properties against nosocomial MDR strains of A. baumannii, by generating ROS and cell membrane and DNA damage. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study indicates that repurposing phenothiazine derivatives for treating recalcitrant infections by A. baumannii could be promising.

No signs of inbreeding despite long-term isolation and habitat fragmentation in the critically endangered Montseny brook newt (Calotriton arnoldi).

Endemic species with restricted geographic ranges potentially suffer the highest risk of extinction. If these species are further fragmented into genetically isolated subpopulations, the risk of extinction is elevated. Habitat fragmentation is generally considered to have negative effects on species survival, despite some evidence for neutral or even positive effects. Typically, non-negative effects are ignored by conservation biology. The Montseny brook newt (Calotriton arnoldi) has one of the smallest distribution ranges of any European amphibian (8 km2) and is considered critically endangered by the International Union for Conservation of Nature. Here we apply molecular markers to analyze its population structure and find that habitat fragmentation owing to a natural barrier has resulted in strong genetic division of populations into two sectors, with no detectable migration between sites. Although effective population size estimates suggest low values for all populations, we found low levels of inbreeding and relatedness between individuals within populations. Moreover, C. arnoldi displays similar levels of genetic diversity to its sister species Calotriton asper, from which it separated around 1.5 million years ago and which has a much larger distribution range. Our extensive study shows that natural habitat fragmentation does not result in negative genetic effects, such as the loss of genetic diversity and inbreeding on an evolutionary timescale. We hypothesize that species in such conditions may evolve strategies (for example, special mating preferences) to mitigate the effects of small population sizes. However, it should be stressed that the influence of natural habitat fragmentation on an evolutionary timescale should not be conflated with anthropogenic habitat loss or degradation when considering conservation strategies.

Instability of streaks in pipe flow of shear-thinning fluids.

This study is motivated by recent experimental results dealing with the transition to turbulence in a pipe flow of shear-thinning fluids, where a streaky flow with an azimuthal wave number n=1 is observed in the transitional regime. Here, a linear stability analysis of pipe flow of shear-thinning fluids modulated azimuthally by finite amplitude streaks is performed. The shear-thinning behavior of the fluid is described by the Carreau model. The streaky base flows considered are obtained from two-dimensional direct numerical simulation using finite amplitude longitudinal rolls as the initial condition and by extracting the velocity field at time t(max), where the amplitude of the streaks reaches its maximum, denoted by A(max). It is found that the amplitude A(max) increases with increasing Reynolds number as well as with increasing amplitude E(0) of the initial longitudinal rolls. For sufficiently large streaks amplitude, streamwise velocity profiles develop inflection points, leading to instabilities. Depending on the threshold amplitude A(c), two different modes may trigger the instability of the streaks. If A(c) exceeds approximately 41.5% of the centerline velocity, the instability mode is located near the axis of the pipe, i.e., it is a "center mode." For weaker amplitude A(c), the instability mode is located near the pipe wall, in the region of highest wall normal shear, i.e., it is a "wall mode." The threshold amplitude A(c) decreases with increasing shear-thinning effects. The energy equation analysis indicates that (i) wall modes are driven mainly by the work of the Reynolds stress against the wall normal shear and (ii) for center modes, the contribution of the normal wall shear remains dominant; however, it is noted that the contribution of the Reynolds stress against the azimuthal shear increases with increasing shear-thinning effects.

Evolutionary patterns of the mitochondrial genome in the Moorish gecko, Tarentola mauritanica.

A previous study on the evolutionary patterns of Tarentola mauritanica demonstrated that low levels of mitochondrial diversity observed in the European populations relative to nuclear markers were consistent with a selective sweep hypothesis. In order to unravel the mitochondrial evolutionary history in this European population and two other lineages of T. mauritanica (Iberian and North African clades), variation within 22 nearly complete mitogenomes was analyzed. Surprisingly, each clade seems to have a distinct evolutionary history; with both the European and Iberian clades presenting a decrease of polymorphism, which in the former is consistent with departure from neutrality of the mtDNA (positive or background selection), but in the latter seems to be the result of a bottleneck after a population expansion. The pattern exhibited by the North African clade seems to be a consequence of adaptation to certain mtDNA variants by positive selection.

Molecular phylogenetics and historical biogeography of the west-palearctic common toads (Bufo bufo species complex).

In most pan-Eurasiatic species complexes, two phenomena have been traditionally considered key processes of their cladogenesis and biogeography. First, it is hypothesized that the origin and development of the Central Asian Deserts generated a biogeographic barrier that fragmented past continuous distributions in Eastern and Western domains. Second, Pleistocene glaciations have been proposed as the main process driving the regional diversification within each of these domains. The European common toad and its closest relatives provide an interesting opportunity to examine the relative contributions of these paleogeographic and paleoclimatic events to the phylogeny and biogeography of a widespread Eurasiatic group. We investigate this issue by applying a multiproxy approach combining information from molecular phylogenies, a multiple correspondence analysis of allozyme data and species distribution models. Our study includes 304 specimens from 164 populations, covering most of the distributional range of the Bufo bufo species complex in the Western Palearctic. The phylogenies (ML and Bayesian analyses) were based on a total of 1988 bp of mitochondrial DNA encompassing three genes (tRNAval, 16S and ND1). A dataset with 173 species of the family Bufonidae was assembled to estimate the separation of the two pan-Eurasiatic species complexes of Bufo and to date the main biogeographic events within the Bufo bufo species complex. The allozyme study included sixteen protein systems, corresponding to 21 presumptive loci. Finally, the distribution models were based on maximum entropy. Our distribution models show that Eastern and Western species complexes are greatly isolated by the Central Asian Deserts, and our dating estimates place this divergence during the Middle Miocene, a moment in which different sources of evidence document a major upturn of the aridification rate of Central Asia. This climate-driven process likely separated the Eastern and Western species. At the level of the Western Palearctic, our dating estimates place most of the deepest phylogenetic structure before the Pleistocene, indicating that Pleistocene glaciations did not have a major role in splitting the major lineages. At a shallow level, the glacial dynamics contributed unevenly to the genetic structuring of populations, with a strong influence in the European-Caucasian populations, and a more relaxed effect in the Iberian populations.

When selection deceives phylogeographic interpretation: the case of the Mediterranean house gecko, Hemidactylus turcicus (Linnaeus, 1758).

A previous study on Hemidactylus turcicus based on mtDNA makers indicated that this gecko has a Middle-East origin, and that the current phylogeographic pattern is the result of a very rapid spread from the east to the west of the species' range. The same study identified two distinct mitochondrial lineages with low differentiation and genetic diversity. Since H. turcicus is known to be closely associated to humanized environments, its present distribution range and phylogeography is frequently interpreted to be the result of recurrent human-mediated introductions. These conclusions used to be the same as those used to interpret the results obtained for the European populations of another gecko, Tarentola mauritanica. However, a recent study has revealed that the phylogeographic pattern of T. mauritanica is not solely the result of a recent colonization, but also of a mitochondrial selective sweep. Could the same be occurring in H. turcicus? To answer this question, two mitochondrial (12S rRNA and cytochrome b) and two nuclear genes (ACM4 and Rag2) were used in this study. From the mtDNA data we confirmed the existence of two distinct phylogeographic lineages; one occurring exclusively in the northern Mediterranean (Clade A), and another one more widespread that is the only lineage present in North Africa (Clade B). In light of these results, we could hypothesize that H. turcicus had its origin in Turkey, and from there Clade A moved to Europe and Clade B to North Africa spreading latter into Europe. However, Clade A presents significantly higher nucleotide diversity for the nuclear DNA compared to the mtDNA, and neutrality tests gave significant results for the mitochondrial data. These results suggest that the lack of mtDNA genetic diversity and structure in the European population of H. turcicus could also be due to a selective sweep, and not only because of a recent colonization. Together with the situation reported in T. mauritanica, the identification of a hitch-hiking process occurring in H. turcicus, represents two unprecedented cases of a selective sweep taking place in the same geographic area shaping the phylogeographic patterns of two unrelated genera of geckos.

Conflicting patterns of nucleotide diversity between mtDNA and nDNA in the Moorish gecko, Tarentola mauritanica.

The gecko Tarentola mauritanica is a Mediterranean species that is widely distributed across southern Europe and North Africa. Initial phylogeographic studies based on mtDNA concluded that the European populations originated from recent colonizations from North Africa, possibly involving man-mediated introductions. A distinct lineage, found to be a sister taxon to the widespread European lineage, was subsequently reported from parts of the Iberian Peninsula. Like many gecko species, T. mauritanica contains several deep intraspecific genetic lineages within North Africa. However, in contrast to this diversity, a single mtDNA haplotype is widespread across a large part of its European range. In this work, we analysed a total of 834 base pairs from two mtDNA genes (12SrRNA and 16SrRNA) for 154 specimens, and a total of 1876bp from three nuclear genes (ACM4, MC1R and Rag2) for 51 specimens to test the phylogenetic and phylogeographic patterns proposed for T. mauritanica based on mtDNA sequences. The mtDNA results reveal a Moroccan lineage basal to the common "European" haplotype, thus suggesting that the latter probably originated in Morocco. The remaining lineages obtained are the same as those observed in previous studies. In contrast, the nDNA data do not support the majority of the mtDNA phylogenetic relationships, possibly due to incomplete lineage sorting of these markers. Moreover, the mtDNA data suggest that the Iberian clade seems to have undergone a population expansion. Surprisingly, the European clade presents a higher nucleotide diversity for the nuclear genes when compared to the combined mtDNA dataset. These analyses suggest that the low mtDNA variability that characterises the European populations of T. mauritanica is the result of a selective sweep process and not solely due to a recent colonization event.

Systematics and biogeography of the Neotropical genus Mabuya, with special emphasis on the Amazonian skink Mabuya nigropunctata (Reptilia, Scincidae).

Phylogenetic analyses using up to 1532 base pairs (bp) of mitochondrial DNA from 106 specimens of Neotropical Mabuya, including 18 of the 19 recognized South American and Mesoamerican species, indicate that most species of the genus are monophyletic, including M. nigropunctata that had previously been reported to be paraphyletic. The present results shows that this species includes three highly divergent and largely allopatric lineages restricted to occidental, meridional, and oriental Amazonia. Our dataset demonstrates that previous claims regarding the paraphyletic status of M. nigropunctata and the phylogenetic relationships within this species complex based on the analysis of three mitochondrial and four nuclear genes (approx. 5000bp) were erroneous and resulted from two contaminated cytochrome b sequences. The phylogenetic results indicate that diversification in the Neotropical genus Mabuya started approximately in the Middle Miocene (15.5-13.4Ma). The divergence dates estimated for the Mabuya nigropunctata species complex suggest that the major cladogenetic events that produced the three main groups (occidental (oriental+meridional)) occurred during the Late Miocene. These estimations show that diversification within the M. nigropunctata species complex was not triggered by the climatic changes that occurred during the Pleistocene, as has been suggested by several authors. Rather, our data support the hypothesis that the late tertiary (essentially Miocene epoch) was a period that played a very important role in the generation of biological diversity in the Amazonian forests. Speciation between Mabuyacarvalhoi, endemic to the coastal mountain range of Venezuela, and M. croizati, restricted to the Guiana Shield, occurred during the Middle Miocene and may have been as the result of a vicariant event produced by the formation of the present day Orinoco river drainage basin and the consequent appearance of the Llanos del Orinoco, which acted as a barrier to dispersal between these two species. The split between M. bistriata and M. altamazonica and between the occidental and (meridional+oriental) clades of M. nigropunctata fits very well with the biogeographic split between the eastern and western Amazon basins reported for several other taxa.

Radiation, multiple dispersal and parallelism in the skinks, Chalcides and Sphenops (Squamata: Scincidae), with comments on Scincus and Scincopus and the age of the Sahara Desert.

Phylogenetic analysis using up to 1325 base pairs of mitochondrial DNA from 179 specimens and 30 species of Chalcides, Sphenops, Eumeces, Scincopus and Scincus indicates that Sphenops arose twice independently within Chalcides. It is consequently synonymized with that genus. Chalcides in this broader sense originated in Morocco, diversifying into four main clades about 10 Ma, after which some of its lineages dispersed widely to cover an area 40 times as large. Two separate lineages invaded the Canary Islands and at least five main lineages colonized southern Europe. At least five more spread across northern Africa, one extending into southwest Asia. Elongate bodies with reduced limbs have evolved at least four times in Chalcides, mesic 'grass-swimmers' being produced in one case and extensive adaptation to life in loose desert sand in two others. In clade, Chalcides striatus colonized SW Europe from NW Africa 2.6 Ma and C. chalcides mainland Italy 1.4 Ma, both invasions being across water, while C. c. vittatus reached Sardinia more recently, perhaps anthropogenically, and C. guentheri spread 1200km further east to Israel. C. minutus is a composite, with individuals from the type locality forming a long independent lineage and the remaining ones investigated being most closely related to C. mertensi. In the Northern clade, C. boulengeri and C. sepsoides spread east through sandy habitats north of the Sahara about 5 Ma, the latter reaching Egypt. C. bedriagai invaded Spain around the same time, perhaps during the Messinian period when the Mediterranean was dry, and shows considerable diversification. Although it is currently recognized as one species, the C. ocellatus clade exhibits as much phylogenetic depth as the other main clades of Chalcides, having at least six main lineages. These have independently invaded Malta and Sardinia from Tunisia and also southwest Arabia C. o. humilis appears to have spread over 4000 km through the Sahel, south of the Sahara quite recently, perhaps in the Pleistocene. In the Western clade of Chalcides, C. delislei appears to have dispersed in a similar way. There were also two invasions of the Canary Islands: one around 5 Ma by C. simonyi, and the other about 7 Ma by the ancestor of C. viridanus+C. sexlineatus. C. montanus was believed to be related to C. lanzai of the Northern clade, but in the mtDNA tree it is placed within C. polylepis of the Western clade, although this may possibly be an artifact of introgression. The Eumeces schneideri group, Scincopus and Scincus form a clade separate from Chalcides. Within this clade, the geographically disjunct E. schneideri group is paraphyletic. One of its members, E. algeriensis is the sister taxon to Scincopus, and Scincus may also be related to these taxa. The phylogeny suggests Scincopus entered desert conditions in Africa, up to 9.6 Ma and the same may have been true of Scincus up to 11.7 Ma. Scincus appears to have diversified and spread into Arabia around 6 Ma. Dates of origin and divergence of these skinks, desert Chalcides and other squamates agree with recent geological evidence that the Sahara is at least 5-7 My old. The subspecies Chalcides viridanus coeruleopunctatus is upgraded to the species level as C. coeruleopunctatus stat nov., on the basis of its large genetic divergence from C. v. viridanus.

Phylogeny, biogeography, and evolution of two Mediterranean snakes, Malpolon monspessulanus and Hemorrhois hippocrepis (Squamata, Colubridae), using mtDNA sequences.

Variation in 815bp of mitochondrial DNA from two gene fragments (300bp of cytochrome b and 395-515bp of 12S rRNA) for 26 Malpolon monspessulanus, and cytochrome b for a further 21 individuals, indicates that this species originated in the Maghreb area of Northwest Africa. Here, an estimated 3.5-6Mya, it divided into the western M. m. monspessulanus, and an eastern clade including M. m. insignitus and M. m. fuscus. The very limited genetic differentiation between Maghreb and Southwest European populations of this form suggests that it arrived in the Iberian Peninsula only recently. Population genetics and demographic tests indicate subsequent expansion in this area around 83,000-168,000 year ago. Because present populations of Malpolon arrived recently, mid-Pliocene and at least some Pleistocene fossils of the genus Malpolon in Southwest Europe are probably derived from an earlier invasion from the Maghreb, possibly as early as the end of the Miocene period, 5.3-5.9Mya, when there was a temporary land bridge across the site of the Strait of Gibraltar and the Mediterranean Sea desiccated. The descendants of this earlier invasion must have eventually become extinct, perhaps during one of the Pleistocene glaciations. In contrast to the western M. m. monspessulanus, the greater genetic divergence found in the eastern clade of M. monspessulanus suggests that it dispersed at an earlier date and probably over a longer period, spreading eastwards through northern Libya and Egypt to Syria, Iraq, and Iran, and around the Mediterranean Sea through Turkey into the Aegean archipelagos and the Balkan peninsula. The western and eastern units of M. monspessulanus have different dorsal color pattern, differences in skull structure and exhibit an 8.4% uncorrected genetic divergence in the combined gene fragments investigated here. It is consequently recommended that they should be treated as separate species: M. monspessulanus (sensu stricto) and Malpolon insignitusstat. nov., the latter including the subspecies Malpolon insignitus fuscuscomb. nov. The same combined mitochondrial gene fragments used in Malpolon were investigated in 20 individuals of Hemorrhois hippocrepis, and of cytochrome b alone in a further 17. They indicate that this species also originated in the Maghreb and again invaded the Iberian Peninsula quite recently. Some of the most recent invasions of the Iberian Peninsula by reptiles and amphibian taxa could probably be anthropogenic in origin. Some other species including M. monspessulanus and H. hippocrepis, may have crossed naturally, by "hopping" across the Strait of Gibraltar via temporary islands on the shallowest parts that were exposed during sea-level fall associated with Pleistocene glaciations.

Systematics, biogeography, and evolution of Hemidactylus geckos (Reptilia: Gekkonidae) elucidated using mitochondrial DNA sequences.

With more than 80 species inhabiting all warm continental land masses and hundreds of intervening continental and oceanic islands, Hemidactylus geckos are one of the most species-rich and widely distributed of all reptile genera. They consequently represent an excellent model for biogeographic, ecological, and evolutionary studies. A molecular phylogeny for Hemidactylus is presented here, based on 702 bp of mtDNA (303 bp cytochrome b and 399 bp 12S rRNA) from 166 individuals of 30 species of Hemidactylus plus Briba brasiliana, Cosymbotus platyurus, and several outgroups. The phylogeny indicates that Hemidactylus may have initially undergone rapid radiation, and long-distance dispersal is more extensive than in any other reptilian genus. In the last 15 My, African lineages have naturally crossed the Atlantic Ocean at least twice. They also colonized the Gulf of Guinea, Cape Verde and Socotra islands, again sometimes on more than one occasion. Many extensive range extensions have occurred much more recently, sometimes with devastating consequences for other geckos. These colonizations are likely to be largely anthropogenic, involving the 'weedy' commensal species, H. brookii s. lat, H. mabouia, H. turcicus, H. garnotii, and H. frenatus. These species collectively have colonized the Mediterranean region, tropical Africa, much of the Americas and hundreds of islands in the Pacific, Indian, and Atlantic oceans. Five well-supported clades are discernable in Hemidactylus, with the African H. fasciatus unallocated. 1. Tropical Asian clade: (Cosymbotus platyurus (H. bowringii, H. karenorum, H. garnotii)) (H. flaviviridis (Asian H. brookii, H. frenatus)). 2. African H. angulatus and Caribbean H. haitianus. 3. Arid clade, of NE Africa, SW Asia, etc.: (H. modestus (H. citernii, H. foudai)) (H. pumilio (H. granti, H. dracaenacolus) (H. persicus, H. macropholis, H. robustus, H. turcicus (H. oxyrhinus (H. homoeolepis, H. forbesii))). 4. H. mabouia clade (H. yerburii, H. mabouia). 5. African-Atlantic clade: H. platycephalus ((H. agrius, H. palaichthus) (H. longicephalus, H. greeffi, H. bouvieri, Briba brasiliana))). Cosymbotus and Briba are synonymized with Hemidactylus, and African populations of H. brookii separated as H. angulatus, with which H. haitianus may be conspecific. Some comparatively well-sampled widespread species show high genetic variability (10-15% divergence) and need revision, including Cosymbotus platyurus, H. bowringii, Asian H. brookii, H. frenatus, H. angulatus, and H. macropholis. In contrast, most populations of H. mabouia and H. turcicus are very uniform (1-2% divergence). Plasticity of some of the morphological features of Hemidactylus is confirmed, although retention of primitive morphologies also occurs.

Phylogeography of the false smooth snakes, Macroprotodon (Serpentes, Colubridae): mitochondrial DNA sequences show European populations arrived recently from Northwest Africa.

Mitochondrial DNA (1075 bp: cytochrome b, 300 bp; 12S rRNA, 393 bp; and 16S rRNA, 382 bp) corroborates the monophyly of the genus Macroprotodon and of the species M. mauritanicus, M. abubakeri, and M. brevis. The subspecies M. brevis ibericus is also monophyletic. The mtDNA tree presented here indicates that M. cucullatus consists of at least two separate units and may possibly represent a primitive morphology rather than a species in its own right. However, this hypothesis is tentative since it is only reflects the history of a single evolutionary unit (mtDNA). A definitive understanding of the evolution of M. cucullatus will not be possible until informative nuclear markers are added to the mitochondrial data. Macroprotodon appears to have originated in the Maghreb region of NW Africa and speciated there around 4-5.5 million years ago around the end of the Miocene period, after which its three main lineages may each have expanded north into more mesic conditions. The group also spread eastwards into coastal areas of Libya quite recently and on to Egypt and Israel. Later still, M. b. ibericus from extreme north Morocco reached the Iberian Peninsula, and M. mauritanicus from Tunisia or Algeria colonised the Balearic Islands of Menorca and Mallorca. Both these range extensions may result from very recent natural colonisations or even from accidental human introduction. Recency of origin of Iberian and Balearic populations is indicated by uniformity of their mtDNA even across large distances, and its great similarity to that of populations in source regions. Isolated populations assigned to M. cucullatus in the Hoggar mountains (southern Algeria) and Western Sahara are probably relicts from quite recent periods of climatic amelioration in the North African desert.

Relationships and evolution of the North African geckos, Geckonia and Tarentola (Reptilia: Gekkonidae), based on mitochondrial and nuclear DNA sequences.

Mitochondrial (cytochrome b and 12S rRNA) and nuclear (c-mos) genes, analyzed by a variety of methods, indicate that the distinctive northwest African gecko Geckonia chazaliae is a member of the Tarentola clade, being most closely related to the species of the western Canary and Cape Verde islands. Relationships in Tarentola as a whole are as follows: (T. americana ((T. mauritanica, T. angustimentalis) ((T. deserti, T. boehmei) ((T. b. boettgeri-South (T. b. boettgeri-North (T. b. bischoffi, T. b. hierrensis))) ((T. annularis, T. ephippiata) (Geckonia, T. delalandii, T. gomerensis, Cape Verde species)))))); nearly all nodes have high bootstrap support. Results confirm that T. americana of Cuba and the Bahamas separated at the most basal dichotomy of the phylogeny and give no positive support for the monophyly of the subgenera Tarentola s. str. and Makariogecko. The latter includes Geckonia and the subgenus Sahelogecko. Continental Tarentola appear to have invaded the Sahara desert from its northern edge. They have also colonized groups of Atlantic islands five times: a single invasion of the West Indies and three of the Canary islands, one of which then went on to invade the Cape Verde archipelago. The phylogeny corroborates anatomical evidence that the ground-dwelling Geckonia had a climbing ancestry, something that is paralleled in some southern African terrestrial gekkonids related to Pachydactylus. Distinctive derived features of Geckonia occur in other gekkonids that are ground dwelling in arid habitats and may be functionally related to this environment. The evolution of such features indicates that, although Tarentola is generally very uniform and may have been so for over 10 million years, this is not due to any overwhelming phylogenetic constraint. G. chazaliae should be included in Tarentola, as Tarentola chazaliae.

Parallel gigantism and complex colonization patterns in the Cape Verde scincid lizards Mabuya and Macroscincus (Reptilia: Scincidae) revealed by mitochondrial DNA sequences.

The scincid lizards of the Cape Verde islands comprise the extinct endemic giant Macroscincus coctei and at least five species of Mabuya, one of which, Mabuya vaillanti, also had populations with large body size. Phylogenetic analysis based on DNA sequences derived from the mitochondrial cytochrome b, cytochrome oxidase I and 12S rRNA genes (711, 498 and 378 base pairs (bp), respectively) corroborates morphological evidence that these species constitute a clade and that Macroscincus is unrelated to very large skinks in other areas. The relationships are ((M. vaillanti and Mabuya delalandii) (Mabuya spinalis and Macroscincus coctei (Mabuya fogoensis nicolauensis (Mabuya fogoensis antaoensis and Mabuya stangeri)))). The Cape Verde archipelago was colonized from West Africa, probably in the Late Miocene or Early Pliocene period. The north-eastern islands were probably occupied first, after which the ancestor of M. vaillanti and M. delalandii may have originated on Boavista, the ancestor of the latter species arriving on Santiago or Fogo later. The M. fogoensis--M. stangeri clade colonized the islands of Branco, Razo, Santa Luzia and São Vicente from São Nicolau and reached Santo Antão after this. Colonization of these northeastern islands was slow, perhaps because the recipient islands had not developed earlier or because colonization cut across the path of the Canary Current and the Northeast Trade Winds, the main dispersing agents in the region. Rapid extension of range into the southwestern islands occurred later in M. spinalis and then in M. vaillanti and M. delalandii. The long apparent delay between the origin of these species and their southwestern dispersal may have been because there were earlier colonizations of the southern islands which excluded later ones until the earlier inhabitants were exterminated by volcanic or climatic events. The evolution of large size in Macroscincus occurred in the northwestern islands and was paralleled in the eastern and southern islands by populations of M. vaillanti. Both cases of size increase in Cape Verde skinks were accompanied by the development of herbivory.

Mesigyna once-a-month combined injectable contraceptive: experience in Latin America.

A phase III clinical study was carried out among 534 fertile Latin American women to evaluate cycle control, side effects, and contraceptive efficacy of a once-a-month combined injectable, Mesigyna, consisting of 50 mg norethisterone enanthate and 5 mg estradiol valerate. The pregnancy rate at 1 year was 0 per 100 woman-years for a total experience of 4688 woman-months. The overall discontinuation rate at one year was 17.9%. Discontinuation rate for bleeding problems was 5.1%. The Colombian women had a significant increase (p <0.001) in bleeding problems compared to other countries. The discontinuation rate for amenorrhea was 1.1%. There were no significant differences between the groups regarding discontinuation for other medical or non-medical reasons. Mean weight gain after one year of use was 1.02 kg. Mesigyna is an appropiate once-a-month injectable contraceptive for Latin American women since it is highly effective and its perception of normal menstrual bleeding is of importance in the Latin American population.

Long-distance colonization and radiation in gekkonid lizards, Tarentola (Reptilia: Gekkonidae), revealed by mitochondrial DNA sequences.

Morphological systematics makes it clear that many non-volant animal groups have undergone extensive transmarine dispersal with subsequent radiation in new, often island, areas. However, details of such events are often lacking. Here we use partial DNA sequences derived from the mitochondrial cytochrome b and 12S rRNA genes (up to 684 and 320 bp, respectively) to trace migration and speciation in Tarentola geckos, a primarily North African clade which has invaded many of the warmer islands in the North Atlantic Ocean. There were four main invasions of archipelagos presumably by rafting. (i) The subgenus Neotarentola reached Cuba up to 23 million years (Myr) ago, apparently via the North Equatorial current, a journey of at least 6000 km. (ii) The subgenus Tarentola invaded the eastern Canary Islands relatively recently covering a minimum of 120 km. (iii) The subgenus Makariogecko got to Gran Canaria and the western Canary Islands 7-17.5 Myr ago, either directly from the mainland or via the Selvages or the archipelago of Madeira, an excursion of 200-1200 km. (iv) A single species of Makariogecko from Gomera or Tenerife in the western Canaries made the 1400 km journey to the Cape Verde Islands tip to 7 Myr ago by way of the south-running Canary current. Many journeys have also occurred within archipelagos, a minimum of five taking place in the Canaries and perhaps 16 in the Cape Verde Islands. Occupation of the Cape Verde archipelago first involved an island in the northern group, perhaps São Nicolau, with subsequent spread to its close neighbours. The eastern and southern islands were colonized from these northern islands, at least two invasions widely separated in time being involved. While there are just three allopatric species of Makariogecko in the Canaries, the single invader of the Cape Verde Islands radiated into five, most of the islands being inhabited by two of these which differ in size. While size difference may possibly be a product of character displacement in the northern islands, taxa of different sizes reached the southern islands independently.

Origin and evolution of paralogous rRNA gene clusters within the flatworm family Dugesiidae (Platyhelminthes, Tricladida).

Analysis of the 18S rDNA sequences of five species of the family Dugesiidae (phylum Platyhelminthes, suborder Tricladida, infraorder Paludicola) and eight species belonging to families Dendrocoelidae and Planaridae and to the infraorder Maricola showed that members of the family Dugesiidae have two types of 18S rDNA genes, while the rest of the species have only one. The duplication event also affected the ITS-1, 5.8S, ITS-2 region and probably the 28S gene. The mean divergence value between the type I and the type II sequences is 9% and type II 18S rDNA genes are evolving 2.3 times more rapidly than type I. The evolutionary rates of type I and type II genes were calibrated from biogeographical data, and an approximate date for the duplication event of 80-120 million years ago was calculated. The type II gene was shown, by RT-PCR, to be transcribed in adult individuals of Schmidtea polychroa, though at very low levels. This result, together with the fact that most of the functionally important positions for small-subunit rRNA in prokaryotes have been conserved, indicates that the type II gene is probably functional.

Phylogeny of the arachnid order Opiliones (Arthropoda) inferred from a combined approach of complete 18S and partial 28S ribosomal DNA sequences and morphology.

The phylogenetic relationships among the main evolutionary lines of the arachnid order Opiliones were investigated by means of molecular (complete 18S rDNA and the D3 region of the 28S rDNA genes) and morphological data sets. Equally and differentially weighted parsimony analyses of independent and combined data sets provide evidence for the monophyly of the Opiliones. In all the analyses, the internal relationships of the group coincide in the monophyly of the following main groups: Cyphophthalmi, Eupnoi Palpatores, Dyspnoi Palpatores, and Laniatores. The Cyphophthalmi are monophyletic and sister to a clade that includes all the remaining opilionid taxa (=Phalangida). Within the Phalangida the most supported hypothesis suggests that Palpatores are paraphyletic, as follows: (Eupnoi (Dyspnoi + Laniatores)), but the alternative hypothesis (Laniatores (Eupnoi + Dyspnoi)) is more parsimonious in some molecular data analyses. Relationships within the four main clades are also addressed. Evolution of some morphological characters is discussed, and plesiomorphic states of these characters are evaluated using molecular data outgroup polarization. Finally, Martens' hypothesis of opilionid evolution is assessed in relation to our results.