Count Procedure Research Articles

Percutaneous transhepatic biliary drainage (PTBD) with or without stenting—complications, re-stent rate and a new risk stratification score

To review the success rate and number of complications in patients with obstructive jaundice treated with percutaneous transhepatic biliary drainage (PTBD), and to stratify the procedural risk of both PTBD and biliary stenting. 948 procedures performed in 704 consecutive patients with obstructive jaundice over a 7 year period were reviewed: 345 male; 359 females, mean age 70.1 years (range 48-96 years). Statistical analysis included X ( 2 ) test and multivariate logistic regression analysis. The technical success rate was 99%. The mortality related to the procedure was 2% and the 30-day mortality 13%. 91 (13%) stents inserted occluded during the study period. Predictors for stent failure and re-stenting were a diagnosis of cholangiocarcinoma, a lesion in the distal CBD, a high bilirubin, high urea and high white cell count and post procedure cholangitis. Factors significantly related to complications and 30-day mortality were retrospectively reviewed to devise a risk stratification score. PTBD and stenting offer a safe and effective method in providing palliative treatment for patients with biliary obstruction. Patients likely to have high levels of morbidity and mortality can be predicted before PTBD, using a risk stratification score, highlighting the need for closer clinical observation and delayed stent placement.

International collaboration and counting inflation in the assessment of national research productivity

In this paper we describe how different accounting procedures affected the counting of scientific paper numbers at the country level and the country ranks based on paper production quantity in physics. Using 1989--2008 citation data, we also report the counting inflation ratio between different accounting procedures. We found that, in general, different accounting procedures yielded relatively similar and stable rankings. But for certain clusters of countries, the normal count procedure tended to favor the more advanced Western countries. In contrast, the newly developed countries received more credit in the adjusted and straight count procedures.

When Counts Count: Improving Practice and Documentation

The Association of Perioperative Registered Nurses recommended practice for sponge, sharp, and instrument counts provides guidance to the perioperative nurse to account for all items and lessen the potential for injury from a retained item. A complete and accurate counting procedure helps to promote optimal patient outcomes and demonstrates a commitment to patient safety. Legislation does not propose who, how, or when counts are conducted, therefore, all team members should be committed to and involved in establishing a count policy and procedure

Improving the AOAC Use-Dilution Method by Establishing a Minimum Log Density Value for Test Microbes on Inoculated Carriers

The AOAC Use-Dilution methods, 955.14 (Salmonella enterica), 955.15 (Staphylococcus aureus), and 964.02 (Pseudomonas aeruginosa), are used to measure the efficacy of disinfectants on hard inanimate surfaces. The methods do not provide procedures to assess log density of the test microbe on inoculated penicylinders (carrier counts). Without a method to measure and monitor carrier counts, the associated efficacy data may not be reliable and repeatable. This report provides a standardized procedure to address this method deficiency. Based on carrier count data collected by four laboratories over an 8 year period, a minimum log density value is proposed to qualify the test results. Carrier count data were collected concurrently with 242 Use-Dilution tests. The tests were conducted on products bearing claims against P. aeruginosa and S. aureus with and without an organic soil load (OSL) added to the inoculum (as specified on the product label claim). Six carriers were assayed per test for a total of 1452 carriers. All 242 mean log densities were at least 6.0 (geometric mean of 1.0 x 10(6) CFU/carrier). The mean log densities did not exceed 7.5 (geometric mean of 3.2 x 10(7) CFU/carrier). For all microbes and OSL treatments, the mean log density (+/- SEM) was 6.7 (+/- 0.07) per carrier (a geometric mean of 5.39 x 10(6) CFU/carrier). The mean log density for six carriers per test showed good repeatability (0.29) and reproducibility (0.32). A minimum mean log density of 6.0 is proposed as a validity requirement for S. aureus and P. aeruginosa. The minimum level provides for the potential inherent variability that may be experienced by a wide range of laboratories and the slight effect due to the addition of an OSL. A follow-up report is planned to present data to support the carrier count procedure and carrier counts for S. enterica.

The application of viable count procedures for measuring viable cells in the various marine environments

To investigate whether the use of direct viable count (DVC), quantitative viable count (qDVC), colony-forming units and the contribution of capsule-bearing bacteria to the total number of bacteria and esterase-active bacteria could be used to clearly differentiate viable cells in various trophic status of seawater. Hundred and four marine isolates from various marine environments in Turkey (Western Black Sea, northern part of the Sea of Marmara, Northern Aegean Sea and eastern part of the Sea of Marmara) were screened. Seawater samples were taken from the surface (the upper 0-30 cm) and deeper layers (from 5 to 500 m) of the sea at different time periods between February 2002 and June 2007. For the assessment of cell elongation, minor modifications were made on DVC procedure in order to optimize the concentration of yeast extract and incubation time for enumeration of bacteria in response to nutrient addition. The best results were obtained when the yeast extract was used at a final concentration of 250 mg l(-1) (at 35 degrees C 24 h incubation) for bacteria isolated from eutrophic areas and a final concentration of 50 mg l(-1) for those selected from oligotrophic areas. A positive correlation was found between the trophic level and the level of metabolically active bacteria. Among these methods, the bacterial number obtained by qDVC is higher than those gained by other methods. The results indicate that the qDVC procedure could easily differentiate between viable cells and dormant or dead cells. We suggest that this method may be applicable to detecting the level of metabolic potential of bacterial communities in marine environments. The study resulted in increased knowledge on the applicability of the qDVC method that arranges the substrate amount and incubation time as well as on the comparison of various viable bacteria count procedures related to trophic situation of seawater samples.

Direct Viable Count Combined with Fluorescence in situ Hybridization (DVC-FISH) for Specific Enumeration of Viable Escherichia coli in Cow Manure

A direct viable count procedure combined with fluorescence in situ hybridization (DVC-FISH) was developed for the specific detection and enumeration of viable Escherichia coli in cow manure. The DVC method was performed by trapping bacterial cells, extracted from cow manure samples, onto Nucleopore filters followed by incubation on a DVC medium containing yeast extract and four gyrase inhibitors. E. coli cells were identified by using the probe ES445. The DVC method efficiently promoted the elongation of E. coli cells and allowed for the recognition of individual cell division events, by observing microcolonies. Cell recovery by DVC-FISH together with bacterial extraction, was 53% with an inoculum of 10(7) to 10(10) cells g(-1) dry weight, when the manure samples were inoculated with a fresh culture of E. coli and determinations were made immediately. An examination of the survival of E. coli in a cow manure microcosm showed that an increasing fraction of E. coli became non-culturable but were still detectable by DVC-FISH. All these results suggest that DVC-FISH is useful for enumerating viable, even non-culturable, E. coli in cow manure.

Modelling count and growth data with many zeros

We discuss the problem of modelling survival/mortality and growth data that are skewed with excess zeros. This type of data is a common occurrence in biological and environmental studies. The method presented here allows us to utilize both the survival/mortality and growth data when both data sets contain a large proportion of zeros. The method consists of four stages. Firstly the original data is divided into two sets; one contains all the surviving organisms and the other all of the mortalities. Secondly we calculate the actual growth of the surviving organisms and of the mortalities. Thirdly we count the number of surviving organisms for which growth has occurred and the number where no growth occurred, and the same count procedure is carried out on the mortalities. Next we model the survival/mortality data and growth/no growth data using logistic regression, and separately model the growth data using an ordinary regression. Finally we combine the three models to estimate the expected growth for a specific set of values of the explanatory variables. If we used another statistical method that did not involve the dead mussels or the ones with no growth, some of the information provided by these mussels would be lost. However, using the method we propose, all of the data collected are used to achieve an optimal estimation of the mussel growth. A case study of survival and growth of blue mussels ( Mytilus galloprovincialis) and ribbed mussels ( Aulacomya atra maoriana) trans-located from their natural distribution to different depths and sites along the axis of Doubtful Sound, New Zealand, is used for illustration.

Molecular typing of industrial strains of Pseudomonas spp. isolated from milk and genetical and biochemical characterization of an extracellular protease produced by one of them

P. fluorescens is responsible for the highest depredation of milk because of its capacity to synthesize extracellular lipase and protease which hydrolyze milk fat and proteins. Several P. fluorescens synthesize an extracellular caseinolytic metalloprotease, called AprX. It is important to rapidly detect the presence of a contamination of raw milk by a strain, especially a P. fluorescens strain, having a high potential of depredation. If standard plate count procedures are often employed, they are time consuming and do not permit to rapidly evaluate the potential of depredation. An alternative method consists to search the aprX gene, but such a method remains of low sensitivity and does not allow evaluating the real potential of depredation of the contaminant. After a milk depredation event, three strains of Pseudomonas spp. (F, 2312 and 2313) have been isolated from a dairy plant. Using molecular and phenotypic approaches, these strains were identified as P. fluorescens strains. Their respective extracellular caseinolytic potential was characterized as well as that of several collection strains of P. fluorescens. It appeared that these strains secreted one protease of about 45 kDa, that their extracellular caseinolytic potential was highly variable for one strain to another and that the one of strain F was the highest. The protease secreted by the strain F was purified and its N-terminal sequence established. It shared 100% identity with the domain 14–34 of extracellular alkaline endoprotease sequences which are called AprX for some of them. Its gene was sequenced as well as that of two collection strains of P. fluorescens having a significant lower extracellular caseinolytic potential. The genomic environment of the aprX gene as well as its expression during the strain growth was investigated. It appears that the difference of extracellular caseinolytic potential which has been observed between the three strains does not mainly result from the AprX sequence/structure but it might rather result from the aprX level of expression.

Studies of Photokilling of Bacteria Using Titanium Dioxide Nanoparticles

Metal pins used to apply skeletal traction or external fixation devices protruding through skin are susceptible to the increased incidence of pin site infection. In this work, we tried to establish the photokilling effects of titanium dioxide (TiO2) nanoparticles on an orthopedic implant with an in vitro study. In these photocatalytic experiments, aqueous TiO2 was added to the tested microorganism. The time effect of TiO2 photoactivation was evaluated, and the loss of viability of five different bacteria suspensions (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae, and Bacteroides fragilis) was examined by the viable count procedure. The bactericidal effect of TiO2 nanoparticle-coated metal plates was also tested. The ultraviolet (UV) dosage used in this experiment did not affect the viability of bacteria, and all bacteria survived well in the absence of TiO2 nanoparticles. The survival curve of microorganisms in the presence of TiO2 nanoparticles showed that nearly complete killing was achieved after 50 min of UV illumination. The formation of bacterial colonies above the TiO2 nanoparticle-coated metal plates also decreased significantly. In this study, we clearly demonstrated the bactericidal effects of titanium dioxide nanoparticles. In the presence of UV light, the titanium dioxide nanoparticles can be applicable to medical facilities where the potential for infection should be controlled.

AOAC Method 966.04: Preliminary Evaluation of Cooked Meat Medium with Manganese Sulfate for the Cultivation of Clostridium sporogenes: Precollaborative Study

AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. For regulatory purposes, Method 966.04 is accepted by the U.S. Environmental Protection Agency (EPA) and the U.S. Food and Drug Administration (FDA) for the generation of product performance data for sporicides and sterilants. In this study, we report on findings associated with proposed improvements (modifications) to the Clostridium component of the method. Egg meat medium (EMM), the culture medium for C. sporogenes currently specified in the method, is no longer commercially available and finding a suitable replacement is critical. In addition, the use of a nonstandardized extract of raw soil as an amendment to EMM, as stipulated in the current method, may result in a highly variable spore suspension. The primary focus of this study was to find replacements for EMM and soil extract. A carrier count procedure, the establishment of target carrier counts (spores/carrier), and a neutralization confirmation procedure were also evaluated. The study was limited to liquid products tested against Clostridium on a hard surface carrier (porcelain penicylinder). Spore suspensions of C. sporogenes were generated using: (1) EMM with soil extract (EMM/SE), (2) cooked meat medium with soil extract (CMM/SE), and (3) cooked meat medium with 5 microg/mL manganese sulfate (CMM/MnSO4). The titer of the spore suspension, carrier counts, resistance to hydrochloric acid (HCI), and efficacy against 3 liquid sporicidal agents were used to evaluate the potential of CMM and MnSO4 as replacements. The study was performed by the EPA Office of Pesticide Programs Microbiology Laboratory, Fort Meade, MD. Use of CMM/SE and CMM/MnSO4 resulted in comparable results for titer of spore suspensions (approximately 10(8) spores/mL) and carrier counts (approximately 3 x 10(6) spores/carrier). The carrier counts for the EMM/SE were approximately 1 log lower than CMM-based treatments; however, no attempt was made to dilute the CMM spore suspensions prior to carrier inoculation to reduce the carrier counts for CMM. Resistance of spores to 2.5 M HCI was acceptable across the 3 media types. Treatments for comparative efficacy testing were designed to provide a range of sporicidal activity, i.e., high and low efficacy treatments. Sodium hypochlorite (bleach), hydrogen peroxide/peracetic acid, and glutaraldehyde were used as test chemicals. The number of carriers resulting in growth (positive) for the low treatments for all 3 chemicals ranged from 9 to 59 out of 60 across the 3 media types--EMM exhibited fewer positives overall. The high efficacy treatments for sodium hypochlorite and hydrogen peroxide/peracetic acid yielded a range of 0 to 2 positives out of 60 across the 3 media. However, the high glutaraldehyde treatment generated 3, 20, and 20 positives out of 60 for the EMM/SE, CMM/SE, and CMM/MnSO4, respectively. The lower number of positive carriers for EMM/SE may be due to the reduced carrier counts. CMM, either with SE or MnSO4, appears to be a suitable replacement for EMM/SE. On the basis of the results of this study, the Study Director recommends that CMM/MnSO4 and the spore enumeration target carrier count and neutralization procedures be considered for collaborative study to officially modify the Clostridium x porcelain component of Method 966.04.

Lotka's distribution and distribution of co-author pairs’ frequencies

The original Lotka's Law refers to single scientist distribution, i.e. the frequency of authors A i with i publications per author is a function of i: A i = f( i). However, with increasing collaboration in science and in technology the study of the frequency of pairs or triples of co-authors is highly relevant. Starting with pair distribution well-ordered collaboration structures of co-author pairs will be presented, i.e. the frequency of co-author pairs N ij between authors with i publications per author and authors with j publications per author is a function of i and j: N ij = f( i, j) using the normal count procedure for counting i or j. We have assumed that the distribution of co-author pairs’ frequencies can be considered to be reflection of a social Gestalt and therefore can be described by the corresponding mathematical function based on well-known general characteristics of structures in interpersonal relations in social networks. We have shown that this model of social Gestalts can better explain the distribution of co-author pairs than by a simple bivariate function in analogy to Lotka's Law. This model is based on both the Gestalt theory and the old Chinese Yin/Yang theory.

The Line Method for Petrographic Determination of the Quantity of Fly Ash and Ground-granulated Blast Furnace Slag in Hardened Concrete and Blended Cement

Abstract Research was undertaken using thin sections and immersion mounts with the objective of using a modified line count procedure for the quantitative petrographic determination of fly ash and ground-granulated blast furnace slag (GGBFS) in hardened concrete and blended cements. The line method used in this study is a variation of standard microscopic point count methods and yields an average number frequency that is a linear function of the weight percent of fly ash or GGBFS of total cementitious materials. Multiple traverses were counted on thin sections of concrete over areas of paste only and in immersion slides of blended cements containing known amounts of fly ash and GGBFS. The average number of particles in a traverse was computed, the traverse length being equal to the width of the field of view of the petrographic microscope. For hardened concrete, only pozzolan particles were counted. For blended cements, all particles were counted and the ratio of pozzolan particles to total particles was determined. A linear relationship between the average number of pozzolan particles for a traverse line and the weight percent of pozzolan makes it possible to estimate the fly ash and GGBFS content of unknowns. Results of the research indicate that the line method is a rapid and accurate method for petrographic determination of fly ash and GGBFS in hardened concrete and blended cements. This technique and variations of it have been used by other researchers and it has the potential to be a standard test procedure.

Enumeration Procedure for Monitoring Test Microbe Populations on Inoculated Carriers in AOAC Use-Dilution Methods

The AOAC Use-Dilution methods do not provide procedures to enumerate the test microbe on stainless steel carriers (penicylinders) or guidance on the expected target populations of the test microbe (i.e., a performance standard). This report describes the procedures used by the U.S. Environmental Protection Agency to enumerate the test microbe (carrier counts) associated with conducting the Use-Dilution method with Staphylococcus aureus (Method 955.15) and Pseudomonas aeruginosa (Method 964.02) and the examination of historical data. The carrier count procedure involves the random selection of carriers, shearing bacterial cells from the carrier surface through sonication, and plating of serially diluted inoculum on trypticase soy agar. For each Use-Dilution test conducted, the official AOAC method was strictly followed for carrier preparation, culture initiation, test culture preparation, and carrier inoculation steps. Carrier count data from 78 Use-Dilution tests conducted over a 6-year period were compiled and analyzed. A mean carrier count of 6.6 logs (approximately 4.0 x 10(6) colony-forming units/carrier) was calculated for both S. aureus and P. aeruginosa. Of the mean values, 95% fell within +/- 2 repeatability standard deviations. The enumeration procedure and target carrier counts are desirable for standardizing the Use-Dilution methods, increasing their reproducibility, and ensuring the quality of the data.

EXTENDING STORAGE LIFE OF KING COCONUT, COCOS NUCIFERA VAR. AURANTA

This study was conducted to facilitate low temperature storage and distribution of fresh king coconut, Cocos nucifera var. auranta, for periods up to 28 days. Problems addressed included incidence of disease, perianth drop, discolouration of coconut water, chilling injury and prevention of moisture loss during refrigerated storage. Trials were conducted using leaf count and tagging procedures to determine maturity indices. A comparison of organoleptic and physico-chemical parameters indicated that nuts harvested at 7 and 8 months after flowering were at the optimum stage of maturity for extending storage life for up to 28 days at 13.5°C. Four wax formulations developed at the Industrial Technology Institute - W1, W2, W3, and W4 - were then tested on nuts harvested at the 7 to 8 stage of maturity. Treated nuts were stored at ambient (28±2°C) and 13.5°C. Nuts were observed to respond best to a dip treatment in wax formulation Wl, when stored at 13.5°C for 28 days. Nuts stored at 28±2°C, showed complete deterioration after 7 days. There was no significant difference in quality irrespective of the wax formulations tested.

Distribution and Statistical Analysis of Bacteria in Lake Alau in the Arid Northern Nigeria

The direct microscopic count using bright field illumination was subjected to a statistical comparison with four different microbial cell viability procedures viz., pour plate, spread plate, most probable number (MPN) and membrane filtration (MF) for the analysis of the distribution of bacteria in water samples from Lake Alau Dam in the arid Northern Nigeria. A total of 27 samples were collected from three locations of the dam site and analysed. The total coliform represented 32% of the 22 isolates and of this 10% represented the fecal coliform bacteria. The fecal coliform level of 6.0 x 102 to 8.8 x 102/100ml placed lake Alau Dam water samples in the EEC Grade A2 and thus, the water is recommended for further treatments before it can be distributed to municipalities for domestic use. Statistical analysis using one-way analysis of variance (ANOVA) showed significant differences in counts between three locations, based on the direct microscopic counts. There were no significant differences in counts between locations using the four cultural methods. There was an honestly significant difference (hsd, 5% level, Tukeys test) between the direct microscopic counts and the four cultural methods, but there was not honestly significant difference (hsd, 5% level) between the four cultural methods. It is concluded that in the absence of more sophisticated procedures, the four cultural methods together with the direct bright field microscopic count procedure served to establish a good measure of the microbiological quality of the Lake Alau water, this is strengthened by the statistical correlations. Journal of Applied Sciences and Environmental Management Vol. 8 (1) 2004: 5-9

Does Counting Count for 3‐ to 4‐year‐olds? Parental Assumptions about Preschool Children's Understanding of Counting and Cardinality

AbstractResearch indicates that children do not typically understand the connection between counting and cardinality for several months after learning to count, yet parents speak to 3‐year‐olds as though they already understood the significance of counting. The present research was designed to investigate mothers’ awareness of the discrepancy between children's procedural and conceptual mastery of counting. In Study 1 mothers of a hundred 3‐ to 41/2‐year‐olds completed an anonymous questionnaire asking them to anticipate how their child would respond to a series of real‐life vignettes based on widely used experimental measures of cardinal understanding. Most anticipated that their child, irrespective of age, would (1) understand the significance of the last word of a count, and (2) be able accurately to give a specified non‐subitizable number of objects. Comparison with the performance of 54 children from the same local population supported the hypothesis that parents overestimate children's understanding of the cardinal significance of counting. Mothers reported a range of impromptu number‐related activities in which their child had recently participated at home; most of these involved simple procedural counting. In Study 2, 35 mothers of 3‐ to 41/2‐year‐olds completed a modified questionnaire concerning procedural aspects of counting as well as cardinality; their responses were then compared with the performance of their own children. Again, mothers overestimated their children's cardinal understanding, but this was shown not to be a result of a general tendency to overestimate their counting abilities. It is suggested that preschoolers’ counting generally occurs during joint activities in which caregivers may be unaware of the support that they provide, and, provided that the jointly executed count procedures are error‐free, parents implicitly assume a ‘common knowledge’ regarding the cardinal significance of counting.

Cadmium Leachability and Microbial Population Dynamics in a Stabilized and Bioaugmented Model Contaminated Soil

This article presents a laboratory study on the consequences of the application of combined soil stabilization and bioaugmentation in the remediation of a model contaminated soil. Stabilization and bioaugmentation are two techniques commonly applied independently for the remediation of heavy metal and organic contamination respectively. However, for a cocktail of contaminants combined treatments are currently being considered. The model soil was contaminated with a cocktail of organics and heavy metals based on the soil and contaminant conditions in a real contaminated site. The soil stabilization treatment was applied using either zeolite or green waste compost as additives and a commercially available hydrocarbon degrading microbial consortium was used for the bioaugmentation treatment. The effects of stabilization with or without bioaugmentation on the leachability of cadmium and copper was observed using an EU batch leaching test procedure and a flow-through column leaching test, both using deionized water at a pH of 5.6. In addition, the population of hydrocarbon degrading microorganisms was monitored using a modified plate count procedure in cases where bioaugmentation was applied. It was found that while the stabilization treatment reduced the metal leachability by up to 60%, the bioaugmentation treatment increased it by up to 100% Microbial survival was also higher in the stabilized soil samples.

Healthcare Safety: The Impact of Disabling “Safety” Protocols

With increasing attention to patient safety, hospitals and other clinical facilities are developing practice guidelines and protocols with the specific intent of reducing harm to patients. However, the introduction of these protocols can have unanticipated negative consequences and if followed rigidly can become disabling. We use the manual count procedure that was designed to improve patient safety by reducing the likelihood of leaving an object (e.g., needle, sponge, or instrument) inside a patient body cavity during a surgical procedure to illustrate this point. Using results from a focus group of seven operating room nurses and an observational study of nine complex operations, we show that the count protocol has unanticipated negative consequences that need to be considered in evaluating the net positive gain in patient safety. The study highlights the importance of evaluating the overall impact of proposed protocols in assessing its potential benefits to patient safety.

Combining direct viable count (DVC) and fluorescent in situ hybridisation (FISH) to enumerate viable E. coli in rivers and wastewaters

In this study, a combination of the direct viable count procedure (DVC) and the FISH method was used to monitor by epifluorescence microscopy the abundance of viable E. coli in river water and wastewater samples. The DVC procedure consisted of exposing bacterial cells to a resuscitation medium containing antibiotics preventing cellular division and, thus, inducing an elongation of the viable cells. The FISH was performed using the "Colinsitu" probe specific for E. coil 16S r-RNA. Accuracy and detection limit of the epifluorescence microscopic DVC-FISH procedure were investigated. The method was then applied to river-water and wastewater samples. A good correlation was found in a log-log plot between the abundance of E. coil enumerated by a classical culture-based method (MPN method) and the DVC-FISH procedure. However, the DVC-FISH procedure gave consistently higher numbers. The ratio between both enumerations (DVC-FISH/MPN), which also indicated the ratio between viable and culturable E. coli, ranged between 2 and >30. It increased with decreasing abundance of culturable E. coli.

Variation patterns in the Phlomis × composita (Lamiaceae) hybrid complex in the Iberian Peninsula

Hybridization seems to be common in the genus Phlomis (Lamiaceae) in the Iberian Peninsula, especially in the P. × composita complex. In order to detect patterns of morphological variation linked to eco- and geographical variation, ecological (cluster and canonical correspondence) and morphometric (principal component and discriminant function) analyses were performed. Character count procedure was applied to discern between divergence and reticulate events for the origin of the morphologically intermediate plants. Following these analyses clear patterns were detected suggesting the existence of four independent morphological groups also supported by the ecogeographical data. These are P. crinita ssp. crinita (Levante, eastern Spain), P. crinita ssp. malacitana (Andalusia, southern Spain) and P. lychnitis (widespread) being the extremes of the morphological variation, and P. × composita including all the morphologically intermediate individual plants. Furthermore, at the population level significant differences in hybrid plant frequencies between areas were also found. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 145, 97–108.