Application of central composite rotatable design to lipase catalysed synthesis of m-cresyl acetate

Esterification of m-cresol with acetic acid using porcine pancreas lipase (PPL) was investigated by response surface methodology (RSM). A central composite rotatable design (CCRD) involving 32 experiments of five variables at five levels was employed to analyse the esterification behaviour. The effect of five variables studied namely, m-cresol concentrations (0.005–0.025 mol), enzyme/substrate ratios (0.18–1.22 activity units/millimol, AU/mmol), incubation periods (6–54 h), pH (4–8) and buffer volumes (0–0.2 ml) was useful in arriving at an optimum ester yield. The methodology projected conditions for higher yields up to 6.0 mmol. Validation experiments carried out under these predicated conditions showed good correspondence between experimental and predicted yields. CCRD treatment clearly showed the inhibitory nature of m-cresol in the esterification process. The reaction required the presence of buffer for better conversions and a minimum amount of 0.1 ml buffer was found necessary for this reaction. Buffer pH values around 6.0 and below appeared to favour better esterification than those at pH values in the range 6.0–8.0. However an optimum condition for maximum yield was: incubation period: 54 h; buffer volume: 0.2 ml; pH: 8; E/S ratio: 0.83 AU/mmol; m-cresol: 0.02 mol; predicted yield: 6.0 mmol; experimental yield: 6.4 mmol.     

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol

Crystal structures of cyclomaltohexaose (α-cyclodextrin) complexes with p-chlorophenol and p-cresol have been determined by single-crystal X-ray diffraction studies. The space group of the α-cyclodextrin–p-chlorophenol complex is P2₁2₁2₁ with unit cell dimensions of a=15.299(3), b=24.795(5), c=13.447(5) Å, and that of the α-cyclodextrin–p-cresol complex is P2₁ with unit cell dimensions of a=7.927(7), b=13.568(7), c=24.54(1) Å, β=90.41(8)°. In spite of the similar structures of guest molecules, both complexes have different inclusion modes and packing structures.     

Successful oviposition and reproductive biology of Aprostocetus vaquitarum (Hymenoptera: Eulophidae): A predator of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Aprostocetus vaquitarum (Wolcott) causes 78–91 percent mortality to eggs of Diaprepes abbreviatus (L.), under field conditions in southern Florida. In the laboratory, A. vaquitarum was reared on D. abbreviatus eggs at 25 °C, a photoperiod of 12:12 (L:D) and with abundant hosts, A. vaquitarum adult females lived around 15 days. Oviposition was significantly affected by the age of the host egg mass. Egg masses aged 0- to 3-day-old were accepted significantly better than those aged 4–6 days. The mean number of eggs deposited per female was around 53, with extreme values of 124 and 19 eggs per female. Using these data in combination with the sex ratio observed in the field (0.16) and the duration of the preimaginal stages, r_m (0.168–0142 day⁻¹), T (22.39–22.89 days), and R₀ (43.03–25.81 females per female) were calculated.     

Optimum growth conditions and light utilization efficiency of Spirulina platensis (= Arthrospira fusiformis) (Cyanophyta) from Lake Chitu, Ethiopia

Spirulina platensis (= Arthrospira fusiformis) was isolated from Lake Chitu, a saline, alkaline lake in Ethiopia, where it forms an almost unialgal population. Optimum growth conditions were studied in a turbidostat. Cultures grown in modified Zarrouk's medium and exposed to a range of light intensities (20–500 µmol photons m^–2s^–1) showed a maximum specific growth rate (µ_max) of 1.78 d^–1. Quantum yield for growth (µ) was 3.8% at the optimum light for growth of 330 µmol photons m^–2s^–1, and ranged from 2.8 to 9.4%. With increase in irradiance, the chlorophyll a concentration decreased, and the carotenoids/chlorophyll a ratio increased by a factor of 2.4. The phosphorus to carbon ratio (P/C) showed some variation, while the nitrogen to carbon ratio (N/C) remained relatively constant, thus causing fluctuations in the N:P ratio (7–11) of cells. An optimum N:P ratio of about 7 was attained in cells growing at the optimum light for growth. Results from the continuous culture experiments agreed well with maximum values of photosynthetic efficiency given in the literature for natural populations of S. platensis in the soda lakes of East Africa, Lake Arenguade (Ethiopia), and Lake Simbi (Kenya).     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Crustacean assemblage and environmental characteristics of a man-made solar saltwork in southern France,with emphasis on anostracan (Branchiopoda) population dynamics

Physical and chemical variables, anostracan populations (Artemia parthenogenetica and Branchinella spinosa) and other biota were studied during 1996–1997 in a Camargue saltern (max. depth 1 m). The taxonomic composition and density of macroinvertebrates were investigated twice monthly, based on benthic substrate and water column samples. Fauna was composed of three groups in terms of numerical importance. The benthic macroinvertebrates were represented only by nematodes (< 50 ind. m^–2 to > 500 ind. m^–2 in November–December and May respectively). The zooplankton was dominated by crustaceans, one cladoceran, Moina salina (ranging from 670 to 2350 ind. m^–2 in spring), two anostracans, Artemia parthenogenetica (< 50 ind. m^–2 in autumn), and Branchinella spinosa (max. 190 ind. m^–2 in December to absent in April), and two copepods, Cletocamptus retrogressus (max. density 2000 ind. m^–2 in November), and Eurytemora velox (max. density 650 ind. m^–2 in February–March). Insects (Chironomidae, Culicidae) were rare, with mean densities < 1 ind. m^–2. The phenology of each crustacean population is discussed in relation to physical and chemical water variables. Salinity appeared to be of greatest importance regulating the population abundance.     

Ulva rigida (Ulvales,Chlorophyta) tank culture as biofilters for dissolved inorganic nitrogen from fishpond effluents

Ulva rigida was cultivated in 7501 tanks at different densities with direct and continuous inflow (at 2, 4, 8 and 12 volumes d^–1) of the effluents from a commercial marine fishpond (40 metric tonnes, Tm, of Sparus aurata, water exchange rate of 16 m³ Tm^–1) in order to assess the maximum and optimum dissolved inorganic nitrogen (DIN) uptake rate and the annual stability of the Ulva tank biofiltering system. Maximum yields (40 g DW m^–2 d^–1) were obtained at a density of 2.5 g FW 1^–1 and at a DIN inflow rate of 1.7 g DIN m^–2 d^–1. Maximum DIN uptake rates were obtained during summer (2.2 g DIN M^–2 d^–1), and minimum in winter (1.1 g DIN m^–2 d^–1) with a yearly average DIN uptake rate of 1.77 g DIN m^–2 d^–1 At yearly average DIN removal efficiency (2.0 g DIN m^–2 d^–1, if winter period is excluded), 153 m² of Ulva tank surface would be needed to recover 100% of the DIN produced by 1 Tm of fish.Abbreviations DIN= dissolved inorganic nitrogen (NH _inf4 ^sup+ + NO _inf3 ^sup– + NO _inf2 ^sup– ); - FW= fresh weight; - DW= dry weight; - PFD= photon flux density; - V= DIN uptake rate     

The multiple soluble malate dehydrogenase of Hoplias malabaricus(Erythrinidae, Characiformes)

To explain the six-banded pattern obtained upon electrophoresis of the soluble form of malate dehydrogenase (sMDH, EC 1.1.1.37) from the characiform Hoplias malabaricus, a recent locus duplication of its A isoform (sMDH-A*), in addition to its sMDH-B* isoform, was proposed. Klebe’s serial dilutions carried out using skeletal muscle, heart and liver extracts showed that the A1 and A2 subunits have the same visual end-points, indicating that these A-duplicated genes have a nondivergent pattern. Since there is no evidence of polyploidy in the Erythrinidae family, the MDH-A* loci have probably evolved from regional gene duplication. While these sMDH-A* loci encode nondivergent thermostable isoforms, the sMDH-B* encodes a thermolabile one. Thermostable sMDHs differ from the thermolabile sMDHs in that they have a higher K_m of oxaloacetate. Liver, muscle and heart unfractionated sMDH levels at three different temperature and two pH regimens were analysed and the results showed that, in the adaptative temperature range of Hoplias, the variation in K_m under conditions of constant pH (imidazole buffer) was less (approximately threefold) than that measured in the presence of temperature-dependent pH imidazole buffer (sevenfold). Estimation of the ratio of both isoforms in these tissues by Klebe’s method showed that, in unfractionated liver – where K_m values were the highest and the minimum K_m was obtained at 30^C (both for temperature-dependent pH and constant-pH imidazole buffer) – the duplicate A (thermostable, A1 and A2) and B (thermolabile) subunits were detected in a ratio of 2:1. On the other hand, in muscle extracts – in which the lowest K_m values were measured, with the minimum K_m at 10–20^C (temperature-dependent pH and constant-pH imidazole buffer, respectively) – a ratio of two thermolabile to one thermostable subunits was observed.     

Development and reproduction of Stratiolaelaps scimitus (Acari: Laelapidae) with fungus gnat larvae (Diptera: Sciaridae), potworms (Oligochaeta: Enchytraeidae) or Sancassania aff. sphaerogaster (Acari: Acaridae) as the sole food source

Stratiolaelaps ( = Hypoaspis) miles (Berlese) (Acari: Mesostigmata: Laelapidae) is a polyphagous soil-dwelling predatory mite that is widely marketed for use in greenhouse production systems to manage populations of dark-winged fungus gnats, Bradysia spp. (Diptera: Sciaridae) and for supplemental control of thrips. The suggestion by Walter and Campbell (2003, Biol. Control 26: 253–269) that North American commercial cultures of S. miles may actually be S. scimitus was confirmed. The development and reproduction at 21–23 °C of S. scimitus provided ad libidum with one of three different prey – the fungus gnat Bradysia aff. coprophila (Lintner), potworms (Enchytraeidae), or Sancassania aff. sphaerogaster (Zachvatkin, 1937) (Acari: Astigmata: Acaridae) – were compared. Developmental duration of the egg and non-feeding larval stages were 2.47 and 1.11 days, respectively; mortalities were 8.3 and 5.5%. Stratiolaelaps scimitus failed to develop beyond the protonymphal stage when provided with S. aff. sphaerogaster alone, although some feeding was observed. Development and reproduction of S. scimitus was successful on both fungus gnat larvae and enchytraeids, with no influence of prey on protonymphal duration (4.70 days) and mortality (8.3%), or on deutonymphal duration (4.61 days) and mortality (6.1%). Adult female S. scimitus feeding on potworms, compared to those feeding on fungus gnat larvae, had a significantly shorter pre-oviposition period (2.69 vs. 4.59 days). However, diet did not influence other adult female developmental or reproductive characteristics (oviposition period, 18.6 days; post-oviposition period, 6.2 days; total adult longevity, 27.3 days; total number of eggs, 26.5). S. scimitus reared on potworms tended (p = 0.06) to have a higher intrinsic rate of increase, a higher finite rate of increase and a shorter doubling time (r _m = 0.142 day⁻¹, λ = 1.153, Dt = 4.85 days) than those reared on fungus gnat larvae (r _m = 0.105 day⁻¹, λ = 1.110, Dt = 6.58 days), but differences in net reproductive rate (R ₀) and generation time (G) were not significant.     

Differentiation between the two subspecies of Atherosperma moschatum Labill. (Atherospermataceae) from their leaf oils

The leaf oils of both subspecies of Atherosperma moschatum have been investigated to determine if there were any chemical differences between the two subspecies. While both subspecies of A. moschatum contained methyl eugenol (55–87%), safrole (5–20%) and linalool (1–6%), camphor (5–10%) was found to be only in the oils of A. moschatum subsp. moschatum and to be either absent or, at most, less than 0.5% in the oils of A. moschatum subsp. integrifolium. There was also a range of monoterpene hydrocarbons and 1,8-cineole, all in the range of 0.1–5%, that were present in subspecies moschatum but virtually absent in subspecies integrifolium.     

Enzyme inhibition by p-cresol and lacticacid in lipase-mediated synthesis of p-cresyl acetate and stearoyl lactic acid: a kinetic study

A detailed kinetic study was carried out to investigate the porcine pancreatic lipase-catalysed esterification reactions of p-cresol–acetic acid and lactic acid–stearic acid. The kinetic data were in agreement with a Ping Pong Bi–Bi mechanism being followed by the enzyme, where inhibition is indicated in the presence of p-cresol and lactic acid in the respective reactions. Mathematical analyses of experimentally observed initial rates yielded various kinetic parameters, K _m(p-cresol) = 0.1, K _{m(acetic acid)} = 0.54, K _{m(lactic acid)} = 0.059 M, K _{m(stearic acid)} = 0.04 M, V _{max(p-cresol–acetic acid)} = 13.2(h^–1), V _{max(lactic acid–stearic acid)} = 0.00163 M/h, K _i(p-cresol) = 0.59 and K _{i(lactic acid)} = 0.079 M. The K _m and K _i values of p-cresol and lactic acid observed in the respective reactions showed both the competitive nature of binding between the substrates p-cresol and acetic acid on the one hand and lactic acid and stearic acid on the other and the inhibitory nature of p-cresol and lactic acid.     

Observations on the distribution patterns,behaviour, diets and reproductive cycles of sand-dwelling clinids (Perciformes: Clinidae) from South Africa

Synopsis Twenty-two samples of sand-dwelling fishes were collected from sublittoral sandy substrata off the Cape Peninsula, South Africa using a novel quantitative technique in which the ichthyocide, rotenone, was introduced beneath 6.25 m² plastic sheets weighted around the perimeter with chain. A total of 94 fish of eight species and four families were recorded. Of these, four species of the family Clinidae made up 92% of the material. Fish densities varied from 0.10–2.96 fish m^–2 (0.17–1.14 g m^–2). Distribution patterns amongst the Clinidae were explained by sediment particle size, with all of the newly discovered species, Cancelloxus longior, inhabiting fine sand (median grain size 0.25–0.50 mm), and all Xenopoclinus leprosus, C. elongatus and Pavoclinus smalei occurring in gravel (> 1 mm). Xenopoclinus kochi was found in all sediment types, but was most abundant in coarse substrata. Diets of all four species were similar, consisting chiefly of amphipods and isopods. However, small differences in prey preference were evident. All four species reached sexual maturity at 25–30 mm standard length, were viviparous, exhibited superembryonation and gave birth to live young of approximately 14 mm. Breeding seasonality occurred in all species except P. smalei.     

Life cycle and growth ofTrichostegia minor (Curtis) in temporary woodland pools (Trichoptera: Phryganeidae)

Larvae of the caddisTrichostegia minor (Curtis) were collected from four woodland pools in The Netherlands, three of which are temporary, from August 1986 till June 1987. Eggs and larvae of this species proved to be very well adapted to drought, freezing, strongly fluctuating pH and alkalinity levels and prolonged oxygen deficit. The life cycle ofT.minor in a small woodland marsh overgrown byCalla palustris took one year. Adult flight period started at the end of May. Oviposition took place independent of water. Hatching of the eggs started in September and was probably induced by immersion. During the larval stage from September until May, 5 instars could be distinguished by the size of the head capsule. Growth of instars I, II and III during autumn was moderate. Most larvae overwintered as instar III or IV. Possibly there was a larval diapause during winter. In spring rapid growth to instar V took place prior to pupation. Growth rate, expressed as the increase of mean individual dry weight was highest from March to April (2.05±0.75% DW.m^–2.d^–1). In extremely shallow water growth in spring was initially more rapid compared to growth in deeper water. During winter the growth rate decreased to 0.038±0.071% DW.m^–2.d^–1. Net annual production based on the changes of momentary biomass was 183.2±31.7 mg DW.m^–2.y^–1 or 177.2±31.3 mg AFDW.m^–2.y^–1. Production loss during the winter season was 75.1±10.8 mg DW.m^–2.y^–1 or 72.3±10.6 mg AFDW.m^–2.y^–1.     

Biology ofChironomus piger Str. (Diptera: Chironomidae) and its role in the self-purification of a river

Chironomus piger larvae are widespread in small rivers and canals strongly polluted with domestic sewage. Despite this, almost nothing is known concerning the biology of the species under natural conditions and its role in the process of river self-purification. For two years, benthic samples were collected in the Sestra River at a site about 250 m downstream of the effluent discharge drains of a sewage treatment installation where the greatest concentrations of the larvae occurred. The number and biomass ofC. piger larvae were subject to marked fluctuations,viz. 96,000–348,000 ind m^–2 and 420–1,800 g m^–2. 460 degreedays (on average) was required for development of one generation.C. piger has 5 or 6 generations per year depending on the hydrometeorological conditions during the growing season.C. piger larvae play an important role in self-purification of the river. They utilize precipitating seston for food and for building their dwelling tubes. According to our calculations the amount of organic matter assimilated in the area of maximum larval concentration ranged 80–177 g wet weight m^–2 day^–1, and 32–71 g wet weight m^–2day^–1 was mineralized.     

Biodegradation of phenol and <Emphasis Type="Italic">m</Emphasis>-cresol by <Emphasis Type="Italic">Candida albicans</Emphasis> PDY-07 under anaerobic condition

Strain Candida albicans PDY-07 was used to study the anaerobic biodegradation of phenol and m-cresol as single and dual substrates in batch cultures. The strain had a higher potential to degrade phenol than m-cresol. The cell growth kinetics of batch cultures with various initial m-cresol concentrations was investigated, and the Haldane kinetic model adequately described the dynamic behavior of cell growth on m-cresol. When cells grew on the mixture of phenol and m-cresol, substrate interactions were observed. Phenol inhibited the utilization of m-cresol; on the other hand, m-cresol also inhibited the degradation of phenol. However, the presence of low-concentration phenol enhanced m-cresol biodegradation; 100 mg/l m-cresol could be completely degraded within a shorter period of time than m-cresol alone in the presence of 150–300 mg/l phenol. The maximum m-cresol biodegradation rate was obtained at the existence of 200 mg/l phenol. Phenol was preferably utilized by the strain as a carbon and energy source. In addition, a sum kinetics model was used to describe the cell growth behavior in binary mixture of phenol and m-cresol, and the interaction parameters were determined. The model adequately predicted the growth kinetics and the interaction between the substrates.     

Annual growth rate of the calcareous red alga Lithothamnion corallioides (Corallinales,Rhodophyta) in the Bay of Brest,France

Lithothamnion corallioides Crouan et Crouan (Rhodophyta, Corallinales) is the main constituent of the maerl beds of the Bay of Brest (Atlantic coast of western Brittany). Its growth rate was measured monthly in situ during one year. Growth rates were obtained by an adaptation of the buoyant weight technique. The highest daily growth rate was observed in July and reached 0.26 % d^–1 (S.D. = 0.06), when expressed as the increase of calcium carbonate weight. The average daily growth rate was 0.12% d^–1 (S.D. = 0.04) for a period of 275 days (summer and autumn 1988, winter 1989). Using this preliminary data, the calcium carbonate accretion rate can be estimated provisionally: 876 g m^–2 year^–1, a rate much lower than that of tropical reef coralline algae, but higher than that of Lithophyllum incrustans, the well known temperate European reef-builder.     

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography–mass spectrometry

We report here the development and validation of an LC–MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C₁₈ reversed-phase column using an acetonitrile–water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS–MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477→266), DMLOP (m/z 463→252) and A-LOP (m/z 519→266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3±1.50% (range: 70.7–73.7%) and 79.4±12.8% (64.9–88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.     

The effect of environmental factors on the growth rate of Karenia brevis (Davis) G. Hansen and Moestrup

The red tide dinoflagellate Karenia brevis (Davis) G. Hansen and Moestrup is noted for causing mass mortalities of marine organisms in the Gulf of Mexico. Most research has focused on culture isolates from the eastern Gulf of Mexico. In this investigation, we examine the effects of light, temperature and salinity on the growth rate of K. brevis from the western Gulf of Mexico. Growth rates of K. brevis were determined under various combinations of irradiance (19, 31, 52, 67, and 123 μmol m⁻² s⁻¹), salinity (25, 30, 35, 40 and 45), and temperature (15, 20, 25, and 30 °C). Maximum growth rates varied from 0.17 to 0.36 div day⁻¹ with exponential growth rates increasing with increasing irradiance. Little or no growth was supported at 19 μmol photons m⁻² s⁻¹ for any experiment. Maximum growth rates at 15 °C were much lower than at other temperatures. Maximum growth rates of the Texas clone (SP3) fell within the range of Florida clones reported in the literature (0.17–0.36 div day⁻¹ versus 0.2–1.0 div day⁻¹). The Texas clone SP3 had a very similar light saturation point compared to that of a Florida isolate (Wilson's clone) (67 μmol m⁻² s⁻¹ versus 65 μmol m⁻² s⁻¹), and light compensation (20–30 μmol m⁻² s⁻¹1). The upper and lower salinity tolerance of the Texas clone was similar than that of some Florida clones (45 versus 46 and 25 versus 22.5, respectively). In our study, the Texas clone had the same temperature tolerance reported for Florida clones (15–30 °C). While individual clones can vary considerably in maximum growth rates, our results indicate only minor differences exist between the Texas and Florida strains of K. brevis in their temperature and salinity tolerance for growth. While the literature notes lower salinity occurrences of K. brevis in nearby Louisiana, our isolate from the southern Texas coast has the higher salinity requirements typical of K. brevis in the eastern Gulf of Mexico.     

Molecular phylogeny of the genus Stereocaulon (Stereocaulaceae, lichenized ascomycetes)

In the present study phylogenetic relationships of the genus Stereocaulon (lichenized ascomycetes) were examined using DNA sequences from the ITS1–5.8 S–ITS2 rDNA gene cluster and from the protein-coding β-tubulin gene. In addition to the fruticose species traditionally classified in Stereocaulon, representatives of the crustose species that have recently been transferred to the genus were included. Muhria, a monotypic genus that is morphologically similar to Stereocaulon, differing only in apothecia ontogeny, was also incorporated. The analyses included 101 specimens from the ingroup representing 49 taxa. Sequences from both DNA regions were analysed simultaneously using direct optimization under the parsimony optimality criterion. The results support the inclusion of the crustose species and Muhria in Stereocaulon, while the current infrageneric classification is not supported. As Muhria is securely nested within Stereocaulon the new combination Stereocaulon urceolatum comb. nov. (syn. Muhria urceolata) is made. Further, species concepts need to be re-examined, as some species do not appear as monophyletic entities in the phylogeny.