Normal and abnormal appearance of fetal ganglionic eminence on second-trimester three-dimensional ultrasound.

OBJECTIVES: To describe the appearance and size of the ganglionic eminence (GE) in normal fetuses on midtrimester three-dimensional (3D) neurosonography and to report on the association between GE alterations (cavitation or enlargement) and malformation of cortical development (MCD). METHODS: This was a prospective multicenter cohort study of normal fetuses and a retrospective analysis of pathological cases with MCD. From January 2022 to June 2022, patients attending our tertiary centers for an expert fetal brain scan were recruited for the purpose of the study. A 3D volume of the fetal head, starting from the sagittal plane, was acquired in apparently normal fetuses using a transabdominal or transvaginal approach. Stored volume datasets were then evaluated independently by two expert operators. Two measurements (longitudinal diameter and transverse diameter) of the GE in the coronal view were obtained twice by each operator. Intra- and interobserver measurement variation was calculated. Reference ranges for GE measurements were calculated in the normal population. A previously stored volume dataset of 60 cases with MCD was also analyzed independently by the two operators using the same method in order to assess if GE abnormalities (cavitation or enlargement) were present. Postnatal follow-up was obtained in all cases. RESULTS: In the study period, 160 normal fetuses between 19 and 22 weeks of gestation were included in the study. The GE was visible in the coronal plane on 3D neurosonography in 144 (90%) cases and was not clearly visible in the remaining 16 (10%) cases. The intra- and interobserver agreement was almost perfect for the longitudinal diameter, with an intraclass correlation coefficient (ICC) of 0.90 (95% CI, 0.83-0.93) and 0.90 (95% CI, 0.86-0.92), respectively, and substantial for the transverse diameter, with an ICC of 0.80 (95% CI, 0.70-0.87) and 0.64 (95% CI, 0.53-0.72), respectively. A retrospective analysis of 50 cases with MCD in the second trimester showed that GE enlargement was present in 12 cases and GE cavitation was present in four cases. CONCLUSIONS: Systematic assessment of the GE in fetuses at 19-22 weeks of gestation is feasible on 3D neurosonography, with good reproducibility in normal cases. Cavitation or enlargement of the GE can be demonstrated in fetuses with MCD. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Ethanol addition enhances acid treatment to eliminate Lactobacillus fermentum from the fermentation process for fuel ethanol production.

Fermentation is one of the most critical steps of the fuel ethanol production and it is directly influenced by the fermentation system, selected yeast, and bacterial contamination, especially from the genus Lactobacillus. To control the contamination, the industry applies antibiotics and biocides; however, these substances can result in an increased cost and environmental problems. The use of the acid treatment of cells (water-diluted sulphuric acid, adjusted to pH 2·0-2·5) between the fermentation cycles is not always effective to combat the bacterial contamination. In this context, this study aimed to evaluate the effect of ethanol addition to the acid treatment to control the bacterial growth in a fed-batch system with cell recycling, using the industrial yeast strain Saccharomyces cerevisiae PE-2. When only the acid treatment was used, the population of Lactobacillus fermentum had a 3-log reduction at the end of the sixth fermentation cycle; however, when 5% of ethanol was added to the acid solution, the viability of the bacterium was completely lost even after the first round of cell treatment. The acid treatment +5% ethanol was able to kill L. fermentum cells without affecting the ethanol yield and with a low residual sugar concentration in the fermented must. SIGNIFICANCE AND IMPACT OF THE STUDY: In Brazilian ethanol-producing industry, water-diluted sulphuric acid is used to treat the cell mass at low pH (2·0) between the fermentative cycles. This procedure reduces the number of Lactobacillus fermentum from 107 to 104  CFU per ml. However, the addition of 5% ethanol to the acid treatment causes the complete loss of bacterial cell viability in fed-batch fermentation with six cell recycles. The ethanol yield and yeast cell viability are not affected. These data indicate the feasibility of adding ethanol to the acid solution replacing the antibiotic use, offering a low cost and a low amount of residue in the biomass.

The endocannabinoid 2-arachidonoylglycerol dysregulates the synthesis of proteins by the human syncytiotrophoblast.

In recent years, endocannabinoids emerged as new players in various reproductive events. Recently, we demonstrated the involvement of 2-arachidonoylglycerol (2-AG) in human cytotrophoblast apoptosis and syncytialization. However, 2-AG impact in hormone production by the syncytiotrophoblast (hST) was never studied. In this work, we demonstrate that 2-AG activates cannabinoid (CB) receptors, exerting an inhibitory action on cyclic AMP/protein kinase A (cAMP/PKA) and mitogen-activated protein kinase (MAPK) p38 pathways, and enhancing ERK 1/2 phosphorylation. Furthermore, 2-AG affects the synthesis of human chorionic gonadotropin (hCG), leptin, aromatase, 3-ß-hydroxysteroid dehydrogenase (3-ß-HSD), and placental protein 13 (PP13). These 2-AG effects are mediated by the activation of CB receptors, in a mechanism that may involve p38, ERK 1/2 and cAMP/PKA pathways, which participate in the regulation of placental proteins expression. To our knowledge, this is the first study that associates the endocannabinoid signalling and endocrine placental function, shedding light on a role for 2-AG in the complex network of molecules that orchestrate the production of placental proteins essential for the gestational success.

The endocannabinoid anandamide affects the synthesis of human syncytiotrophoblast-related proteins.

The human syncytiotrophoblast (hST) has a major role in the production of important placental hormones. Several molecules regulate hST endocrine function but the role of endocannabinoids in this process is still unknown. Here, we report that the endocannabinoid anandamide (AEA) decreased cAMP levels, impaired human chorionic gonadotropin secretion, placental alkaline phosphatase activity and decreased aromatase mRNA levels and protein expression, through cannabinoid (CB) receptor activation. AEA also downregulated leptin and placental protein 13 transcription, though via a CB receptor-independent mechanism. All this evidence suggests AEA is a novel modulator of hormone synthesis by the syncytiotrophoblast, supporting the importance of the endocannabinoid signalling in placental function.

Rat spontaneous foetal resorption: altered α2-macroglobulin levels and uNK cell number.

During rat pregnancy, some of the foetoplacental units undergo complete spontaneous resorption while the adjacent units remain unaffected. In an attempt to clarify the mechanisms implicated in this spontaneous resorption, implantation units from days 14 and 16 of pregnancy were examined. The number of implantation sites and resorption units was recorded, and uterine paraffin sections were stained with haematoxylin and eosin for the evaluation of tissue morphology. The incidence of resorption was about 9.2 % on day 14 and 8.2 % on day 16. Perforin and active caspase-3 immunostaining were performed for localization and characterization of uterine natural killer (uNK) and apoptotic cells, respectively. The α2-macroglobulin (α2-MG) expression was examined by in situ hybridization, immunohistochemistry and its levels quantified by enzyme-linked immunosorbent assay. A reduction in α2-MG decidual levels in resorpted units was observed when compared to normal implantation units in both days. This potent protease inhibitor is the major product secreted by the mesometrial decidual tissue and may constitute an indicator of maternal tissues remodelling abnormalities. Besides the decreased α2-MG levels, an increase in uNK cell number was found in resorption units. The decreased α2-MG levels may be related to the aberrant control of trophoblast invasion that may activate uNK cells. The elucidation of the mechanisms underlying natural pregnancy loss in rat may contribute for the clarification of the "vanishing twin" phenomenon that occurs in human pregnancy.

2-arachidonoylglycerol effects in cytotrophoblasts: metabolic enzymes expression and apoptosis in BeWo cells.

The major endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a member of the endocannabinoid system (ECS) that participates in cell proliferation and apoptosis, important events for the homoeostasis of biological systems. The formation of placenta is one of the most important stages of pregnancy and its development requires highly regulated proliferation, differentiation and apoptosis of trophoblasts. Anomalies in these processes are associated with gestational pathologies. In this work, we aimed to study the involvement of 2-AG in cytotrophoblast cell turnover. We found that 2-AG biosynthetic (diacylglycerol lipase A) and degradative (monoacylglycerol lipase) enzymes are expressed in human cytotrophoblasts and in BeWo cells. We also found that 2-AG induces a decrease in cell viability in a time- and concentration-dependent manner and exerts antiproliferative effects. The loss of cell viability induced by a 48-h treatment with 2-AG (10 µM) was accompanied by chromatin fragmentation and condensation, morphological features of apoptosis. Additionally, 2-AG induced an increase in caspase 3/7 and 9 activities, a loss of mitochondrial membrane potential (Δψm) and an increase in reactive oxygen species (ROS)/reactive nitrogen species (RNS) generation, suggesting the activation of the mitochondrial pathway. Moreover, whereas Δψm loss and ROS/RNS generation were significantly attenuated by the antagonists of both the cannabinoid receptors 1 and 2 (CB1 and CB2), the increase in caspase 3/7 and 9 activities and loss of cell viability were reversed only by the antagonist of CB2 receptor; the blockage of the eCB membrane transporter and the depletion of cholesterol failed to reverse the effects of 2-AG. Therefore, this work supports the importance of cannabinoid signalling during cytotrophoblast cell turnover and that its deregulation may be responsible for altered placental development and poor pregnancy outcomes.

Genetic diversity of Angiopolybia pallens (Lepeletier) (Hymenoptera, Vespidae, Polistinae) explained by the disjunction of South American rainforests.

Angiopolybia pallens is a swarm-founding wasp that occurs in Atlantic and Amazonian rainforests, which have been isolated from each other by a large arid corridor in central Brazil since the Tertiary. In this study, we used data from polymerase chain reaction-restriction fragment length polymorphism of 16S rDNA to infer some aspects of the genetic diversity and evolutionary history of this social wasp population. Samples collected across the species range in both rainforests showed remarkable differences that separated them into two distinct haplogroups. However, some samples from isolated areas in northeastern Bahia were more related to Amazonian lineages than to southern Bahia populations. Vicariant divergenc is discussed under South American rainforests biogeographical proposals.

Specialist nurse in Europe: education, regulation and role.

BACKGROUND: The concept of a 'specialist nurse' has existed for many years and related education programmes are proliferating. However, while literature clearly outlines the roles and practice of registered nurses and advanced practice nurses, those of specialist nurses remain unclear and nursing specializations across Europe need clarifying. AIM: This pilot study aimed to explore the competencies, education requirements and regulation of specialist nurses in Europe. DESIGN: A descriptive cross-sectional survey. METHODS: An online questionnaire named 'Specialist nurse and specialization in Europe' was sent to 550 members of the European Federation of Nurse Educators and ten members of the European Specialist Nurses Organizations. Snowball sampling was then used to build a convenience sample of nurse educators, clinical nurses and specialist nurses, national nursing association members, and chief nursing officers from all European countries. Besides quantitative aspects, responses to open-ended questions were analysed using a qualitative content analysis process. RESULTS: A total of 77 experts from 29 European countries responded to the questionnaire. Findings highlighted variations in titles, levels and length of education, certification, regulation and scope of practice for specialized nurses in Europe. Analysis of the promoted competencies revealed dominant clinical and technical aspects of the role with a high level of knowledge. CONCLUSIONS: The study emphasized the need to improve standards for education, certification and regulation for specialist nurses. Interpretation of the role and competencies is diverse with a weak presence of health policy that would enhance and develop the specialities. IMPLICATIONS FOR NURSING AND HEALTH POLICY: To address the current lack of provisions for automatic recognition of specialist nurses, common training frameworks corresponding to the relevant level of the European Qualifications Framework should promote lifelong learning and mobility, and enhance levels of health care and patient safety.

Endogenous cannabinoids revisited: a biochemistry perspective.

Marijuana is the most commonly used illegal drug, particularly in Western societies. The discovery of an endogenous cannabinoid system (ECS) highlighted new molecules in various physiological processes. The ECS consists of G-protein-coupled cannabinoid receptors that can be activated by small lipid mediators, termed endocannabinoids (eCBs) and cannabis-derived drugs, plus the associated biochemical machinery (precursors, synthesis and degradative enzymes, and transporters). Several biochemical, pharmacological and physiological studies have shown that endocannabinoid system elements are widely distributed throughout the body, with regional variations and organ-specific actions. This review portrays the endocannabinoid "family" on new studies concerning eCB storage, release and functional roles and on the growing importance of its bioactive metabolites. Those findings reinforce and confirm the importance of ECS. Strategies for manipulating the system for the treatment of human disease will require a thorough understanding of the roles of the different eCBs and their sources.

Molecular cytogenetics of nucleolar organizer regions in Phyllomedusa and Phasmahyla species (Hylidae, Phyllomedusinae): a cytotaxonomic contribution.

Chromosome numbers, morphology, and nucleolus organizer region (NOR) locations are useful cytological characters for taxonomic and evolutionary studies. In this study, we provide the first cytogenetic analysis of Phyllomedusa bahiana and Phasmahyla spectabilis, and report new cytogenetic data on variation in NOR numbers and positions in Phyllomedusa rohdei and Phyllomedusa nordestina using conventional staining, AgNOR-banding, and 45S rDNA fluorescence in situ hybridization. All 4 species showed 2n = 26 chromosomes. P. spectabilis and P. bahiana had only 1 pair of NOR-carrying chromosomes. P. bahiana showed an NOR length polymorphism, and a rare homomorphic self-compatibility for both NOR lengths in anurans. Variation in the number of NOR-bearing chromosomes was found between the sampled populations of P. nordestina, ranging from 3 to 4. This study also clarified previous conflicting results concerning the occurrence of inter- and intra-population NOR variation in P. rohdei. The variation, confirmed by 45S rDNA fluorescence in situ hybridization analysis, was congruent with results obtained from AgNOR-banding in all species.

Mitochondrial DNA variability in populations of Centris aenea (Hymenoptera, Apidae), a crop-pollinating bee in Brazil.

Centris spp are oil-collecting solitary bees that are valuable pollinators of crops such as Brazil nut, cashew, and acerola. We investigated the genetic variability of populations of C. aenea in the northeastern region of Brazil. Total DNA was extracted from 59 individuals from 6 locations in the States of Pernambuco and Bahia and a 600-650-bp fragment of the mitochondrial COI/COII region amplified by PCR, followed by digestion with the restriction enzymes DraI and SspI. PCR-RFLP analysis revealed eight different haplotypes among the populations. Haplotype A1, revealed by DraI, was the most frequent (50%), and haplotypes A3 and A4 were exclusive to Feira de Santana, Bahia and Morro do Chapéu, Bahia, respectively. Among the haplotypes revealed by SspI, B2 was the most frequent (37%) and B3 was exclusive to Feira de Santana. This information revealing high haplotype diversity will be useful for developing management strategies for Centris, especially because of increasing interest in the rearing and/ or relocation of these bees for crop pollination.

Role of ecto-NTPDases on UDP-sensitive P2Y(6) receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women.

This study aimed at investigating the expression and function of uracil nucleotide-sensitive receptors (P2Y(2), P2Y(4), and P2Y(6)) on osteogenic differentiation of human bone marrow stromal cells (BMSCs) in culture. Bone marrow specimens were obtained from postmenopausal female patients (68 ± 5 years old, n = 18) undergoing total hip arthroplasty. UTP and UDP (100 µM) facilitated osteogenic differentiation of the cells measured as increases in alkaline phosphatase (ALP) activity, without affecting cell proliferation. Uracil nucleotides concentration-dependently increased [Ca(2+)](i) in BMSCs; their effects became less evident with time (7 > 21 days) of the cells in culture. Selective activation of P2Y(6) receptors with the stable UDP analog, PSB 0474, mimicked the effects of both UTP and UDP, whereas UTPγS was devoid of effect. Selective blockade of P2Y(6) receptors with MRS 2578 prevented [Ca(2+)](i) rises and osteogenic differentiation caused by UDP at all culture time points. BMSCs are immunoreactive against P2Y(2), P2Y(4), and P2Y(6) receptors. While the expression of P2Y(6) receptors remained fairly constant (7∼21 days), P2Y(2) and P2Y(4) became evident only in less proliferative and more differentiated cultures (7 < 21 days). The rate of extracellular UTP and UDP inactivation was higher in less proliferative and more differentiated cell populations. Immunoreactivity against NTPDase1, -2, and -3 rises as cells differentiate (7 < 21 days). Data show that uracil nucleotides are important regulators of osteogenic cells differentiation predominantly through the activation of UDP-sensitive P2Y(6) receptors coupled to increases in [Ca(2+)](i) . Endogenous actions of uracil nucleotides may be balanced through specific NTPDases determining whether osteoblast progenitors are driven into proliferation or differentiation.

Characterization of a Par j 1/Par j 2 mutant hybrid with reduced allergenicity for immunotherapy of Parietaria allergy.

BACKGROUND: Parietaria pollen is one of the major cause of pollinosis in the southern Europe. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease restoring a normal immunity against allergens. METHODS: We designed a recombinant molecule (PjEDloop1) comprised of genetic-engineered variants of the major allergens of the Parietaria pollen (Par j 2/Par j 1). Purity and chemical-physical properties of the derivative were analysed by RP-HPLC chromatography and Photon Correlation Spectroscopy. Immunological activity was evaluated by means of Western blotting, ELISA inhibition and PBMC proliferation assay in 10 Parietaria allergic patients. Basophil activation was studied in six subjects. The immunogenicity of the hybrid was studied looking at the immune responses induced in a mouse model of sensitization. RESULTS: The PjEDloop1 hybrid was produced as a purified recombinant protein with high stability in solution. Western blot, ELISA inhibition and basophil activation test showed that the PjEDloop1 displays a remarkable reduced IgE binding and anaphylactic activity. CD3 reactivity was conserved in all patients. Mice immunization with the rPjEDloop1 induced antibodies and T cell responses comparable to that obtained by the wild type allergens. Such antibodies shared the specificities to rPar j 1 and rPar j 2 with human IgE antibodies. CONCLUSION: Our results demonstrated that a mutant hybrid expressing genetically engineered forms of the major P. judaica allergens displayed reduced allergenicity and retained T cell reactivity for the induction of protective antibodies in vaccination approaches for the treatment of Parietaria pollinosis.

Molecular genetic analysis of the yellow-breasted capuchin monkey: recommendations for ex situ conservation.

The yellow-breasted capuchin monkey, Cebus xanthosternos, is one of the most endangered species of the Brazilian Atlantic Forest. In situ conservation for this species is problematic due to habitat destruction; therefore, captive conservation has been considered as an alternative strategy. A Studbook for C. xanthosternos has been kept for more than 20 years; however, no genetic data has been collected. Our aim was to provide a preliminary assessment of the genetic variability of C. xanthosternos in captivity in Brazil and compare it with data from the wild. Microsatellite and mtDNA sequencing were carried out in 40 samples from five Brazilian institutions registered in the international Studbook and compared with 8 samples collected in a wild population from REBIO-Una/BA. DNA for analysis was extracted from hair, feces and blood. Our results showed that two of the five captive groups assessed had a genetic variability comparable to wild animals. However, the other three groups apparently require urgent management to improve its genetic variability. Considering that inbreeding effects are more pronounced in captivity due to lack of gene flow, our data indicate a need to increase population size by introducing newly rescued individuals into these captive groups. Our results are the first attempt to provide genetic information for captive C. xanthosternos in Brazil.

Physical location of the carotenoid biosynthesis genes Psy and ß-Lcy in Capsicum annuum (Solanaceae) using heterologous probes from Citrus sinensis (Rutaceae).

Carotenoids are responsible for a range of fruit colors in different hot pepper (Capsicum) varieties, from white to deep red. Color traits are genetically determined by three loci, Y, C1, and C2, which are associated with carotenogenic genes. Although such genes have been localized on genetic maps of Capsicum and anchored in Lycopersicon and Solanum, physical mapping in Capsicum has been restricted to only a few clusters of some multiple copy genes. Heterologous probes from single copy genes have been rarely used. Fluorescent in situ hybridization was performed in Capsicum annuum varieties with different fruit colors, using heterologous probes of Psy and ß-Lcy genes obtained from a BAC library of the sweet orange (Citrus sinensis). The probes hybridized in the terminal portion of a chromosome pair, confirming the location of these genes in genetic maps. The hybridized segments showed variation in size in both chromosomes.

Genotyping in the Brazilian Criollo Horse Stud Book: resources and perspectives.

The goal of this research was to evaluate the ability of the genotyping information available in the Brazilian Criollo Horse Stud Book to describe the genetic variability of the breed and the exclusion probability determined in comparative tests. Altogether, two softwares were used in the analyses of the available genotypes: Cervus 3.0.3 and Genepop 4.0. Eight microsatellite markers totaled 109 alleles, with an average of 13.6 +/- 0.6 alleles per locus. Large differences between expected and observed heterozygosity were ubiquitous (0.821 +/- 0.07 and 0.470 +/- 0.17, respectively). Although the estimated null allele frequency caused initial concern (0.284 +/- 0.199), it is likely that it was a reflection of the inbreeding coefficients found (0.432 +/- 0.184). All loci showed significant deviation from Hardy-Weinberg equilibrium, with heterozygote deficit (P < 0.0001) and genotypic linkage disequilibrium with at least one marker. The high polymorphic information content (0.798 +/- 0.088) could not warrant exclusion power for three loci (HMS7, HMS6 and HTG4) above 50% (0.491 +/- 0.158). However, combined exclusion probability reached 99.61%, a level close to ideal. The results demonstrate the excellent performance of the markers assessed in describing the genetic status of the breed and suggest the considerable ability to establish parentage.

Adenosine A1 and A2a receptors modulate the nitrergic system in cell culture from dorsomedial medulla oblongata.

Adenosine and nitric oxide act on the fine-tuning regulation of neural cardiovascular control in the nucleus tractus solitarius (NTS). Although the interaction between adenosine and NO is well known in the periphery, the mechanisms by which adenosine interferes in the dynamics of nitrergic neurotransmission, related to neural control of circulation, are not completely understood and might be relevant for individuals predisposed to hypertension. In this study we evaluate the interaction between adenosinergic and nitrergic systems in cell culture from the dorsomedial medulla oblongata of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Using quantification of nitrite levels, RT-PCR analysis and RNA interference we demonstrate that adenosine A1 (A1R) and A2a receptor (A2aR) agonists induce a concentration-dependent decrease and increase of nitrite and nNOS mRNA levels in cultured cells from WKY and SHR, respectively. These effects in nitrite levels are attenuated by the administration of A1R and A2aR selective antagonists, CPT and ZM 241385. Furthermore, knockdown of A1R and A2aR show an increase and decrease of nNOS mRNA levels, respectively. Pretreatment with the nonselective inhibitor of NOS, L-NAME, abolishes nitrite-increased levels triggered by CGS 21680 in WKY and SHR cells. Finally, it is shown that the cAMP-PKA pathway is involved in A1R and A2aR-mediated decrease and increase in nitrite levels in SHR and WKY cells. Our results highlight the influence of adenosine on nitric oxide levels in cultured cells from dorsal medulla oblongata of neonate WKY and SHR rats. In part, the modulatory profile is different in the SHR strain.

Occurrence of multiple nucleolus organizer regions and intraspecific karyotype variation in Scaptotrigona xanthotricha Moure (Hymenoptera, Meliponini).

Scaptotrigona xanthotricha has a wide geographic distribution in the Brazilian Atlantic rainforest. One population from southeast and two from northeast Brazil were analyzed and were found to have chromosome polymorphisms. Although the chromosome number 2n = 34 is conserved in this species, karyotypic analysis revealed clear differences between the three populations. Congruent and ubiquitous multiple nucleolus organizer regions, heterochromatin and CMA(3)-positive blocks were found. The variations suggest that this species is in a process of genetic differentiation. This differentiation process might have been enhanced by restricted nesting preferences, combined with recent extensive fragmentation of the Atlantic rainforest, which limits gene flow between populations.

Leaf carbon assimilation and molecular phylogeny in Cattleya species (Orchidaceae).

We examined leaf CO(2) assimilation and how it varied among species within the orchid genus Cattleya. Measurements of CO(2) assimilation and maximum quantum yield of PS II (Fv/Fm) were made for mature leaves of nine species using a portable system for photosynthesis measurement and a portable fluorometer. Leaf area was measured with an area meter, and the specific leaf mass was determined. DNA of nine Cattleya species and two species of Hadrolaelia was extracted using the CTAB protocol. Each sample was amplified and sequenced using primers for the trnL gene. The phylogenetic analyses, using neighbor-joining and maximum parsimony methods, retrieved a group that included Cattleya and Hadrolaelia species, in which the unifoliate species were separated from the bifoliates. The topologies of the two cladograms showed some similarities. However, C. guttata (bifoliate) was placed in the unifoliate clade in the neighbor-joining tree, while C. warneri (unifoliate) was not placed in this clade in the maximum parsimony tree. Most Cattleya species keep the leaf stomata closed from 6 am to 4 pm. We suggest that C. elongata, C. tigrina and C. tenuis have C(3)-crassulacean acid metabolism since they open their stomata around 12 am. The Fv/Fm values remained relatively constant during the measurements of CO(2) assimilation. The same was observed for the specific leaf mass values, although great variations were found in the leaf area values. When the species were grouped using molecular data in the neighbor-joining analysis, no relation was observed with CO(2) assimilation.