Activation of C-C motif chemokine receptor 2 modulates testicular macrophages number, steroidogenesis, and spermatogenesis progression.

The monocyte chemoattractant protein 1 (MCP-1) belongs to the CC chemokine family and acts in the recruitment of C-C motif chemokine receptor 2 (CCR2)-positive immune cell types to inflammation sites. In testis, the MCP-1/CCR2 axis has been associated with the macrophage population's functional regulation, which presents significant functions supporting germ cell development. In this context, herein, we aimed to investigate the role of the chemokine receptor CCR2 in mice testicular environment and its impact on male sperm production. Using adult transgenic mice strain that had the CCR2 gene replaced by a red fluorescent protein gene, we showed a stage-dependent expression of CCR2 in type B spermatogonia and early primary spermatocytes. Several parameters related to sperm production were reduced in the absence of CCR2 protein, such as Sertoli cell efficiency, meiotic index, and overall yield of spermatogenesis. Daily sperm production decreased by almost 40%, and several damages in the seminiferous tubules were observed. Significant reduction in the expression of important genes related to the Sertoli cell function (Cnx43, Vim, Ocln, Spna2) and meiosis initiation (Stra8, Pcna, Prdm9, Msh5) occurred in comparison to controls. Also, the number of macrophages significantly decreased in the absence of CCR2 protein, along with a disturbance in Leydig cell steroidogenic activity. In summary, our results show that the non-activation of the MCP-1/CCR2 axis disturbs the testicular homeostasis, interfering in macrophage population, meiosis initiation, blood-testis barrier function, and androgen synthesis, leading to the malfunction of seminiferous tubules, decreased testosterone levels, defective sperm production, and lower fertility index.

The identification of the Rosa S-locus and implications on the evolution of the Rosaceae gametophytic self-incompatibility systems.

In Rosaceae species, two gametophytic self-incompatibility (GSI) mechanisms are described, the Prunus self-recognition system and the Maleae (Malus/Pyrus/Sorbus) non-self- recognition system. In both systems the pistil component is a S-RNase gene, but from two distinct phylogenetic lineages. The pollen component, always a F-box gene(s), in the case of Prunus is a single gene, and in Maleae there are multiple genes. Previously, the Rosa S-locus was mapped on chromosome 3, and three putative S-RNase genes were identified in the R. chinensis 'Old Blush' genome. Here, we show that these genes do not belong to the S-locus region. Using R. chinensis and R. multiflora genomes and a phylogenetic approach, we identified the S-RNase gene, that belongs to the Prunus S-lineage. Expression patterns support this gene as being the S-pistil. This gene is here also identified in R. moschata, R. arvensis, and R. minutifolia low coverage genomes, allowing the identification of positively selected amino acid sites, and thus, further supporting this gene as the S-RNase. Furthermore, genotype-phenotype association experiments also support this gene as the S-RNase. For the S-pollen GSI component we find evidence for multiple F-box genes, that show the expected expression pattern, and evidence for diversifying selection at the F-box genes within an S-haplotype. Thus, Rosa has a non-self-recognition system, like in Maleae species, despite the S-pistil gene belonging to the Prunus S-RNase lineage. These findings are discussed in the context of the Rosaceae GSI evolution. Knowledge on the Rosa S-locus has practical implications since genes controlling floral and other ornamental traits are in linkage disequilibrium with the S-locus.

Lights4Violence: a quasi-experimental educational intervention in six European countries to promote positive relationships among adolescents.

BACKGROUND: Preventing intimate partner violence or dating violence (DV) among adolescents is a public health priority due to its magnitude and damaging short and long-term consequences for adolescent and adult health. In our study protocol, we complement prior experiences in DV prevention by promoting protective factors (or assets) against gender violence such as communication skills, empathy and problem-solving capability through "Cinema Voice", a participatory educational intervention based on adolescents' strengths to tackle DV. METHODS/DESIGN: A longitudinal quasi-experimental educational intervention addressed to boys and girls ages 13-17 years, enrolled in secondary education schools in Alicante (Spain), Rome (Italy), Cardiff (UK), Iasi (Romania), Poznan (Poland) and Matosinhos (Portugal). Both process and results evaluations will be carried out with 100-120 intervention and 120-150 control group students per city at three time periods: before, after and 6 months after the implementation of the following interventions: 1) Training seminar with teachers to promote knowledge and skills on the core issues of intervention; 2) Workshops with intervention groups, where participants produce their own digital content presenting their perspective on DV; and 3) Short film exhibitions with participants, their families, authorities and other stakeholders with the objective of share the results and engage the community. Outcome measures are self-perceived social support, machismo, sexism, tolerance towards gender violence, social problem-solving and assertiveness as well as involvement in bullying/cyberbullying. Other socio-demographic, attitudes and violence-related co-variables were also included. DISCUSSION: This study may provide relevant information about the effectiveness of educational interventions that combine a positive youth development framework with educational awareness about the importance of achieving gender equality and preventing and combating gender violence. To our knowledge, this is the first study that involves six European countries in an educational intervention to promote violence protective assets among enrolled adolescents in secondary schools. This study may provide the needed tools to replicate the experience in other contexts and other countries. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03411564 . Unique Protocol ID: 776905. Date registered: 18-01-2018.

Do different tendons exhibit the same response following chronic exposure to statins?

Over the past few years, a number of cases of tendon injuries associated with statin therapy have been reported. In this study, we assessed whether statins can affect the extracellular matrix (ECM) of the deep digital flexor tendon (DDFT) and patellar tendon (PT). Wistar rats were assigned to groups treated with atorvastatin (A20, A80), treated with simvastatin (S20, S80), and control. Zymography, Western blotting for collagen I, non-collagenous proteins (NCP), glycosaminoglycans (GAGs), and hydroxyproline quantifications were performed. DDFT findings: NCP were increased in A20 and A80; higher concentration of hydroxyproline was found in S80; levels of GAGs was increased in all statin-treated groups; collagen I was increased in S80 and pro-MMP-2 activity was reduced in A80, S20, and S80. PT findings: NCP were reduced in A20, A80, and S80; GAGs was reduced in A80 and S20; collagen I was increased in A20 and pro-MMP-2 activity was reduced in the S20. Both the statins provoked marked changes in both tendons. All these changes may make the tendons more prone to microdamage and ruptures. Therefore, a better understanding of the behavior of the tendon ECM components under statin therapy may provide important insights into the mechanisms behind statin-induced tendon injuries.

Extracts of Caesalpinia ferrea and Trichoderma sp. on the control of Colletotrichum sp. transmission in Sideroxylon obtusifolium seeds / Extratos de Caesalpinia ferrea e Trichoderma spp. no controle da tramissibilidade de Colletotrichum sp. em sementes de Sideroxylon obtusifolium (Sapotaceae)

ABSTRACT Recent research reports the importance of preserving plants in Brazilian semiarid regions, in this context, the scientific literature has reported different pharmacological studies from plant extracts with an antifungal potential, coming from forest species that can contribute as a control and management strategy in the transmission of phytopathogens. This study aimed to evaluate the effect of biotech treatments in controlling the transmission of Colletotrichum sp. in seeds of S. obtusifolium. In this study, 100 seeds were subjected to the following preventive treatments: fungicide Captan®, extract of Caesalpinia ferrea Mart. Ex. Tul., and biological control with Trichoderma spp. The biological control with Trichoderma spp. and the alternative control using C. ferrea extract provided a greater protection to seeds and seedlings of S. obtusifolium facing the transmissibility of Colletotrichum sp.The treatment based on plant extract is more efficient for this purpose only in large seeds and does not interfere on the germination percentage and speed. Therefore it is necessary to perform other studies with Trichoderma spp. and C. ferrea extract to test different doses of these products.

RESUMO Recentes pesquisas relatam a importância da preservação de plantas do semiárido brasileiro. Neste contexto, a literatura científica tem relatado diferentes estudos farmacológicos com extratos vegetais com potencial antifúngico proveniente de espécies florestais que podem contribuir como estratégia de controle e gerenciamento na transmissão de fitopatógenos. No presente estudo o objetivo foi avaliar o efeito de tratamentos biotecnológicos no controle da transmissibilidade de Colletotrichum sp. em sementes de S. obtusifolium. Neste estudo foram utilizadas 100 sementes submetidas aos seguintes tratamentos preventivos: fungicida Captan®, extrato de Caesalpinia ferrea Mart. Ex. Tul. e controle biológico com Trichoderma spp. O controle biológico com Trichoderma spp. e o alternativo com extrato de C. ferrea proporcionam maior proteção às sementes e plântulas S. obtusifolium quanto a transmissibilidade do Colletotrichum sp. O tratamento à base de extrato vegetal foi o mais eficiente para este fim, apenas em sementes de maior tamanho, por não interferir na porcentagem e velocidade de germinação. Portanto, faz-se necessário à realização de outros trabalhos com Trichoderma spp. e extrato de C. ferrea para testar doses diferentes desses produtos.

Kefir Grains Change Fatty Acid Profile of Milk during Fermentation and Storage.

Several studies have reported that lactic acid bacteria may increase the production of free fatty acids by lipolysis of milk fat, though no studies have been found in the literature showing the effect of kefir grains on the composition of fatty acids in milk. In this study the influence of kefir grains from different origins [Rio de Janeiro (AR), Viçosa (AV) e Lavras (AD)], different time of storage, and different fat content on the fatty acid content of cow milk after fermentation was investigated. Fatty acid composition was determined by gas chromatography. Values were considered significantly different when p<0.05. The highest palmitic acid content, which is antimutagenic compost, was seen in AV grain (36.6g/100g fatty acids), which may have contributed to increasing the antimutagenic potential in fermented milk. Higher monounsaturated fatty acid (25.8 g/100g fatty acids) and lower saturated fatty acid (72.7 g/100g fatty acids) contents were observed in AV, when compared to other grains, due to higher Δ9-desaturase activity (0.31) that improves the nutritional quality of lipids. Higher oleic acid (25.0 g/100g fatty acids) and monounsaturated fatty acid (28.2g/100g fatty acids) and lower saturated fatty acid (67.2g/100g fatty acids) contents were found in stored kefir relatively to fermented kefir leading to possible increase of antimutagenic and anticarcinogenic potential and improvement of nutritional quality of lipids in storage milk. Only high-lipidic matrix displayed increase polyunsaturated fatty acids after fermentation. These findings open up new areas of study related to optimizing desaturase activity during fermentation in order to obtaining a fermented product with higher nutritional lipid quality.

Effect of Calendula officinalis cream on achilles tendon healing.

In recent years, the scientific community has undertaken research on plant extracts, searching for compounds with pharmacological activities that can be used in diverse fields of medicine. Calendula officinalis L. is known to have antioxidant, anti-inflammatory, antibacterial, and wound healing properties when used to treat skin burns. Therefore, the purpose of this study was to analyze the effects of C. officinalis on the initial phase of Achilles tendon healing. Wistar rats were separated in three groups: Calendula (Cal)-rats with a transected tendon were treated with topical applications of C. officinalis cream and then euthanized 7 days after injury; Control (C)-rats were treated with only vehicle after transection; and Normal (N)-rats without tenotomy. Higher concentrations of hydroxyproline (an indicator of total collagen) and non-collagenous proteins were observed in the Cal group in relation to the C group. Zymography showed no difference in the amount of the isoforms of metalloproteinase-2 and of metalloproteinase-9, between C and Cal groups. Polarization microscopy images analysis showed that the Cal group presented a slightly higher birefringence compared with the C group. In sections of tendons stained with toluidine blue, the transected groups presented higher metachromasy as compared with the N group. Immunocytochemistry analysis for chondroitin-6-sulfate showed no difference between the C and Cal groups. In conclusion, the topical application of C. officinalis after tendon transection increases the concentrations of collagen and non-collagenous proteins, as well as the collagen organization in the initial phase of healing.

Green tea and glycine aid in the recovery of tendinitis of the Achilles tendon of rats.

PURPOSE: Green tea (GT) is widely used due to its anti-inflammatory properties. Previous studies have shown beneficial effects of a glycine diet on the remodeling process in inflamed tendons. Tendinitis is commonly observed in athletes and is of concern to surgeons due to the slowness of the recovery process. Our hypothesis is that GT + a glycine diet may improve tendinitis. AIM OF THE STUDY: To analyze the effect of GT and/or glycine in the diet on tendinitis. MATERIALS AND METHODS: Wistar rats were divided into seven groups (G): control group (C); G1 and G4, tendinitis; G2 and G5, tendinitis supplied with GT; and G3 and G6, tendinitis supplied with GT and a glycine diet for 7 or 21 days, respectively. We performed zymography for metalloproteinase, biochemical, morphological and biomechanics tests. RESULTS: G2, G3 and G5 showed high levels of hydroxyproline in relation to G1, while G4 showed high levels of glycosaminoglycans. High activity of metalloproteinase-2 was detected in G3. The organization of collagen bundles was better in G2 and G3. G5 showed similar birefringence measurements compared with C. G5 withstood a larger load compared with G4. CONCLUSIONS: The presence of metalloproteinase-2 indicates that a tissue is undergoing a remodeling process. High birefringence suggests a better organization of collagen bundles. After 21 days, G5 sustained a high load before rupture, unlike G4. The results suggest that GT + a glycine diet has beneficial effects that aid in the recovery process of the tendon after tendinitis.

Structural and biomechanical changes in the Achilles tendon after chronic treatment with statins.

Cases of tendinopathy and tendon ruptures have been reported as side effects associated with statin therapy. This work assessed possible changes in the structural and biomechanical properties of the tendons after chronic treatment with statins. Wistar rats were divided into the following groups: treated with atorvastatin (A-20 and A-80), simvastatin (S-20 and S-80) and the group that received no treatment (C). The doses of statins were calculated using allometric scaling, based on the doses of 80 mg/day and 20 mg/day recommended for humans. The morphological aspect of the tendons in A-20, S-20 and S-80 presented signals consistent with degeneration. Both the groups A-80 and S-80 showed a less pronounced metachromasia in the compression region of the tendons. Measurements of birefringence showed that A-20, A-80 and S-80 groups had a lower degree of organization of the collagen fibers. In all of the groups treated with statins, the thickness of the epitenon was thinner when compared to the C group. In the biomechanical tests the tendons of the groups A-20, A-80 and S-20 were less resistant to rupture. Therefore, statins affected the organization of the collagen fibers and decreased the biomechanical strength of the tendons, making them more predisposed to ruptures.

Niche evolution and thermal adaptation in the temperate species Drosophila americana.

The study of ecological niche evolution is fundamental for understanding how the environment influences species' geographical distributions and their adaptation to divergent environments. Here, we present a study of the ecological niche, demographic history and thermal performance (locomotor activity, developmental time and fertility/viability) of the temperate species Drosophila americana and its two chromosomal forms. Temperature is the environmental factor that contributes most to the species' and chromosomal forms' ecological niches, although precipitation is also important in the model of the southern populations. The past distribution model of the species predicts a drastic reduction in the suitable area for the distribution of the species during the last glacial maximum (LGM), suggesting a strong bottleneck. However, DNA analyses did not detect a bottleneck signature during the LGM. These contrasting results could indicate that D. americana niche preference evolves with environmental change, and thus, there is no evidence to support niche conservatism in this species. Thermal performance experiments show no difference in the locomotor activity across a temperature range of 15 to 38 °C between flies from the north and the south of its distribution. However, we found significant differences in developmental time and fertility/viability between the two chromosomal forms at the model's optimal temperatures for the two forms. However, results do not indicate that they perform better for the traits studied here in their respective optimal niche temperatures. This suggests that behaviour plays an important role in thermoregulation, supporting the capacity of this species to adapt to different climatic conditions across its latitudinal distribution.

Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells.

Low CD8(+) T lymphocyte numbers have long been described in hereditary haemochromatosis (HH). Recently, two conserved haplotypes localized near the microsatellite D6S105 at the major histocompatibility complex (MHC) class I region were described predicting the clinical expression of HH and the CD8(+) T lymphocyte numbers. The A-A-T haplotype was associated with a severe clinical expression of HH and low CD8(+) T lymphocyte numbers, while the G-G-G haplotype was associated with a milder clinical expression of HH and high CD8(+) T lymphocyte numbers. As CD8(+) T lymphocytes are a very heterogeneous population, in this study we analysed the CD8(+) subpopulations of naive, central memory (T(CM)) and effector memory (T(EM)), and further subsets of CD8(+) T(EM) cells in 47 HH patients and 68 controls. In addition, association studies were conducted between the conserved haplotypes and the CD8(+) T cell subpopulations in HH. Variations of the numbers of naive and central memory cells with age were similar between HH patients and controls. For T(EM) cells and the T(EM) CD27(-)CD28(-) subset no effect of age was observed in HH [R(2) = 0.001, not significant (n.s.) and R(2) = 0.01, n.s., respectively] contrasting with the increasing of these subpopulations with age in controls (R(2) = 0.09, P = 0.017 and R(2) = 0.22, P = 0.0005, respectively). Interestingly, patients homozygous for the A-A-T haplotype have lower numbers of CD8(+) T(EM) cells due especially to lower numbers of T(EM) CD27(-)CD28(-) (0.206 +/- 0.119 and 0.066 +/- 0.067 x 10(6) cells/ml, respectively) than patients carrying the G-G-G haplotype (0.358 +/- 0.195 and 0.246 +/- 0.202 x 10(6) cells/ml, respectively). This may suggest an inability of HH patients to differentiate the CD8(+) T cells into the most mature phenotype.

Comparative analysis of five immunity-related genes reveals different levels of adaptive evolution in the virilis and melanogaster groups of Drosophila.

Five immunity-related genes previously reported to be evolving under positive selection in Drosophila melanogaster and D. simulans have been analysed across the Drosophila genus using two types of approaches, random-site and branch-site likelihood models as well as the proportion of synonymous and non-synonymous variation within and between species. Different selective pressures have been detected in the sample of genes, one showing evidence for adaptive evolution across the phylogeny of Drosophila and two showing lineage-specific positive selection. Furthermore, amino-acid sites identified as being under positive selection in the melanogaster and the virilis groups are different, suggesting that the evolution of the proteins in these two divergent groups may have been shaped by different pathogens.

Inferences on the number and frequency of S-pollen gene (SFB) specificities in the polyploid Prunus spinosa.

In flowering plants, self-incompatibility is a genetic mechanism that prevents self-fertilization. In gametophytic self-incompatibility (GSI), pollen specificity is encoded by the haploid genotype of the pollen tube. In GSI, specificities are maintained by frequency-dependent selection, and for diploid species, at equilibrium, equal specificity frequencies (isoplethy) are expected. This prediction has been tested in diploid, but never in polyploid self-incompatible species. For the latter, there is no theoretical expectation regarding isoplethy. Here, we report the first empirical study on specificity frequencies in a natural population of a polyploid self-incompatible species, Prunus spinosa. A total of 32 SFB (the pollen S gene) putative specificities are observed in a large sample from a natural population. Although P. spinosa is polyploid, the number of specificities found is similar to that reported for other diploid Rosaceae species. Unequal specificity frequencies are observed.

Dynamics and function of intron sequences of the wingless gene during the evolution of the Drosophila genus.

To understand the function and evolution of genes with complex patterns of expression, such as the Drosophila wingless gene, it is essential to know how their transcription is regulated. However, extracting the relevant regulatory information from a genome is still a complex task. We used a combination of comparative genomics and functional approaches to identify putative regulatory sequences in two introns (1 and 3) of the wingless gene and to infer their evolution. Comparison of the sequences obtained from several Drosophila species revealed colinear and well-conserved sequence blocks in both introns. Drosophila willistoni showed a rate of evolution, in both introns, faster than expected from its phylogenetic position. Intron 3 appeared to be composed of two separate modules, one of them lost in the willistoni group. We tested whether sequence conservation in noncoding regions is a reliable indicator of regulatory function and, if this function is conserved, by analyzing D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. melanogaster (Sophophora subgenus) and the species from the Drosophila subgenus presenting the most divergent sequence, D. americana. The analysis indicated that intron 3 contains pupal enhancers conserved during the evolution of the genus, despite the fact that only 30% of the D. melanogaster intron 3 sequences lie in conserved blocks. Additional analysis of D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. willistoni revealed the absence of an abdomen-specific expression pattern, probably due to the above-mentioned loss of a regulatory module in this species.

Evidence for rare recombination at the gametophytic self-incompatibility locus.

The gametophytic self-incompatibility locus has been thought to be a nonrecombining genomic region. Inferences have been made, however, about the functional importance of different parts of the S-locus, based on differences in the levels of variability along the gene, and this is valid only if recombination occurs. It is thus important to test whether recombination occurs within and near the S-locus. Several recent attempts to test this have reached conflicting conclusions. In this study, we examine a large data set on sequence variation at the S-locus in several species with gametophytic self-incompatibility systems, in the Solanaceae, Rosaceae and Scrophulariaceae. We use the longest sequences available to test for recombination based on linkage disequilibrium between polymorphic sites in the S-locus. The relationship between linkage disequilibrium and physical distance between the sites suggests rare intragenic exchange in the evolutionary history of four species of Solanaceae and two species of Rosaceae.

Polymorphic microsatellites in Antirrhinum (Scrophulariaceae), a genus with low levels of nuclear sequence variability.

In Antirrhinum, reproductive systems range from self-compatible to self-incompatible, but the actual outcrossing rates of self-compatible populations are not known. Thus the extent to which levels of variability and inbreeding differ among Antirrhinum populations is not known. In order to address this issue we isolated nine Antirrhinum nuclear microsatellite loci. In contrast to several nuclear genes that show low levels of sequence variation, six of the microsatellite loci indicate high levels of variability within and between Antirrhinum species. The highly self-compatible Antirrhinum majus ssp. cirrhigerum population has high levels of variability and no significant deviation from Hardy-Weinberg equilibrium, suggesting substantial rates of outcrossing.

Molecular variation at the self-incompatibility locus in natural populations of the genera Antirrhinum and Misopates.

The self-incompatibility system of flowering plants is a classic example of extreme allelic polymorphism maintained by frequency-dependent selection. We used primers designed from three published Antirrhinum hispanicum S-allele sequences in PCR reactions with genomic DNA of plants sampled from natural populations of Antirrhinum and Misopates species. Not surprisingly, given the polymorphism of S-alleles, only a minority of individuals yielded PCR products of the expected size. These yielded 35 genomic sequences, of nine different sequence types of which eight are highly similar to the A. hispanicum S-allele sequences, and one to a very similar unpublished Antirrhinum S-like RNase sequence. The sequence types are well separated from the S-RNase sequences from Solanaceae and Rosaceae, and also from most known "S-like" RNase sequences (which encode proteins not involved in self-incompatibility). An association with incompatibility types has so far been established for only one of the putative S-alleles, but we describe evidence that the other sequences are also S-alleles. Variability in these sequences follows the pattern of conserved and hypervariable regions seen in other S-RNases, but no regions have higher replacement than silent diversity, unlike the results in some other species.

Low rates of silent substitution in nuclear genes of two distantly related Scrophulariaceae (Antirrhinum and Verbascum).

Low levels of genetic diversity and divergence at nuclear loci have previously been observed for cycloidea and fil1-like genes within and between several Antirrhinum species, and divergence at these loci is also low between species in genera at different levels of relatedness in the former family Scrophulariaceae (Digitalis and Verbascum). The low divergence values are surprising, because (based on the sequences of chloroplast loci) the Scrophulariaceae are thought to be polyphyletic, with two anciently diverged clades, and the species we compared belonged to the two different clades. Here, we extend our studies of sequence divergence to more nuclear genes: fil2, far, globosa, and ADH: Detailed studies revealed that in Antirrhinum these genes belong to gene families. Low levels of divergence between Antirrhinum and Verbascum were observed for four of the loci studied, fil2-1, fil2-2, far-L, and globosa, similar to our previous observations. We discuss hypotheses to explain these low synonymous divergence values. For Adh, no cases of very similar sequences were found, but, rather, our sequences from the three different genera (Antirrhinum, Digitalis, and Verbascum) were all very diverged. Repeated gene duplication and loss of elements in the Adh gene family is likely in these lineages, making it impossible to determine orthology of the Adh genes.

Low diversity and divergence in the fil1 gene family of Antirrhinum (Scrophulariaceae).

Detailed nucleotide diversity studies revealed that the fil1 gene of Antirrhinum, which has been reported to be single copy, is a member of a gene family composed of at least five genes. In four Antirrhinum majus populations with different mating systems and one A. graniticum population, diversity within populations is very low. Divergence among Antirrhinum species and between Antirrhinum and Digitalis is also low. For three of these genes we also obtained sequences from a more divergent member of the Scrophulariaceae, Verbascum nigrum. Compared with Antirrhinum, little divergence is again observed. These results, together with similar data obtained previously for five cycloidea genes, suggest either that these gene families (or the Antirrhinum genome) are unusually constrained or that there is a low rate of substitution in these lineages. Using a sample of 52 genes, based on two measures of codon usage (ENC and GC3 content), we show that cyc and fil1 are among the least biased Antirrhinum genes, so that their low diversity is not due to extreme codon bias.