Storage protein activator controls grain protein accumulation in bread wheat in a nitrogen dependent manner.

The expression of cereal grain storage protein (GSP) genes is controlled by a complex network of transcription factors (TFs). Storage protein activator (SPA) is a major TF acting in this network but its specific function in wheat (Triticum aestivum L.) remains to be determined. Here we generated an RNAi line in which expression of the three SPA homoeologs was reduced. In this line and its null segregant we analyzed GSP accumulation and expression of GSP and regulatory TF genes under two regimes of nitrogen availability. We show that down regulation of SPA decreases grain protein concentration at maturity under low but not high nitrogen supply. Under low nitrogen supply, the decrease in SPA expression also caused a reduction in the total quantity of GSP per grain and in the ratio of GSP to albumin-globulins, without significantly affecting GSP composition. The slight reduction in GSP gene expression measured in the SPA RNAi line under low nitrogen supply did not entirely account for the more significant decrease in GSP accumulation, suggesting that SPA regulates additional levels of GSP synthesis. Our results demonstrate a clear role of SPA in the regulation of grain nitrogen metabolism when nitrogen is a limiting resource.

High hedgehog signaling is transduced by a multikinase-dependent switch controlling the apico-basal distribution of the GPCR smoothened.

The oncogenic G-protein-coupled receptor (GPCR) Smoothened (SMO) is a key transducer of the hedgehog (HH) morphogen, which plays an essential role in the patterning of epithelial structures. Here, we examine how HH controls SMO subcellular localization and activity in a polarized epithelium using the Drosophila wing imaginal disc as a model. We provide evidence that HH promotes the stabilization of SMO by switching its fate after endocytosis toward recycling. This effect involves the sequential and additive action of protein kinase A, casein kinase I, and the Fused (FU) kinase. Moreover, in the presence of very high levels of HH, the second effect of FU leads to the local enrichment of SMO in the most basal domain of the cell membrane. Together, these results link the morphogenetic effects of HH to the apico-basal distribution of SMO and provide a novel mechanism for the regulation of a GPCR.

A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study.

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

Engrailed, Suppressor of fused and Roadkill modulate the Drosophila GLI transcription factor Cubitus interruptus at multiple levels.

Morphogen gradients need to be robust, but may also need to be tailored for specific tissues. Often this type of regulation is carried out by negative regulators and negative feedback loops. In the Hedgehog (Hh) pathway, activation of patched (ptc) in response to Hh is part of a negative feedback loop limiting the range of the Hh morphogen. Here, we show that in the Drosophila wing imaginal disc two other known Hh targets genes feed back to modulate Hh signaling. First, anterior expression of the transcriptional repressor Engrailed modifies the Hh gradient by attenuating the expression of the Hh pathway transcription factor cubitus interruptus (ci), leading to lower levels of ptc expression. Second, the E-3 ligase Roadkill shifts the competition between the full-length activator and truncated repressor forms of Ci by preferentially targeting full-length Ci for degradation. Finally, we provide evidence that Suppressor of fused, a negative regulator of Hh signaling, has an unexpected positive role, specifically protecting full-length Ci but not the Ci repressor from Roadkill.

Smaug1 membrane-less organelles respond to AMPK and mTOR and affect mitochondrial function.

Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule fluorescence in situ hybridization (FISH) assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 (also known as SAMD4A and SAMD4B, respectively) affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA-binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial complex I inhibition upon exposure to rotenone, but not strong mitochondrial uncoupling upon exposure to CCCP, rapidly induced the dissolution of Smaug1 MLOs. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMP-activated protein kinase (AMPK). Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK-mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins. This article has an associated First Person interview with the first authors of the paper.

Yield of CT angiography in penetrating lower extremity trauma.

PURPOSE: CT angiography (CTA) has become a valuable tool in the assessment of suspected arterial injury in patients with penetrating lower extremity trauma. However, expensive imaging such as CTA should be judiciously utilized to ensure value-based care. We therefore assessed the yield of CTA in this setting at a level-1 trauma unit and correlated it with the clinical history provided. METHODS: A retrospective descriptive study from 1 July 2013 to 31 June 2018 at a 1386-bed, tertiary-level, public-sector teaching hospital in Cape Town, South Africa.. All patients undergoing CTA for suspected arterial injury following penetrating lower extremity trauma were included. The imaging yield of clinically significant arterial injury and the predictive value of specific clinical signs were determined. RESULTS: A total of 983 patients (median age 27 years, 91% male) were included; 90% (886/983) had gunshots, 9% (89/983) stabs, and 1% (8/983) other injuries. Despite an average 13% year-on-year increase in CTA performed, there was no change in the proportion demonstrating arterial injury. Thirty-four percent (23/68) of patients with strong (hard) signs of arterial injury (active pulsatile bleeding, rapidly expanding hematoma, absent pulse, palpable thrill, or audible bruit), 11% (49/459) with moderate (soft) signs (history of an arterial bleed, excessive non-pulsatile bleeding, large non-expanding hematoma, major neurological deficit, diminished but appreciable pulse, and arterial proximity), and 5% (24/456) with no indication for imaging had clinically significant arterial injuries. Significant positive correlations were rapidly expanding hematoma (p = 0.009), an absent pulse (p < 0.001), and a diminished pulse (p < 0.001). Significant negative correlations were proximity to a major artery (p = 0.005) and no clinical indication provided (p < 0.001). CONCLUSION: There is poor correlation between clinical details provided and the presence of arterial injury at our institution. In this context, CTA serves a pivotal role in the definitive identification of arterial injury.

Chest imaging findings of chronic respiratory disease in HIV-infected adolescents on combined anti retro viral therapy.

Early treatment with combination antiretroviral therapy (cART) has improved survival of children perinatally infected with HIV into adolescence. This population is at risk of long term complications related to HIV infection, particularly chronic respiratory disease. Limited data on chest imaging findings in HIV-infected adolescents, suggest that the predominant disease is of small and large airways: predominantly bronchiolitis obliterans or bronchiectasis. Single cases of emphysema have been reported. Lung fibrosis, lymphocytic interstitial pneumonitis, post tuberculous apical fibrocystic changes and malignancies do not feature in this population. Chest radiograph (CXR) is easily accessible and widely used, especially in resource limited settings, such as sub Saharan Africa, where the greatest burden of HIV disease occurs. Lung ultrasound has been described for the diagnosis of pneumonia in children, pulmonary oedema and interstitial lung disease [1-3]. The use of this modality in chronic respiratory disease in adolescents where the predominant finding is small airway disease and bronchiectasis has however not been described. CXR is useful to evaluate structural/post infective changes, parenchymal opacification and nodules, hyperinflation or extensive bronchiectasis. CXR however, is inadequate for diagnosing small airway disease, for which high resolution computed tomography (HRCT) is the modality of choice. Where available, low dose HRCT should be used early in the course of symptomatic disease in adolescents and for follow up in children who are non responsive to treatment or clinically deteriorating. This article provides a pictorial review of the spectrum of CXR and HRCT imaging findings of chronic pulmonary disease in perinatally HIV-infected adolescents on cART and guidelines for imaging.

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused.

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Quantitative CT analysis for bronchiolitis obliterans in perinatally HIV-infected adolescents-comparison with controls and lung function data.

OBJECTIVE: To compare quantitative chest CT parameters in perinatally HIV-infected adolescents with and without bronchiolitis obliterans compared with HIV-uninfected controls and their association with lung function measurements. MATERIALS AND METHODS: Seventy-eight (41 girls) HIV-infected adolescents with a mean age of 13.8 ± 1.65 years and abnormal pulmonary function tests in the prospective Cape Town Adolescent Antiretroviral Cohort underwent contrast-enhanced chest CT on inspiration and expiration. Sixteen age-, sex-, and height-matched non-infected controls were identified retrospectively. Fifty-one HIV-infected adolescents (28 girls) displayed mosaic attenuation on expiration suggesting bronchiolitis obliterans. Pulmonary function tests were collected. The following parameters were obtained: low- and high-attenuation areas, mean lung density, kurtosis, skewness, ventilation heterogeneity, lung mass, and volume. RESULTS: HIV-infected adolescents showed a significantly higher mean lung density, ventilation heterogeneity, mass, and high- and low-attenuation areas compared with non-infected individuals. Kurtosis and skewness were significantly lower as well. HIV-infected adolescents with bronchiolitis obliterans had a significantly lower kurtosis and skewness compared with those without bronchiolitis obliterans. Lung mass and volume showed the strongest correlations with forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), and alveolar volume. Low-attenuation areas below - 950 HU and ventilation heterogeneity showed the strongest correlation with FEV1/FVC (range, - 0.51 to - 0.34) and forced expiratory flow between 25 and 75% of FVC (range, - 0.50 to - 0.35). CONCLUSION: Quantitative chest CT on inspiration is a feasible technique to differentiate perinatally HIV-infected adolescents with and without bronchiolitis obliterans. Quantitative CT parameters correlate with spirometric measurements of small-airway disease. KEY POINTS: • Perinatally HIV-infected adolescents showed a more heterogeneous attenuation of the lung parenchyma with a higher percentage of low- and high-attenuation areas compared with non-infected patients. • Kurtosis and skewness are able to differentiate between HIV-infected adolescents with and without bronchiolitis obliterans using an inspiratory chest CT. • Quantitative CT parameters of the chest correlate significantly with pulmonary function test. Low-attenuation areas and ventilation heterogeneity are particularly associated with spirometric parameters related to airway obstruction.

Risk-Stratification of Children Presenting to Ambulatory Paediatrics with First-Onset Seizures: Should We Order an Urgent CT Brain?

INTRODUCTION: A computed tomography (CT) brain scan is an often-utilised emergency department imaging modality to detect emergent intra-cranial pathology in a child with a first seizure. Identifying children at low risk of having a clinically significant intra-cranial abnormality could prevent unnecessary radiation exposure and contrast/sedation-related risks. OBJECTIVES: To identify clinical variables which could predict clinically significant CT brain abnormalities and use recursive partitioning analysis to define a low-risk group of children in whom emergent CT brain can be deferred. METHODS: Retrospective cross-sectional review of 468 children who underwent emergent CT brain after presenting to a low- and middle-income paediatric emergency department following first seizure. RESULTS: In total 133/468 (28.4%) of CT brain scans had clinically significant abnormalities. Failure to return to neurological baseline and focal neurological deficit persisting >36 h had statistical significance in a multiple regression analysis. Recursive partitioning analysis, applied to a subgroup without suspected tuberculous meningitis (n = 414), classified 153 children aged between 6 months and 5 years, who had a normal neurological baseline, had returned to baseline post-seizure, and were not in status epilepticus, as non-clinically significant scans and 98% were correctly classified. CONCLUSION: Our study re-inforces the American Academy of Neurology recommendation that children with persistent post-ictal abnormal neurological status and/or post-ictal focal deficit be prioritised for emergent CT brain. Having excluded children with suspected tuberculous meningitis, the remaining subgroup aged 6 months to 5 years presenting with a non-status first seizure, normal neurological baseline and return to baseline post-seizure, are at very low risk of having a clinically significant CT brain abnormality.

High-resolution computed tomography features of lung disease in perinatally HIV-infected adolescents on combined antiretroviral therapy.

INTRODUCTION: Chronic lung disease is common in perinatally HIV-infected children as they increasingly surviving into adolescence. There are few data on the radiologic spectrum of disease in this population. METHODS: Contrasted high-resolution computed tomography (HRCT) was performed in ambulatory South African adolescents enrolled in a prospective study of perinatally-infected adolescents aged 9 to 14 years established on combined antiretroviral therapy (cART) and followed for 36 months. Consecutive participants with reduced lung function (defined by a forced expiratory volume in 1 second [FEV1] of <80% normal and/or lung diffusion capacity [DLCO] <80% normal] underwent HRCT. History, clinical, and laboratory data were collected. Two radiologists blinded to clinical data and to each other, reported scans using standardized methodology; a third radiologist resolved discrepancies. RESULTS: Amongst 100 participants undergoing HRCT, median age was 13.8 years (12.8-15.1). The median duration on cART was 8.4 years (IQR = 5.7-9.8). Mosaic attenuation was the most common finding (73%). Of these 71 (91%) demonstrated associated air trapping radiologically consistent with bronchiolitis obliterans. Bronchiectasis occurred in 39% with significant correlation between extent of bronchiectasis and mosaic attenuation (r = 0.57, P < .001). Prior hospitaliszation for childhood pneumonia at any time before enrollment was associated with mosaic attenuation (OR = 3.9, 95%CI, [1.2-12.5]); prior pulmonary tuberculosis (TB) was associated with the combination of mosaic attenuation and bronchiectasis (OR = 4.9, 95%CI, [1.6-15.7]). Most participants (86%) with mosaic attenuation had stage III or IV HIV disease at time of HIV diagnosis (OR = 3.6; [0.9-14.9]). Inter observer agreement between the two readers was good for bronchiectasis (K = 0.71) and moderate for mosaic attenuation (K = 0.51). DISCUSSION: Despite well-controlled HIV and long duration of cART, HRCT changes were common in perinatally HIV-infected adolescents. There was a high prevalence of small airways disease with and without associated bronchiectasis. These changes were associated with prior pulmonary TB or prior severe pneumonia. Strategies to prevent and treat early life respiratory infection must be strengthened to reduce the burden of chronic lung disease in HIV-infected adolescents.

The bZIP transcription factor SPA Heterodimerizing Protein represses glutenin synthesis in Triticum aestivum.

The quality of wheat grain is mainly determined by the quantity and composition of its grain storage proteins (GSPs). Grain storage proteins consist of low- and high-molecular-weight glutenins (LMW-GS and HMW-GS, respectively) and gliadins. The synthesis of these proteins is essentially regulated at the transcriptional level and by the availability of nitrogen and sulfur. The regulation network has been extensively studied in barley where BLZ1 and BLZ2, members of the basic leucine zipper (bZIP) family, activate the synthesis of hordeins. To date, in wheat, only the ortholog of BLZ2, Storage Protein Activator (SPA), has been identified as playing a major role in the regulation of GSP synthesis. Here, the ortholog of BLZ1, named SPA Heterodimerizing Protein (SHP), was identified and its involvement in the transcriptional regulation of the genes coding for GSPs was analyzed. In gel mobility shift assays, SHP binds cis-motifs known to bind to bZIP family transcription factors in HMW-GS and LMW-GS promoters. Moreover, we showed by transient expression assays in wheat endosperm that SHP acts as a repressor of the activity of these gene promoters. This result was confirmed in transgenic lines overexpressing SHP, which were grown with low and high nitrogen supply. The phenotype of SHP-overexpressing lines showed a lower quantity of both LMW-GS and HMW-GS, while the quantity of gliadin was unchanged, whatever the nitrogen availability. Thus, the gliadin/glutenin ratio was increased, which suggests that gliadin and glutenin genes may be differently regulated.

Unusual Presentation of Pulmonary Hydatidosis Mimicking Thoracic Malignancy in a Paediatric South African Patient.

In this case report, an illustrative case of pulmonary hydatidosis in a young South African girl is presented. The acute symptomatology, rapidly worsening clinical condition and the extremely atypical appearance of the hydatid cysts on imaging were initially misleading and raised suspicion for malignant disease.

"Barbell Sign": A Diagnostic Imaging Finding in Progressive Multifocal Leukoencephalopathy.

The recently proposed diagnostic algorithm for progressive multifocal leukoencephalopathy underscores the importance of imaging and emphasizes the role of the radiologist in the diagnostic workup. We describe a case series of patients with visual symptoms and a typical pattern of brain involvement in definite progressive multifocal leukoencephalopathy, for which we have coined the term barbell sign.

Infantile intrathoracic large airway obstruction in a setting with a high prevalence of tuberculosis/HIV.

Background There has been no detailed analysis of the cause of intrathoracic large airway obstruction in infants in a resource-limited environment with a high prevalence of pulmonary tuberculosis (PTB) and human immunodeficiency virus (HIV). Aim To define the aetiology and severity of intrathoracic large airway obstruction in infants in a tertiary South African hospital with a high prevalence of PTB and HIV. Methods A retrospective study of infants was conducted with computerised tomography (CT) evidence of intrathoracic large airway obstruction from 1 January 2011 to 31 May 2014. CT scans were evaluated for the cause, site and severity of airway narrowing, with severity stratified as 'mild' (<50%), 'moderate' (51-75%) or 'severe' (>75%). Results Forty-four patients (28 males, 64%; median age 145 days, range 5-331), and 79 sites of attenuation were included. Vascular (22/44, 50%) and nodal (18/44, 41%) compressions accounted for over 90% of cases. Thirty-five patients (79.5%) had at least one site of moderate/severe attenuation, and 26 (59%) had multiple such sites. Adenopathy was the commonest cause of moderate/severe compression (18/35, 51%). All cases of nodal compression were of tuberculous origin. HIV-serology was recorded in 32 patients (73%), one of whom, with vascular compression, was HIV-infected. Half of the patients (11/22, 50%) with vascular compression had congenital abnormalities, most commonly cardiac anomalies (7/22, 32%). There were no synchronous vascular and nodal compressions. Conclusion Infantile intrathoracic large airway obstruction where there is a high prevalence of PTB and HIV is characterised by its extrinsic aetiology, severity and multicentricity, with more than half of all moderate/severe obstructions being caused by tuberculous adenopathy.

Biophysical characterisation of the novel zinc binding property in Suppressor of Fused.

Suppressor of Fused (SUFU) is a highly conserved protein that acts as a negative regulator of the Hedgehog (HH) signalling pathway, a major determinant of cell differentiation and proliferation. Therefore, SUFU deletion in mammals has devastating effects on embryo development. SUFU is part of a multi-protein cytoplasmic signal-transducing complex. Its partners include the Gli family of transcription factors that function either as repressors, or as transcription activators according to the HH activation state. The crystal structure of SUFU revealed a two-domain arrangement, which undergoes a closing movement upon binding a peptide from Gli1. There remains however, much to be discovered about SUFU's behaviour. To this end, we expressed recombinant, full-length SUFU from Drosophila, Zebrafish and Human. Guided by a sequence analysis that revealed a conserved potential metal binding site, we discovered that SUFU binds zinc. This binding was found to occur with a nanomolar affinity to SUFU from all three species. Mutation of one histidine from the conserved motif induces a moderate decrease in affinity for zinc, while circular dichroism indicates that the mutant remains structured. Our results reveal new metal binding affinity characteristics about SUFU that could be of importance for its regulatory function in HH.

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused.

Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.

EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments.

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference.

Domestication history and geographical adaptation inferred from a SNP map of African rice.

African rice (Oryza glaberrima Steud.) is a cereal crop species closely related to Asian rice (Oryza sativa L.) but was independently domesticated in West Africa ∼3,000 years ago. African rice is rarely grown outside sub-Saharan Africa but is of global interest because of its tolerance to abiotic stresses. Here we describe a map of 2.32 million SNPs of African rice from whole-genome resequencing of 93 landraces. Population genomic analysis shows a population bottleneck in this species that began ∼13,000-15,000 years ago with effective population size reaching its minimum value ∼3,500 years ago, suggesting a protracted period of population size reduction likely commencing with predomestication management and/or cultivation. Genome-wide association studies (GWAS) for six salt tolerance traits identify 11 significant loci, 4 of which are within ∼300 kb of genomic regions that possess signatures of positive selection, suggesting adaptive geographical divergence for salt tolerance in this species.