Genes

The Stickler syndromes are a group of genetic connective tissue disorders associated with an increased risk of rhegmatogenous retinal detachment, deafness, cleft palate, and premature arthritis. This review article focuses on the molecular genetics of the autosomal dominant forms of the disease. Pathogenic variants in COL2A1 causing Stickler syndrome usually result in haploinsufficiency of the protein, whereas pathogenic variants of type XI collagen more usually exert dominant negative effects. The severity of the disease phenotype is thus dependent on the location and nature of the mutation, as well as the normal developmental role of the respective protein. Full article

Schizophrenia is a heterogeneous and severe psychotic disorder. Epidemiological findings have suggested that the exposure to infectious agents such as Toxoplasma gondii (T. gondii) is associated with an increased risk for schizophrenia. On the other hand, there is evidence involving the catechol-O-methyltransferase (COMT) Val105/158Met polymorphism in the aetiology of schizophrenia since it alters the dopamine metabolism. A case–control study of 141 patients and 142 controls was conducted to analyse the polymorphism, the prevalence of anti-T. gondii IgG, and their interaction on the risk for schizophrenia. IgG were detected by ELISA, and genotyping was performed with TaqMan Real-Time PCR. Although no association was found between any COMT genotype and schizophrenia, we found a significant association between T. gondii seropositivity and the disorder (χ² = 11.71; p-value < 0.001). Furthermore, the risk for schizophrenia conferred by T. gondii was modified by the COMT genotype, with those who had been exposed to the infection showing a different risk compared to that of nonexposed ones depending on the COMT genotype (χ² for the interaction = 7.28, p-value = 0.007). This study provides evidence that the COMT genotype modifies the risk for schizophrenia conferred by T. gondii infection, with it being higher in those individuals with the Met/Met phenotype, intermediate in heterozygous, and lower in those with the Val/Val phenotype. Full article

Prostate adenocarcinoma (PRAD), also named prostate cancer, the most common visceral malignancy, is diagnosed in male individuals. Herein, in order to obtain immune-based subtypes, we performed an integrative analysis to characterize molecular subtypes based on immune-related genes, and further discuss the potential features and differences between identified subtypes. Simultaneously, we also construct an immune-based risk model to assess cancer prognosis. Our findings showed that the two subtypes, C1 and C2, could be characterized, and the two subtypes showed different characteristics that could clearly describe the heterogeneity of immune microenvironments. The C2 subtype presented a better survival rate than that in the C1 subtype. Further, we constructed an immune-based prognostic model based on four screened abnormally expressed genes, and they were selected as predictors of the robust prognostic model (AUC = 0.968). Our studies provide reference for characterization of molecular subtypes and immunotherapeutic agents against prostate cancer, and the developed robust and useful immune-based prognostic model can contribute to cancer prognosis and provide reference for the individualized treatment plan and health resource utilization. These findings further promote the development and application of precision medicine in prostate cancer. Full article

Plant genetic diversity has a significant role in providing traits that can help meet future challenges, such as the need to adapt crops to changing climatic conditions or outbreaks of disease. Our aim in this study was to evaluate the diversity of 61 forage pea specimens (P. sativum ssp. arvense L.) collected from the northeastern Anatolia region of Turkey using 28 simple sequence repeat (SSR) markers. These primers generated a total of 82 polymorphic bands. The number of observed alleles (Na) per primer varied from 2 to 4 with a mean of 2.89 alleles/locus. The mean value of expected heterozygosity (Exp-Het = 0.50) was higher than the mean value of observed heterozygosity (Obs-Het = 0.22). The mean of polymorphic information content (PIC) was 0.41 with a range of 0.03–0.70. The mean number of effective alleles (Ne) was found to be 2.15, Nei’s expected heterozygosity (H) 0.49, and Shannon’s information index (I) 0.81. Cluster analysis through the unweighted pair-group mean average (UPGMA) method revealed that 61 forage pea landraces were divided into three main clusters. Genetic dissimilarity between the genotypes, calculated with the use of NTSYS-pc software, varied between 0.10 (G30 and G34) and 0.66 (G1 and G32). Principal coordinate analysis (PCoA) revealed that three principal coordinates explained 51.54% of the total variation. Moreover, population structure analysis showed that all genotypes formed three sub-populations. Expected heterozygosity values varied between 0.2669 (the first sub-population) and 0.3223 (third sub-population), with an average value of 0.2924. Average population differentiation measurement (Fst) was identified as 0.2351 for the first sub-population, 0.3838 for the second sub-population, and 0.2506 for the third sub-population. In general, current results suggest that SSR markers could be constantly used to illuminate the genetic diversity of forage pea landraces and can potentially be incorporated into future studies that examine the diversity within a larger collection of forage pea genotypes from diverse regions. Full article

The prevalence of germline BRCA1 or BRCA2 pathogenic variants (gBRCA1/2-PV) in patients with primary epithelial ovarian cancer (OC) in a rural area of Japan and their association with clinical characteristics, including treatment response and survival outcome, were investigated. A total of 123 unbiased patients with OC were tested for gBRCA1 and gBRCA2 using next-generation sequencing-based targeted amplicon sequencing. Clinical characteristics of OC patients with and without gBRCA1/2 status were compared. The overall prevalence of gBRCA1/2-PV was 15.4% (19 cases), with gBRCA2-PV (10.5%, 13 cases) being more common than gBRCA1-PV (4.9%, 6 cases). Among the observed gBRCA1/2-PV, several novel variants were included, suggesting that gBRCA1/2-PV unique to the local area exist. gBRCA1/2-PV was significantly more prevalent in OC patients at an older age, with high-grade serous carcinoma, with advanced-stage tumors, and with a family history of breast cancer or hereditary breast and ovarian cancer syndrome (HBOC)-associated cancers. Patients with advanced-stage OC with gBRCA1/2-PV showed a significantly lower recurrence rate and tended to have better progression-free and overall survival than those with wild-type gBRCA1/2. Genetic testing for gBRCA1/2 status in all OC patients is useful not only for diagnosing HBOC in patients and their relatives to assess the risk of HBOC-associated cancers, but also to estimate therapy response and outcomes in patients. Full article

Decreased expression of chicken cholecystokinin A receptor (CCKAR) attenuates satiety, which contributes to increased food intake and growth for modern broilers. The study aims to define the core promoter of CCKAR, and to identify variants associated with expression activity. A 21 kb region around the CCKAR was re-sequenced to detect sequence variants. A series of 5′-deleted promoter plasmids were constructed to define the core promoter of CCKAR. The effects of sequence variants located in promoter (PSNP) and conserved (CSNP) regions on promoter activity were analyzed by comparing luciferase activity between haplotypes. A total of 182 variants were found in the 21 kb region. There were no large structural variants around CCKAR. pNL−328/+183, the one with the shortest insertion, showed the highest activity among the six promoter constructs, implying that the key cis elements regulating CCKAR expression are mainly distributed 328 bp upstream. We detected significant activity differences between high- and low-growth associated haplotypes in four of the six promoter constructs. The high-growth haplotypes of constructs pNL−1646/+183, pNL−799/+183 and pNL−528/+183 showed lower activities than the low-growth haplotypes, which is consistent with decreased expression of CCKAR in high-growth chickens. Lower expression of the high-growth allele was also detected for the CSNP5-containing construct. The data suggest that the core promoter of CCKAR is located the 328 bp region upstream from the transcription start site. Lower expression activities shown by the high-growth haplotypes in the reporter assay suggest that CSNP5 and variants located between 328 bp and 1646 bp upstream form a promising molecular basis for decreased expression of CCKAR and increased growth in chickens. Full article

Background: the impact of knee osteoarthritis (OA) poses a formidable challenge to older adults. Studies have reported that genetic factors, such as MMP1, are one of important risk factors for knee OA. Although the relationship between the genetic polymorphism of MMP1 rs1799750 and the risk of knee OA has been explored, conclusions have been nonunanimous and pending due to research sample sizes, one of determinants in studying genetic polymorphisms associated with disease. Objective: to establish a model to assess whether the genetic polymorphism of MMP1 rs1799750 is associated with knee OA based on an estimation of sample sizes. Methods: samples were collected from a case–control and meta-analysis study. In the case–control study, patients who underwent knee X-ray examinations based on the Kellgren–Lawrence Grading System (KL) as diagnostic criteria were recruited at the Health Examination Center of the Tri-Service General Hospital from 2015 to 2019. Gene sequencing was conducted using iPLEX Gold. Those with unsuccessful gene sequencing were excluded. Finally, there were 569 patients in the knee OA group (KL ≥ 2) and 534 participants in the control group (KL < 2). In the meta-analysis, we used the databases PubMed, EMBASE, and Cochrane to search for studies on the relationship between MMP1 rs1799750 and knee OA. Next, we adopted the trial sequential analysis (TSA) method to assess whether sample sizes were sufficient or not to determine the risk of the genetic polymorphism of MMP1 rs1799750 on knee OA in Caucasians and Asians. Results: in Caucasians, the MMP1 rs1799750 was not significantly associated with knee OA with an odds ratios (OR) of 1.10 (95% confidence interval, CI: 0.45–2.68). Some extra 8559 samples were needed to conclude this relationship in Caucasians by the TSA model. In Asians, neither our case–control study results (n = 1103) nor a combination of samples from the case–control and meta-analysis results showed an association between MMP1 rs1799750 and knee OA. The OR (95% CI) was 1.10 (0.81–1.49) in a combination of Asian samples. Some extra 5517 samples were needed to justify this relationship in Asians by the TSA model. Conclusions: this research shows that an extra 8559 and 5517 samples are needed in Caucasians and Asians, respectively, in order to justify the association between MMP1 rs1799750 and knee OA. Full article

Tubb4b (tubulin β-4b chain) is essential for cell growth and development as a microtubule network protein. Previous studies have shown that TUBB4B affects mouse pronucleus migration, but the gene function has yet to be elucidated. To study TUBB4B-related functions in mouse reproductive development, we designed a single sgRNA in chromosome 2 and generated a knockout spermatogonia cell line of the β-tubulin isoform Tubb4b by the CRISPR/Cas9 system. Tubb4b-KO spermatogonia recognized abnormal lysosomal membranes and cell morphology defects. Compared to control mouse spermatogonia, the proliferation rate was significantly slower and cycling stagnated in the G1/0 population. Although spermatogonia lacking TUBB4B have abnormal divisions, they are not lethal. We detected the mRNA levels of the cell-regulating cyclins CyclinsD1, CyclinsE, Cdk2, Cdk4, P21, Skp2 and the cell growth factors C/EBP α, C/EBP β, and G-CSF in the spermatogonia of Tubb4b-KO and found that the expressions of CyclinsD1, Skp2 and cell growth factors were significantly reduced. Further analysis revealed that 675 genes were expressed differently after Tubb4b deletion and were enriched in negative regulation of cell population proliferation (GO:0008285), negative regulation of cell cycle G2/M phase transition (GO:1902750), and positive regulation of cell death (GO: 0010942). We also found that there is a common gene Cdkn1a (P21) in these three GO pathways related to cell proliferation and cell cycle, and both quantitative analysis and transcriptome sequencing results showed that the expression of this gene was up-regulated in Tubb4b knockout cells. This implies that Tubb4b may be involved in the division of spermatogonia with multiple cell cycle regulatory proteins. Overall, these data indicate that Tubb4b has a specific role in regulating spermatogonia proliferation and cell cycle. Full article

Network and systemic approaches to studying human pathologies are helping us to gain insight into the molecular mechanisms of and potential therapeutic interventions for human diseases, especially for complex diseases where large numbers of genes are involved. The complex human pathological landscape is traditionally partitioned into discrete “diseases”; however, that partition is sometimes problematic, as diseases are highly heterogeneous and can differ greatly from one patient to another. Moreover, for many pathological states, the set of symptoms (phenotypes) manifested by the patient is not enough to diagnose a particular disease. On the contrary, phenotypes, by definition, are directly observable and can be closer to the molecular basis of the pathology. These clinical phenotypes are also important for personalised medicine, as they can help stratify patients and design personalised interventions. For these reasons, network and systemic approaches to pathologies are gradually incorporating phenotypic information. This review covers the current landscape of phenotype-centred network approaches to study different aspects of human diseases. Full article

Stropharia rugosoannulata uses straw as a growth substrate during artificial cultivation and has been widely promoted in China. However, its fruiting body formation and development processes have not been elucidated. In this study, the developmental transcriptomes were analyzed at three stages: the mycelium (G-S), primordium (P-S) and fruiting body (M-F) stages. A total of 9690 differentially expressed genes (DEGs) were identified in the different developmental stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these DEGs were involved mainly in hydrolase activity, structural molecule activity and oxidoreductase activity as well as xenobiotic biodegradation and metabolism and energy metabolism pathways. We further found that the higher expression of most carbohydrate enzyme (i.e., GH, CE, CBM, AA and PL) genes in the hyphal (i.e., G-S) stage was related mainly to substrate degradation, while the upregulation of glycosyltransferase (GT) gene expression in the P-S and M-F stages may be related to cell wall synthesis. In addition, we found that CO₂-sensing-related genes (i.e., CA-2, CA-3, PKA-1 and PKA-2) were upregulated in the P-S and M-F stages, heat shock protein genes (HSP60 and HSP90) were significantly downregulated in the P-S stage and upregulated in the M-F stage and the transcription factors (i.e., steA, MYB, nosA, HAP1, and GATA-4/5/6) involved in growth and development were significantly upregulated in the P-S stage. These results suggest that environmental factors (i.e., CO₂ and temperature) and transcription factors may play a key role in primordium formation. In short, this study provides new insights into the study of stimulating primordia formation affecting the development of fruiting bodies of S. rugosoannulata. Full article

The Dombrock (DO) blood group system has two primary antigens, Do^a and Do^b, which can cause delayed hemolytic transfusion reactions. The paucity of specific monospecific antibodies can hamper the typing based on these antigens. Thus, blood group genotyping (BGG) was investigated as a possible solution. Sequence-specific primers were designed to target a single nucleotide polymorphism (rs11276) on the ART4 gene encoding the DO*A and DO*B alleles. Blood samples (n = 150) from randomly selected volunteer donors were used. DNA was extracted and resulting PCR products were purified and sequenced. The allelic frequencies of DO*A and DO*B were (n = 122, 40.67%) and (n = 178, 59.33%), respectively. The distributions of DO genotypes were as follows: DO*A/DO*A (n = 20), 13.33%; DO*B/DO*B (n = 48), 32.00%; and DO*A/DO*B (n = 82), 54.67%. In conclusion, this study reports on the allelic frequencies of DO*A and DO*B of the DO blood group system in Jazan Province, Kingdom of Saudi Arabia. Furthermore, this study reports on the prevalence of each genotype, of which DO*A/DO*B was the most abundant. This study contributes significantly to build the current blood donor database in Southwestern Saudi Arabia. Moreover, it may assist in providing safe blood to polytransfused patients and reduce the risk of the red cell alloimmunization. Full article

Sweetpotato (Ipomoea batatas (L.) Lam.), which has a complex genome, is one of the most important storage root crops in the world. Sweetpotato blades are considered as a potential source of natural antioxidants owing to their high phenolic content with powerful free radical scavenging ability. The molecular mechanism of phenolic metabolism in sweetpotato blades has been seldom reported thus far. In this work, 23 sweetpotato genotypes were used for the analysis of their antioxidant activity, total polyphenol content (TPC) and total flavonoid content (TFC). ‘Shangshu19’ and ‘Wan1314-6’ were used for RNA-seq. The results showed that antioxidant activity, TPC and TFC of 23 genotypes had significant difference. There was a significant positive correlation between TPC, TFC and antioxidant activity. The RNA-seq analysis results of two genotypes, ‘Shangshu19’ and ‘Wan1314-6’, which had significant differences in antioxidant activity, TPC and TFC, showed that there were 7810 differentially expressed genes (DEGs) between the two genotypes. Phenylpropanoid biosynthesis was the main differential pathway, and upregulated genes were mainly annotated to chlorogenic acid, flavonoid and lignin biosynthesis pathways. Our results establish a theoretical and practical basis for sweetpotato breeding with antioxidant activity and phenolics in the blades and provide a theoretical basis for the study of phenolic metabolism engineering in sweetpotato blade. Full article

Vasa (Ddx4, DEAD box polypeptide 4), an extremely specific marker of germ cells in vivo, is an ATP-dependent RNA helicase that plays an essential role in germ cell development and gametogenesis. However, the expression and function information about this gene in groupers remains lacking. Here, vasa homolog termed Plvasa was isolated and identified Plvasa as a putative germ cell marker in the leopard coral grouper, (Plectropomus leopardus). Results indicated that Plvasa contained 17 exons in the genomic sequence and 9 conserved motifs of the DEAD-box protein by sequence analysis. The sequence comparison, phylogenetic analyses and synteny analyses showed that Plvasa was homologous with other teleosts. Additionally, the expression of Plvasa was significantly higher in gonads than in other tissues in adult individuals (p < 0.05). Further, the distribution of Plvasa revealed that it was only expressed in the germ cells, such as spermatids, germline stem cells and oocytes at different stages, and could not be detected in the somatic cells of gonads. The current study verified that the Plvasa gene is a valuable molecular marker of germ cells in leopard coral grouper, which potentially plays an important role in investigating the genesis and development of teleost germ cells. Full article

Hereditary myopathies are a group of genetically determined muscle disorders comprising more than 300 entities. In Chile, there are no specific registries of the distinct forms of these myopathies. We now report the genetic findings of a series of Chilean patients presenting with limb-girdle muscle weakness of unknown etiology. Eighty-two patients were explored using high-throughput sequencing approaches with neuromuscular gene panels, establishing a definite genetic diagnosis in 49 patients (59.8%) and a highly probable genetic diagnosis in eight additional cases (9.8%). The most frequent causative genes identified were DYSF and CAPN3, accounting for 22% and 8.5% of the cases, respectively, followed by DMD (4.9%) and RYR1 (4.9%). The remaining 17 causative genes were present in one or two cases only. Twelve novel variants were identified. Five patients (6.1%) carried a variant of uncertain significance in genes partially matching the clinical phenotype. Twenty patients (24.4%) did not carry a pathogenic or likely pathogenic variant in the phenotypically related genes, including five patients (6.1%) presenting an autoimmune neuromuscular disorder. The relative frequency of the different forms of myopathy in Chile is like that of other series reported from different regions of the world with perhaps a relatively higher incidence of dysferlinopathy. Full article

Loose smut (LS) disease is a serious problem that affects barley yield. Breeding of resistant cultivars and identifying new genes controlling LS has received very little attention. Therefore, it is important to understand the genetic basis of LS control in order to genetically improve LS resistance. To address this challenge, a set of 57 highly diverse barley genotypes were inoculated with Egyptian loose smut race(s) and the infected seeds/plants were evaluated in two growing seasons. Loose smut resistance (%) was scored on each genotype. High genetic variation was found among all tested genotypes indicating considerable differences in LS resistance that can be used for breeding. The broad-sense heritability (H²) of LS (0.95) was found. Moreover, genotyping-by-sequencing (GBS) was performed on all genotypes and generated in 16,966 SNP markers which were used for genetic association analysis using single-marker analysis. The analysis identified 27 significant SNPs distributed across all seven chromosomes that were associated with LS resistance. One SNP (S6_17854595) was located within the HORVU6Hr1G010050 gene model that encodes a protein kinase domain-containing protein (similar to the Un8 LS resistance gene, which contains two kinase domains). A TaqMan marker (0751D06 F6/R6) for the Un8 gene was tested in the diverse collection. The results indicated that none of the Egyptian genotypes had the Un8 gene. The result of this study provided new information on the genetic control of LS resistance. Moreover, good resistance genotypes were identified and can be used for breeding cultivars with improved resistance to Egyptian LS. Full article

The complete mitochondrial genomes (mitogenomes) of the tribes Coomaniellini and Dicercini were sequenced and described in this study, including Coomaniella copipes (16,196 bp), Coomaniella dentata (16,179 bp), and Dicerca corrugata (16,276 bp). These complete mitogenomes are very similar in length and encoded 37 typical mitochondrial genes, including 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and 13 protein-coding genes (PCGs). Most of PCGs had typical ATN start codons and terminated with TAR. Among these mitogenomes, Leu2 (L2), Ile (I), Ser2 (S2), and Phe (F) were the four most frequently encoded amino acids. Moreover, phylogenetic analyses were performed based on three kinds of nucleotide matrixes (13 PCGs, 2 rRNAs, and 13 PCGs + 2 rRNAs) among the available sequenced species of the family Buprestidae using Bayesian inference and Maximum-likelihood methods. The results showed that a Chrysochroninae species interspersed in Buprestinae, and Coomaniellini is more closely related to Dicercini than Melanophilini. Moreover, the clade of Buprestidae was well separated from outgroups and the monophyly of Agrilinae is confirmed again. Our whole mitogenome phylogenetic results support that the genus Dicerca can be transferred from Chrysochroinae to Buprestinae; whether Dicercini can be completely transferred remains to be further verified after enriching samples. Our results have produced new complete mitogenomic data, which will provide information for future phylogenetic and taxonomic research. Full article

The important roles of machine learning and ferroptosis in bladder cancer (BCa) are still poorly understood. In this study, a comprehensive analysis of 19 ferroptosis-related genes (FRGs) was performed in 1322 patients with BCa from four independent patient cohorts and a pan-cancer cohort of 9824 patients. Twelve FRGs were selected through machine learning algorithm to construct the prognosis model. Significantly differential survival outcomes (hazard ratio (HR) = 2.09, 95% confidence interval (CI): 1.55–2.82, p < 0.0001) were observed between patients with high and low ferroptosis scores in the TCGA cohort, which was also verified in the E-MTAB-4321 cohort (HR = 4.71, 95% CI: 1.58–14.03, p < 0.0001), the GSE31684 cohort (HR = 1.76, 95% CI: 1.08–2.87, p = 0.02), and the pan-cancer cohort (HR = 1.15, 95% CI: 1.07–1.24, p < 0.0001). Tumor immunity-related pathways, including the IL-17 signaling pathway and JAK-STAT signaling pathway, were found to be associated with the ferroptosis score in BCa through a functional enrichment analysis. Further verification in the IMvigor210 cohort revealed the BCa patients with high ferroptosis scores tended to have worse survival outcome after receiving tumor immunotherapy. Significantly different ferroptosis scores could also be found between BCa patients with different reactions to treatment with immune checkpoint inhibitors. Full article

Oculocutaneous albinism (OCA) is an autosomal recessive syndromic and non-syndromic defect with deficient or a complete lack of the melanin pigment. The characteristics of OCA appears in skin, hair, and eyes with variable degree of pigmentation. Clinical manifestations of OCA include nystagmus, photophobia, reduced visual acuity, hypo-plastic macula, and iris trans-illumination. There are eight OCA types (OCA1–8) documented with non-syndromic characteristics. Molecular studies identified seven genes linked to the OCA phenotype (TYR, OCA2, TYRP1, SLC45A2, SLC24A5, C10orf11, and DCT) and one locus (OCA5) in consanguineous and sporadic albinism. The complications of OCA result in skin cancer and variable syndromes such as Hermansky–Pudlak syndrome (HPS) Chediak–Higashi syndrome (CHS). In the Pakistani population, autosomal recessive non-syndromic OCA is common and is associated with a large number of consanguineous families, and mutations in genes of non-syndromic types are reported. This review highlights the updates on the genetic mutation of OCA genes reported from Pakistani families. Several studies reported the genetic mutations in OCA1, OCA2, OCA3, OCA4, and OCA6 albinism in Pakistani families. A locus, OCA5, was also reported from the Pakistani population, but the gene has not been identified. A new type of OCA8 was identified due to the DCT gene mutation, and it is also reviewed here. Full article

Italy hosts a large number of endemic freshwater fish species due to complex geological events which promoted genetic differentiation and allopatric speciation. Among them, the South European roach Sarmarutilus rubilio inhabits various freshwater environments in three different ichthyogeographic districts. We investigated the genetic diversity of S. rubilio using two different mitochondrial markers (COI and CR), aiming to define its relationship with other similar taxa from the Balkan area and, from a phylogeographic perspective, test the effects of past hydrogeological dynamics of Italian river basins on its genetic structure and demographic history. Our analysis highlighted a marked genetic divergence between S. rubilio and all other roach species and, among Italian samples, revealed the existence of three deeply divergent geographic haplogroups, named A, B and C. Haplogroup C likely corresponds to a new putative cryptic species and is located at the northern border of the South European roach range; haplogroup B is restricted to Southern Italy; and haplogroup A is widespread across the entire range and in some sites it is in co-occurrence with C or B. Their origin is probably related to the tectonic uplifting of the Apuan Alps in the north and of the Colli Albani Volcano in the south during the Pleistocene, which promoted isolation and vicariance followed by secondary contacts. Full article