Molecular evolution of chloroplast DNA in fig (Ficus carica L.): Footprints of sweep selection and recent expansion

Here, we report the nucleotide variation in two non-coding regions of the chloroplast DNA (cpDNA) to construct a possible evolutionary scenario in Ficus carica L. Our results suggest the occurrence of haplotypic and nucleotide diversity with a large variation level of chloroplast non-coding regions. Furthermore, our results demonstrated an explicit rejection of the null hypothesis that within F. carica the intron trnL and the spacer trnL-trnF evolved under a strictly neutral model of molecular evolution. Although, recent population expansion could serve as one alternative explanation for the detected excess of singleton, our results imply a positive selection and the genetic hitchhiking effect is unlikely. Parameters performed supported scenario of sweep selection and recent expansion of F. carica across Tunisia. Our results indicate that both positive selection and demographic histories have jointly contributed to the observed patterns of nucleotide diversity and haplotypes structure. Based on the results, we characterize the fig resources and provide several suggestions for effective conservation and improvement programs.     

Chloroplast DNA variation and phylogeographic patterns in the Chinese endemic marsh herb Sagittaria potamogetifolia

We examined chloroplast DNA (cpDNA) atpB–rbcL intergenic spacer sequences variation within Sagittaria potamogetifolia, an endangered and endemic marsh herb in China. Sequence data were obtained from 54 individuals in six extant populations of the species. Sequences appeared to evolve neutral (Tajima's criterion D = −1.59826, 0.1 > P > 0.05 and Fu and Li's tests D* = −1.44484, P > 0.1; F* = −1.83446, P > 0.1). Eleven haplotypes were identified in S. potamogetifolia. A relatively high level of haplotype diversity (h = 0.0.699) and low level of nucleotide diversity (pi = 0.0035 ± 0.0020) were detected in S. potamogetifolia. Pairwise comparisons of F_st and Nm deduced from cpDNA variation suggested no significant genetic differentiation between populations of S. potamogetifolia excepted for the WY-1 population. Low genetic differentiation among populations and also among regions was consistently indicated by both hierarchical analyses of molecular variance (AMOVA) and the structure of a neighbor-joining tree. Lack of population differentiation between populations or between regions in cpDNA sequences may be due to effects of lower substitution rates or lineage sorting. In the minimum spanning network, all tip haplotypes except for the haplotype J were unique to a particular population, while the interior nodes except for the haplotype E were widespread (haplotype A). From nested clade analysis (NCA), the evolutionary events such as restricted gene flow with isolation by distance and allopatric fragmentation were inferred to responsible for the current distribution of S. potamogetifolia populations, as well as their genetic diversity.     

Genetic diversity in endangered Notopterygium forbesii Boissieu based on intraspecies sequence variation of chloroplast DNA and implications for conservation

A thorough understanding of the levels and partitioning of genetic variation across populations and geographical regions of endangered species is a prerequisite to ensure effective conservation and/or restoration activities. Here, we examined chloroplast DNA (cpDNA) trnH-psbA intergenic spacer sequences variation within Notopterygium forbesii, an endangered and endemic perennial herb in China. Sequence data obtained from 141 individuals in 14 populations revealed twenty-two haplotypes. A high level of haplotype diversity (Hd = 0.81) and low level of nucleotide diversity (Pi = 0.0047) were detected. Low genetic differentiation among populations and also among regions was consistently indicated by both hierarchical analyses of molecular variance (AMOVA) and the structure of a neighbor-joining tree. Low level of population differentiation between populations or between regions in cpDNA sequences may be due to effects of the abundance of ancestral haplotype sharing and the high number of private haplotypes fixed for each population. Based on our results, we proposed some conservation strategies.     

Molecular confirmation of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea

The hypothesis of natural hybridization between Lumnitzera racemosa and Lumnitzera littorea, two mangrove species distributed in the Indo-West Pacific region, was proposed in 1970s based on morphological traits; however, no molecular evidence has been reported to support it. In this study, we sequenced two low-copy nuclear genes and one chloroplast intergenic spacer (trnS-trnG) in the two Lumnitzera species and their putative hybrid to test this hypothesis. Our results revealed that there were 9 and 27 nucleotide substitutions at the two nuclear loci, respectively, between one haplotype of L. racemosa and L. littorea, and that the putative hybrid showed additivity in chromatograms at these sites. Sequencing the chloroplast intergenic region trnS-trnG showed that the two Lumnitzera species differed by seven fixed nucleotide substitutions and four fixed insertions/deletions in this region, while the putative hybrid had identical sequences to L. racemosa. Molecular data clearly demonstrated that there indeed existed natural hybridization between L. racemosa and L. littorea and that L. racemosa was the maternal parent in this hybridization event. The uncommon direction of hybridization and F₁ nature of hybrids in this case, and in mangroves in general, is discussed.     

Morphological and allozyme variation in a collection of Cucumeropsis mannii Naudin (Cucurbitaceae) from Côte d'Ivoire

To set up a rational collecting strategy for germplasm of the edible-seeded cucurbit Cucumeropsis mannii, a study was conducted using 24 morphological and seven putative enzyme markers to determine the intra-specific variability from 16 and 22 accessions (representing three cultivars), respectively. The analysis of variance, showed a significant difference between the three cultivars. Principal component analysis pointed out a variation among individuals, mainly on the basis of flower, fruit, and seed size. Dendrogram with UPGMA method allowed clustering of the cultivars. Genetic diversity indices estimated equalled: 9.96% for the proportion of polymorphic loci (P), 1.10 for the number of alleles (A) and 0.023 for observed heterozygosity (H_o). The level of the within accessions genetic diversity (H_S = 0.078) was higher than among accessions (D_ST = 0.042). Nei's genetic distances between the three cultivars were also low (0.079–0.147), indicating a high degree of similarity of the analysed cultivars.     

Genetic diversity of the endangered species Kirengeshoma palmata (Saxifragaceae) in China

In order to investigate the levels of genetic diversity of the endangered species Kirengeshoma palmata (Saxifragaceae), four extant populations were sampled and analyzed using inter-simple sequence repeats (ISSR) markers. We expected a low genetic diversity level, but our results revealed a high level of intraspecific genetic diversity, probably resulting from this species being in a refuge during the last glaciation (at population level: P = 63.25%, A_e = 1.47, H_E = 0.26 and H_O = 0.37; at species level: P = 79.00%, A = 1.5538, H_T = 0.2586 and H_sp = 0.3104). A low level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (16.69%) and AMOVA (19.36%). Populations shared high levels of genetic identity. Insect pollination and seed dispersal by wind may have facilitated extensive gene flow and are likely responsible for this present structure of genetic variation.     

Genetic variations of mitochondrial antiviral signaling gene (MAVS) in domestic chickens

Mitochondrial antiviral signaling (MAVS) gene plays a key role in antiviral regulation in mammals potentially by activating IRF3/7 and NF-κB and leading to the induction of type I interferon (IFN)-mediated antiviral and inflammatory responses. In this study, we screened genetic polymorphisms of the MAVS gene in various Chinese domestic chicken breeds/populations and evaluated its potential effect on gene expression. Among the sequenced fragment (4678 bp), a total of 75 single nucleotide polymorphisms (SNPs) were identified in 46 chickens from 10 breeds/populations, including 30 coding SNPs and 45 non-coding SNPs. Extremely high haplotype diversity (37 nucleotide haplotypes, 18 amino acid haplotypes) was observed in the coding region (CDS), and a similar pattern of high polymorphisms was also observed for the 3′-untranslated region (3′-UTR). Luciferase assays of two representative 3′-UTR haplotypes were performed in both HEK293 cells and DF-1 chicken fibroblast cells, and we found that they were differentially associated with different abilities on regulating mRNA expression level (P < 0.05). Collectively, we observed a considerably high genetic variability of the MAVS gene, and the 3′-UTR variants had an ability to regulate mRNA expression. These results would cast some clues on understanding the potential role of MAVS on viral resistance in chicken.     

The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

Uniformity of organellar DNA in Aldrovanda vesiculosa, an endangered aquatic carnivorous species, distributed across four continents

Organellar DNA from the widely distributed but rare and critically endangered aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) was examined. Six chloroplast intergenic regions (3700 nt in total) were sequenced before analyzing the Southern-RFLP (Restriction Fragment Length Polymorphism) of 2 mt gene flanking regions. Only two different chloroplast haplotypes among 15 A. vesiculosa accessions from Africa, Australia, Europe, and Japan were found, generally distinguishing European and non-European plants, with two exceptions. Genetic variation observed in A. vesiculosa appears to be even lower than in other aquatic species with a similar world-wide distribution. A recent bottleneck followed by long-distance dispersal by water birds or low mutation rates could be responsible for the observed genetic uniformity. Estimation of genetic distances based on six chloroplast intergenic regions led to the conclusion that the chloroplast genome of A. vesiculosa matches more closely to that of Drosera regia than Dionaea muscipula, a sister genus sharing snapping traps. The inconsistency between genetic distance estimates based on nuclear and cytoplasmic markers may reflect a chloroplast capture. In A. vesiculosa, a four amino acids substitution (TGWS) in the amino acid sequence of ATP synthase alpha subunit (ATP1), highly conserved mitochondrial protein, was discovered, unique among all organisms based on current knowledge.     

Phylogenetics,biogeography, and evolutionary trends of the Phalaenopsis sumatrana complex inferred from nuclear and chloroplast DNA

Phylogenetic trees inferred from the internal transcribed spacers 1 and 2 (ITS1 + ITS2) region from nuclear ribosomal DNA (nrDNA) and the intergenic spacer of atpB-rbcL from chloroplast DNA (cpDNA) were used to clarify the phylogenetics and evolutionary trends of the Phalaenopsis sumatrana complex. The P. sumatrana complex includes the two species P. sumatrana and Phalaenopsis corningiana as well as a problem species, Phalaenopsis zebrina, according to the concepts of Sweet (1980) and Christenson (2001). Based on the phylogenetic tree inferred from the ITS sequence, the accessions of P. sumatrana cannot be separated from those of P. corningiana. In contrast, the accessions of P. zebrina can be separated from those of both P. sumatrana and P. corningiana. However, analyses of the sequences of the atpB-rbcL spacer apparently cannot discriminate among these three species of the P. sumatrana complex. An inspection of the morphological characters of the plants of the P. sumatrana complex and the floral fragrances of P. zebrina can be used to separate it from both P. sumatrana and P. corningiana. Based on the molecular data and floral fragrances, P. zebrina perhaps should not be treated as a synonym of P. sumatrana. On the evolutionary trend of the P. sumatrana complex, P. zebrina was suggested to be the relative origin group of the P. sumatrana complex based on the phylogenetic tree and biogeography.     

Analysis of three leafminers' complete mitochondrial genomes

Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16141 bp, 16236 bp and 16183 bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A + T-rich region. The genomes were highly compact with only 60–68 bp of non-coding intergenic spacer. However, considerable differences in the A + T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.     

Genetic diversity and inter-specific relations of western Mediterranean relic Abies taxa as compared to the Iberian A. alba

Several Abies species are currently present in the Mediterranean region and most of them are endemic taxa and tertiary relicts. Using six nuclear microsatellites, we studied the genetic structure and inter-specific relationships among West Mediterranean firs, A. pinsapo (Spain), A. maroccana and A. tazaotana (Morocco). Based on the hypothesis that A. pinsapo could historically exchange genes with A. alba growing in the Pyrenees via secondary contact, we investigated the level of genetic admixture between these species using a Bayesian approach. The studied populations showed moderate genetic diversity (mean H_E = 0.598) and a high level of genetic differentiation (F_ST = 0.225) that was especially pronounced between A. alba and the African firs. All populations experienced a strong bottleneck effect that was likely induced by climatic changes occurring in the West Mediterranean during the last glacial cycle and the Holocene. According to Bayesian clustering, both African taxa grouped together in a single cluster, the two A. pinsapo populations formed a second cluster, and two additional clusters were detected within A. alba. Our results indicate that A. tazaotana is genetically very close to A. maroccana, and hence these two taxa should probably not be considered as separate species. We found no genetic admixture between A. pinsapo and A. alba and only minor between A. pinsapo and the African fir populations suggesting an isolation effect of the Gibraltar Strait. Current limited distributions of firs in the Mediterranean region together with changing climate may lead to further deterioration of the genetic diversity levels. Hence, future efforts should focus on monitoring the demography and genetic threats to existing populations.     

Nuclear and chloroplast DNA sequences data support the origin of Potamogeton intortusifolius J.B. He in China as a hybrid between P. perfoliatus Linn. and P. wrightii Morong

Internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast rbcL gene sequence data were used to test the hypothesis that natural populations of Potamogeton intortusifolius J.B. He in China originated from hybridization between P. perfoliatus Linn. and P. wrightii Morong. Based on ITS sequences data, P. intortusifolius possessed heterozygous rDNA genotypes which confirmed the hybrid origin of P. intortusifolius. Chloroplast rbcL gene sequences of P. intortusifolius from Yichang population revealed the same chloroplast haplotype as P. perfoliatus and the samples of P. intortusifolius from Weinan population had the same chloroplast haplotype as P. wrightii, which indicated that both putative parental species had been the maternal parent and that the two populations of P. intortusifolius had independent origins. This study confirms P. intortusifolius as a reciprocal hybrid. Because P. × intortusifolius in China has the same hybrid origin as P. × anguillanus Koidz. in Japan, it is suggested that P. × intortusifolius should be a synonym of P. × anguillanus.     

Gammaproteobacteria occurrence and microdiversity in Tyrrhenian Sea sediments as revealed by cultivation-dependent and -independent approaches

Bacterial diversity in Tyrrhenian Sea sediments was assessed using cultivation-dependent and -independent approaches. Samples collected from the different sediment layers (up to 30 cm) relative to four seamount and non-seamount stations, at depths from 3425 to 3580 m, were subjected to DNA extraction and 16S rRNA amplification targeting the V3 region. Denaturing gradient gel electrophoresis (DGGE) showed several heterogeneous profiles and 27 single bands were excised and sequenced. Sequence analysis revealed the presence of Firmicutes, Actinobacteria and Chloroflexi in 26% of the DGGE bands and a predominance of sequences affiliated to cultivable and uncultivable clones of Gammaproteobacteria (55%). To corroborate these findings, cultivation attempts were performed that allowed the isolation of 87 strains assigned to the proteobacterial classes. Identification was achieved by means of automated ribosomal intergenic spacer analysis (ARISA) and by 16S rDNA sequencing. The isolates were related to the gamma, alpha and beta subclasses of Proteobacteria with respective percentages of 77, 17 and 6%. The most predominant Gammaproteobacteria isolates, assigned to the Psychrobacter marincola and P. submarinus clade (n = 53) and to Halomonas aquamarina (n = 14), showed a huge intraspecific diversity with 29 distinct ARISA haplotypes. The detection by both approaches of these psychrophilic and moderately halophilic species and their extensive microdiversity indicated their predominance in Tyrrhenian Sea sediments where they constituted the indigenous microflora.     

Genetic diversity of Dioscorea dumetorum (Kunth) Pax using Amplified Fragment Length Polymorphisms (AFLP) and cpDNA

We have utilized Amplified Fragment Length Polymorphisms (AFLP) in conjunction with chloroplast DNA (cpDNA) sequence data to study the genetic diversity in 53 accessions of Dioscorea dumetorum from six countries in West and Central Africa. Our results provide a comparison of the two marker systems with regards to their applicability to differentiate intraspecific genotypes and the grouping of the accessions based on localities of collection. A total of 1052 AFLP fragments (of which 94.1% were polymorphic) produced from twelve primer combinations indicate a relatively high level of polymorphism among the accessions. Three major genetic groups that do not strictly follow a geographic distribution pattern were identified using Neighbour-joining and the principal coordinate (PCo) analyses. Accessions from Togo showed higher numbers of private fragments and the highest percentage polymorphism (59.4%). The detection of highest genetic diversity in accessions from Nigeria and Togo and their relationship to other accessions suggest that these countries are the centre of origin and diversity of D. dumetorum. The moderately high genetic diversity (average of 61%) is suggesting great influence on the D. dumetorum germplasms through exchange and transfer of cultivars among local farmers in the sub-region. In contrast, DNA sequence data from the psbA-trnH and the rpoB-trnC chloroplast regions revealed no variation among accessions from the different localities and clearly differentiated by AFLP patterns. The results demonstrate the usefulness of the AFLP marker in generating high polymorphism in the D. dumetorum accessions from West and Central Africa and hence may be used for agronomic purposes.     

Population genetic structure and historical demography of grey mullet, Mugil cephalus, along the coast of China,inferred by analysis of the mitochondrial control region

Population genetic structure and historical demography of grey mullet, Mugil cephalus, along the coast of China were examined with a 389-bp segment of mtDNA control region. In total, 117 samples were collected from seven locations and 77 haplotypes were obtained. No haplotype was concurrently presented in all the samples, and samples in Southern and Northern regions had distinctly different haplotypes. Sorting of the data cladistically into phylogenetic trees indicated that these haplotypes can be divided into two main clades. Analysis of molecular variance (AMOVA) indicated significant genetic differentiation among subpopulations in different regions (φ_CT = 0.9505, p < 0.01). The average pairwise differences and φ_ST values (0.8943–0.9464) between regional populations were significant. The net Tumura and Nei genetic distance (0.5577 ± 0.1421) between the two groups was large. The deep and unique divergence between the two groups suggested that each group is likely to represent a distinct species. In contrast to the great genetic divergence on a broad scale, small but significant population subdivisions at a less spatial scale were also detected both in Southern and Northern regions, which support the conclusion that M. cephalus has limited dispersal potential. High haplotype diversity (h = 0.8716 ± 0.1462) and low nucleotide diversity (π = 0.0046 ± 0.0030) were found among the three Northern samples. Among the four Southern samples, however, extraordinarily high levels of both haplotype diversity (h = 0.9886 ± 0.0057) and nucleotide diversity (π = 0.0425 ± 0.0212) were detected. The different polymorphisms suggest an apparent distinguishing demographic history between regionally Southern and Northern populations, though mismatch distribution analyses and neutrality tests implied a late Pleistocene population expansion of both Northern and Southern populations.     

Hybrid status of Tibouchina urvilleana Cogn. revealed by ITS sequences of nuclear ribosomal DNA

Tibouchina urvilleana Cogn. is native to southern Brazil and currently cultivated as an important ornamental shrub in frost free areas around the world. Its rapid vegetative growth and sterility suggests that it might be of hybrid origin. In this study, Internal Transcribed Spacer (ITS) of nuclear ribosomal DNA and chloroplast trnL-trnF spacer from T. urvilleana and three other congeneric species were sequenced to test its hybrid status. Cloning sequencing revealed two distinct types of ITS sequences from T. urvilleana, with one type almost identical with Tibouchina aspera. Genetic distance between the two types was much larger than the average interspecific genetic distances calculated from other Tibouchina species. Sequencing of chloroplast trnL-trnF spacer showed that T. urvilleana has identical sequence with T. aspera, but differed from other congeneric species by one nucleotide substitution and two indels. Molecular data demonstrated clearly that T. urvilleana indeed was a hybrid, with T. aspera or closely related species acting as the maternal parent.     

Genetic differentiation of Artemia urmiana from various ecological populations of Urmia Lake assessed by PCR amplified RFLP analysis

This study concentrated on the mitochondrial DNA diversity in adult Artemia urmiana populations. A total of 210 individual specimens were collected from the surface and bottom layers from three different geographical areas, comprising six sampling sites in Urmia Lake (Iran). The mitochondrial rDNA gene region was amplified using the PCR technique followed by RFLP analysis based on using eleven restriction endonucleases. Analysis at the intrapopulation level indicated that the two median and south area bottom layers have somewhat higher proportion of polymorphic sites compare to others. Although floating and sinking cysts did not consistently show genetic differences, there was significant genetic variation among all samples (F_ST = 0.019, P = 0.03) and 98% of the variation was within samples. Our results clearly distinguished nucleotide divergence and genetic structuring patterns, suggest that may be genetic differentiation existed in Artemia populations from Urmia Lake. Pairwise comparisons of the samples showed that the south area surface layer location was the most genetically divergent of the six sampling sites. Genetic characterizations of the various Artemia populations in two defined depths (surface and bottom) revealed differentiation that may be important in understanding the ecology of this commercially important species.     

Characterization of the complete mitochondrial genome of Chilo auricilius and comparison with three other rice stem borers

The mitogenome of Chilo auricilius (Lepidoptera: Pyraloidea: Crambidae) was a circular molecule made up of 15,367 bp. Sesamia inferens, Chilo suppressalis, Tryporyza incertulas, and C. auricilius, are closely related, well known rice stem borers that are widely distributed in the main rice-growing regions of China. The gene order and orientation of all four stem borers were similar to that of other insect mitogenomes. Among the four stem borers, all AT contents were below 83%, while all AT contents of tRNA genes were above 80%. The genomes were compact, with only 121–257 bp of non-coding intergenic spacer. There are 56 or 62-bp overlapping nucleotides in Crambidae moths, but were only 25-bp overlapping nucleotides in the noctuid moth S. inferens. There was a conserved motif ‘ATACTAAA’ between trnS2 (UCN) and nad1 in Crambidae moths, but this same region was ‘ATCATA’ in the noctuid S. inferens. And there was a 6-bp motif ‘ATGATAA’ of overlapping nucleotides, which was conserved in Lepidoptera, and a 14-bp motif ‘TAAGCTATTTAAAT’ conserved in the three Crambidae moths (C. suppressalis, C. auricilius and T. incertulas), but not in the noctuid. Finally, there were no stem-and-loop structures in the two Chilo moths.