Growth-Regulated Expression of a Bacteriocin,Produced by the Sweet Potato Pathogen Streptomyces ipomoeae,That Exhibits Interstrain Inhibition

Certain strains of the bacterial sweet potato pathogen Streptomyces ipomoeae produce the bacteriocin ipomicin, which inhibits other sensitive strains of the same species. Within the signal-sequence-encoding portion of the ipomicin structural gene ipoA exists a single rare TTA codon, which is recognized in Streptomyces bacteria by the temporally accumulating bldA leucyl tRNA. In this study, ipomicin was shown to stably accumulate in culture supernatants of S. ipomoeae in a growth-regulated manner that did not coincide with the pattern of ipoA expression. Similar growth-regulated production of ipomicin in Streptomyces coelicolor containing the cloned ipoA gene was found to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The results here suggest that bldA-dependent translation of the S. ipomoeae ipoA gene leads to growth-regulated production of the ipomicin precursor, which upon processing to the mature form and secretion stably accumulates in the extracellular environment. To our knowledge, this is the first example of bldA regulation of a bacteriocin in the streptomycetes.Streptomyces bacteria are gram-positive spore-forming soil bacteria which display a complex life cycle (4, 6). Spore germination leads to vegetative growth as tangled masses of substrate mycelia on solid surfaces. Nutrient limitation triggers production of secondary metabolites as well as growth of vertically extending aerial hyphae, which are morphologically distinct filaments that derive nutrients from the dying substrate cell layer. Eventually, individual aerial hyphae differentiate into chains of spores. Streptomyces mycelia growing in liquid media display a multiphasic growth curve similar to that of unicellular bacteria. As the culture enters stationary phase, Streptomyces bacteria produce secondary metabolites but do not typically differentiate morphologically (2).Streptomyces ipomoeae is the causative agent of soil rot, the widespread and destructive disease of sweet potatoes. The disease results in decay of fibrous feeder roots and development of necrotic lesions on the edible storage roots (9). Prevention of soil rot currently relies solely on development of resistant sweet potato cultivars; however, alternative approaches, including the use of biocontrol methods, are now beginning to be investigated.Previously, 36 strains of S. ipomoeae were divided into three groups based on interstrain inhibition during their cocultivation on agar plates (7). The group III strains were found to produce a diffusible 10-kDa bacteriocin-like protein (ipomicin), which is inhibitory to group I and II members and which is stable in culture supernatants for at least 1 h at 40°C (in a pH range of 6 to 10) (26). Sequence analyses of the ipomicin structural gene (ipoA) and its protein product revealed that ipomicin is initially expressed in precursor form, which upon processing of an N-terminal signal sequence becomes the 10-kDa mature form (26).Given its potential as a biocontrol agent, we were interested in elucidating the expression characteristics of ipomicin. It was noted previously that TTA, the rarest codon found within the GC-rich Streptomyces genus, is present once in ipoA within the region that designates the ipomicin signal sequence (26). TTA codons in Streptomyces bacteria are recognized by a single leucyl tRNA species encoded by the gene bldA, which is required for normal morphological and physiological differentiation in these organisms (5, 14). The latter effect is due in part to the fact that regulatory genes of antibiotic clusters and aerial hyphae development also contain TTA codons (5). In the model streptomycete, Streptomyces coelicolor, bldA has been shown to be an effective growth phase regulator because it is expressed initially in liquid and surface cultures as an inactive precursor and, upon processing, begins to temporally accumulate as a mature tRNA, at least under certain physiological conditions (15, 24). Interestingly, TTA-containing genes show variation in their dependence on bldA for expression, with some genes demonstrating strong dependence while others show only partial or no apparent dependence (14, 23).Here, stable ipomicin protein is shown to be temporally produced during S. ipomoeae growth in a manner inconsistent with ipoA mRNA levels. Similar contrary results for ipomicin protein and ipoA mRNA concentrations were observed during growth of the heterologous host S. coelicolor expressing a cloned version of the S. ipomoeae ipoA gene, and this effect was shown to be directly dependent on translation of the ipoA TTA codon by the bldA leucyl tRNA. The data here suggest that regulation by bldA leads to temporal production of the ipomicin precursor, which when processed to the mature form is secreted to the external environment, where it accumulates.     

Proteomics analysis of global regulatory cascades involved in clavulanic acid production and morphological development in <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>

The genus Streptomyces comprises bacteria that undergo a complex developmental life cycle and produce many metabolites of importance to industry and medicine. Streptomyces clavuligerus produces the β-lactamase inhibitor clavulanic acid, which is used in combination with β-lactam antibiotics to treat certain β-lactam resistant bacterial infections. Many aspects of how clavulanic acid production is globally regulated in S. clavuligerus still remains unknown. We conducted comparative proteomics analysis using the wild type strain of S. clavuligerus and two mutants (ΔbldA and ΔbldG), which are defective in global regulators and vary in their ability to produce clavulanic acid. Approximately 33.5 % of the predicted S. clavuligerus proteome was detected and 192 known or putative regulatory proteins showed statistically differential expression levels in pairwise comparisons. Interestingly, the expression of many proteins whose corresponding genes contain TTA codons (predicted to require the bldA tRNA for translation) was unaffected in the bldA mutant.     

Use of polymerase chain reaction to identify a leucyl tRNA in Streptomyces coelicolor

Polymerase chain reaction (PCR) was used to amplify a fragment of DNA encoding a tRNA that recognizes the abundant CUC leucine codon from the chromosome of Streptomyces coelicolor. Sequence analysis of the gene, designated leuU, indicated that it codes for a tRNA 88 nucleotides in length that shares 75% identity with the Escherichia coli tRNA^Leu_CUC, while it shares only 65% identity with the only other sequenced leucyl tRNA from S. coelicolor, the bldA encoded tRNA^Leu_UUA. Accumulation of the leuU tRNA was examined by Northern blot analysis and shown to be present at constant levels throughout growth in contrast to the bldA-encoded tRNA which shows a temporal pattern of accumulation [Leskiw et al., 1993. J. Bacteriol., 175, 1995–2005].     

The Use of the Rare TTA Codon in Streptomyces Genes: Significance of the Codon Context?

Streptomycetes, Gram-positive bacteria with huge and GC-rich genomes provide an ample example of codon usage bias taken to the extreme. Particularly, in all sequenced to date streptomycete genomes leucyl codon TTA is the rarest one. It is present (usually once or twice) in 70–200 out of 7000–8000 coding sequences that make up a typical streptomycete genome. tRNA^Leu_UAA of streptomycetes, encoded by the bldA gene, has been shown to be present in mature form only after the onset of morphological differentiation and activation of secondary metabolism. Consequently, during the early stages of cell growth, the translation of genes carrying the TTA codon can be interrupted due to the absence of tRNA^Leu_UAA. Several reports show that mutations of TTA to synonymous codons in certain genes indeed relieve their expression from bldA dependence. However, the deletion of bldA does not always arrest the expression of TTA-containing genes. The nucleotides T/C downstream of TTA were suggested, in 2002, to favor TTA mistranslation. We tested this hypothesis using sizable datasets derived from individual Streptomyces genome and a subset of TTA+ genes for secondary metabolism known for their active expression. Our results revealed nucleotide biases downstream of NNA codons family, such as the preference for C and the avoidance of A. Yet, none of the observed biases was sufficient to claim a special case for TTA codon. Hence, the issue of codon context and TTA codon mistranslation in Streptomyces deserves further elaboration.Electronic supplementary materialThe online version of this article (10.1007/s12088-020-00902-6) contains supplementary material, which is available to authorized users.     

An annotated catalogue of salivary gland transcripts in the adult female mosquito, Ædes ægypti*

Background

The number of completely sequenced plastid genomes available is growing rapidly. This array of sequences presents new opportunities to perform comParative analyses. In comParative studies, it is often useful to compare across wide phylogenetic spans and, within angiosperms, to include representatives from basally diverging lineages such as the genomes reported here: Nuphar advena (from a basal-most lineage) and Ranunculus macranthus (a basal eudicot). We report these two new plastid genome sequences and make comparisons (within angiosperms, seed plants, or all photosynthetic lineages) to evaluate features such as the status of ycf15 and ycf68 as protein coding genes, the distribution of simple sequence repeats (SSRs) and longer dispersed repeats (SDR), and patterns of nucleotide composition.

Results

The Nuphar [GenBank:NC_008788] and Ranunculus [GenBank:NC_008796] plastid genomes share characteristics of gene content and organization with many other chloroplast genomes. Like other plastid genomes, these genomes are A+T-rich, except for rRNA and tRNA genes. Detailed comparisons of Nuphar with Nymphaea, another Nymphaeaceae, show that more than two-thirds of these genomes exhibit at least 95% sequence identity and that most SSRs are shared. In broader comparisons, SSRs vary among genomes in s of abundance and length and most contain repeat motifs based on A and T nucleotides.

Conclusion

SSR and SDR abundance varies by genome and, for SSRs, is proportional to genome size. Long SDRs are rare in the genomes assessed. SSRs occur less frequently than predicted and, although the majority of the repeat motifs do include A and T nucleotides, the A+T bias in SSRs is less than that predicted from the underlying genomic nucleotide composition. In codon usage third positions show an A+T bias, however variation in codon usage does not correlate with differences in A+T-richness. Thus, although plastome nucleotide composition shows "A+T richness", an A+T bias is not apparent upon more in-depth analysis, at least in these aspects. The pattern of evolution in the sequences identified as ycf15 and ycf68 is not consistent with them being protein-coding genes. In fact, these regions show no evidence of sequence conservation beyond what is normal for non-coding regions of the IR.     

The extracellular proteome of Rhizobium etli CE3 in exponential and stationary growth phase

Background

The extracellular proteome or secretome of symbiotic bacteria like Rhizobium etli is presumed to be a key element of their infection strategy and survival. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. To find out the possible role of secreted proteins we analyzed the extracellular proteome of R. etli CE3 in the exponential and stationary growth phases in minimal medium, supplemented with succinate-ammonium.

Results

The extracellular proteins were obtained by phenol extraction and identified by LC-ESI MS/MS. We identified 192 and 191 proteins for the exponential and stationary phases respectively. Using the software Signal P, we predicted signal peptides for 12.95% and 35.60% of the proteins identified in the exponential and stationary phases, respectively, which could therefore be secreted by the Sec pathway. For the exponential growth phase, we found in abundance proteins like the ribosomal proteins, toxins and proteins belonging to the group "defence mechanisms". For the stationary growth phase, we found that the most abundant proteins were those with unknown function, and in many of these we identified characteristic domains of proteases and peptidases.

Conclusions

Our study provided the first dataset of the secretome of R. etli and its modifications, which may lead to novel insights into the adaptive response of different stages of growth. In addition, we found a high number of proteins with unknown function; these proteins could be analyzed in future research to elucidate their role in the extracellular proteome of R. etli.     

Is type 2 diabetes mellitus a vascular disease (atheroscleropathy) with hyperglycemia a late manifestation? The role of NOS, NO, and redox stress.

Background

The impressive correlation between cardiovascular disease and glucose metabolism alterations has raised the likelihood that atherosclerosis and type 2 diabetes may share common antecedents. Inflammation is emerging as a conceivable etiologic mechanism for both. Interleukins are regulatory proteins with ability to accelerate or inhibit inflammatory processes.

Presentation of the hypothesis

A novel interleukins classification is described, based on their role in diabetes and atherosclerosis, hypothesizing that each interleukin (IL) acts on both diseases in the same direction – regardless if harmful, favorable or neutral.

Testing the hypothesis

The 29 known interleukins were clustered into three groups: noxious (the "bad", 8 members), comprising IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-17 and IL-18; protective (the "good", 5 members), comprising IL-4, IL-10, IL-11, IL-12 and IL-13; and "aloof", comprising IL-5, IL-9, IL-14, IL-16 and IL-19 through IL-29 (15 members). Each group presented converging effects on both diseases. IL-3 was reluctant to clustering.

Implications

These observations imply that 1) favorable effects of a given IL on either diabetes or atherosclerosis predicts similar effects on the other; 2) equally, harmful IL effects on one disease can be extrapolated to the other; and 3) absence of influence of a given IL on one of these diseases forecasts lack of effects on the other. These facts further support the unifying etiologic theory of both ailments, emphasizing the importance of a cardiovascular diabetologic approach to interleukins for future research. Pharmacologic targeting of these cytokines might provide an effective means to simultaneously control both atherosclerosis and diabetes.     

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background

Rhizobium leguminosarum is an α-proteobacterial N₂-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.     

The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa

Background

Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown.

Results

The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa.

Conclusions

Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the "hidden overflow pathway" to phenylalanine described previously.     

Transient knockdown and overexpression reveal a developmental role for the zebrafish enosf1b gene

Background

Cancer cells possess unique metabolic phenotypes that are determined by their underlying oncogenic pathways. Activation of the PI3K/Akt/mTOR signaling cascade promotes glycolysis and leads to glucose-dependence in tumors. In particular, cells with constitutive mTORC1 activity secondary to the loss of TSC1/TSC2 function are prone to undergo apoptosis upon glucose withdrawal in vitro, but this concept has not been tested in vivo. This study examines the effects of restricting glucose metabolism by pharmacologic and dietary means in a tuberous sclerosis complex (TSC) tumor xenograft model.

Results

Tumor-bearing mice were randomly assigned to receive unrestricted carbohydrate-free ("Carb-free") or Western-style diet in the absence or presence of 2-deoxyglucose (2-DG) in one of four treatment groups. After 14 weeks, tumor sizes were significantly different among the four treatment groups with those receiving 2-DG having the smallest tumors. Unexpectedly, the "Carb-free" diet was associated with the largest tumors but they remained responsive to 2-DG. PET imaging showed significant treatment-related changes in tumor ¹⁸fluorodeoxyglucose-uptake but the standard uptake values did not correlate with tumor size. Alternative energy substrates such as ketone bodies and monounsaturated oleic acid supported the growth of the Tsc2-/- cells in vitro, whereas saturated palmitic acid was toxic. Correspondingly, tumors in the high-fat, "Carb-free" group showed greater necrosis and liquefaction that contributed to their larger sizes. In contrast, 2-DG treatment significantly reduced tumor cell proliferation, increased metabolic stress (i.e., ketonemia) and AMPK activity, whereas rapamycin primarily reduced cell size.

Conclusions

Our data support the concept of glycolytic inhibition as a therapeutic approach in TSC whereas dietary withdrawal of carbohydrates was not effective.     

Multidimensional protein fractionation using ProteomeLab PF 2D™ for profiling amyotrophic lateral sclerosis immunity: A preliminary report

Background

Trypanosoma cruzi, a flagellate protozoan, is the etiological agent of Chagas disease, a chronic illness that causes irreversible damage to heart and digestive tract in humans. Previous 2-DE analyses of T. cruzi proteome have not focused on basic proteins, possibly because of inherent difficulties for optimizing 2-DE in the alkaline pH range. However, T. cruzi wide pH range 2-DE gels have shown few visible spots in the alkaline region, indicating that the parasite either did not have an appreciable amount of alkaline proteins or that these proteins were underrepresented in the 2-DE gels.

Results

Different IEF conditions using 6–11 pH gradient strips were tested for separation of T. cruzi alkaline proteins. The optimized methodology described here was performed using anodic "paper bridge" sample loading supplemented by increased concentration of DTT and Triton X-100 on Multiphor II (GE Healthcare) equipment and an electrode pad embedded in DTT- containing solution near the cathode in order to avoid depletion of reducing agent during IEF. Landmark proteins were identified by peptide mass fingerprinting allowing the production of an epimastigote 2-DE map. Most identified proteins corresponded to metabolic enzymes, especially those related to amino acid metabolism. The optimized 2-DE protocol was applied in combination with the "two-in-one gel" method to verify the relative expression of the identified proteins between samples from epimastigote and trypomastigote life stages.

Conclusion

High resolution 2-DE gels of T. cruzi life forms were achieved using the optimized methodology and a partial epimastigote alkaline 2-DE map was built. Among 700 protein spots detected, 422 were alkaline with a pI above 7.0. The "two-in-one gel" method simplified the comparative analysis between T. cruzi life stages since it minimized variations in spot migration and silver-stained spot volumes. The comparative data were in agreement with biological traits of T. cruzi life forms and also corroborated previous T. cruzi proteomic studies. For instance, enzymes related to amino acid metabolism and dehydrogenases were more abundant in epimastigote 2-DE gel whilst trans-sialidase and a paraflagellar protein were found specifically in the trypomastigote 2-DE profile.     

Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia

Background

The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia.

Results

We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C₁-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins.

Conclusion

The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria.

Reviewers

This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.     

Genome-wide acceleration of protein evolution in flies (Diptera)

Background

The rate of molecular evolution varies widely between proteins, both within and among lineages. To what extent is this variation influenced by genome-wide, lineage-specific effects? To answer this question, we assess the rate variation between insect lineages for a large number of orthologous genes.

Results

When compared to the beetle Tribolium castaneum, we find that the stem lineage of flies and mosquitoes (Diptera) has experienced on average a 3-fold increase in the rate of evolution. Pairwise gene comparisons between Drosophila and Tribolium show a high correlation between evolutionary rates of orthologous proteins.

Conclusion

Gene specific divergence rates remain roughly constant over long evolutionary times, modulated by genome-wide, lineage-specific effects. Among the insects analysed so far, it appears that the Tribolium genes show the lowest rates of divergence. This has the practical consequence that homology searches for human genes yield significantly better matches in Tribolium than in Drosophila. We therefore suggest that Tribolium is better suited for comparisons between phyla than the widely employed dipterans.     

Sequence and functional analyses of the aldehyde dehydrogenase 7B4 gene promoter in Arabidopsis thaliana and selected Brassicaceae: regulation patterns in response to wounding and osmotic stress

Key message

The core promoter of the antiquitin ALDH7B4 gene was compared between selected Brassicaceae. Conserved cis elements controlling osmotic stress and wound-induced expression were identified and analysed in Arabidopsis thaliana leaves and seeds.

Abstract

Aldehyde dehydrogenases metabolise a wide range of aliphatic and aromatic aldehydes, which become cytotoxic at high levels. Family 7 aldehyde dehydrogenase genes, often described as antiquitins or turgor-responsive genes in plants, are broadly conserved across all domains. Despite the high conservation of the plant ALDH7 proteins and their importance in stress responses, their regulation has not been investigated. Here, we compared ALDH7 genes of different Brassicaceae and found that, in contrast to the gene organisation and protein coding sequences, similarities in the promoter sequences were limited to the first few hundred nucleotides upstream of the translation start codon. The function of this region was studied by isolating the core promoter of the Arabidopsis thaliana ALDH7B4 gene, taken as model. The promoter was found to be responsive to wounding in addition to salt and dehydration stress. Cis-acting elements involved in stress responsiveness were analysed and two conserved ACGT-containing motifs proximal to the translation start codon were found to be essential for the responsiveness to osmotic stress in leaves and in seeds. The integrity of an upstream ACGT motif and a dehydration-responsive element/C-repeat—low temperature-responsive element was found to be necessary for ALDH7B4 expression in seeds and induction by salt, dehydration and ABA in leaves. The comparison of the gene expression in selected Arabidopsis mutants demonstrated that osmotic stress-induced ALDH7B4 expression in leaves and seeds involves both ABA- and lipid-signalling components.     

Chlorophyll-binding proteins revisited - a multigenic family of light-harvesting and stress proteins from a brown algal perspective

Background

Mitochondrial introgression may result in the mitochondrial genome of one species being replaced by that of another species without leaving any trace of past hybridization in its nuclear genome. Such introgression can confuse the species genealogy estimates and lead to absurd inferences of species history. We used a phylogenetic approach to explore the potential mitochondrial genome introgression event(s) between two closely related green pond frog species, Pelophylax nigromaculatus and P. plancyi.

Results

DNA sequence data of one mitochondrial and two nuclear genes from an extensive sampling of the two species were collected, and the genealogies of the three genes were constructed and compared. While the two nuclear genes congruently showed mutual reciprocal monophyly of both species, the mitochondrial phylogeny separated a Korean P. nigromaculatus clade, a paraphyletic central China P. plancyi assemblage, and a large well-supported introgression clade. Within the introgression clade, the mitochondrial haplotypes of the two species were mixed together. This reticulated pattern can be most parsimoniously explained by an ancient mitochondrial introgression event from P. plancyi to P. nigromaculatus that occurred at least 1.36 MYA, followed by multiple recent introgression events from P. nigromaculatus back to P. plancyi within the last 0.63 MY. The re-constitution of previously co-adapted genomes in P. plancyi may be responsible for the recent rampant introgression events. The Korean P. nigromaculatus clade likely represents the only surviving "true" mitochondrial lineage of P. nigromaculatus, and the central China P. plancyi assemblage likely represents the "original" P. plancyi mitochondrial lineage. Refugia in the Korean Peninsula and central China may have played a significant role in preserving these ancient lineages.

Conclusions

The majority of individuals in the two species have either introgressed (P. nigromaculatus) or reclaimed (P. plancyi) mitochondrial genomes while no trace of past hybridization in their nuclear genomes was detected. Asymmetrical reproductive ability of hybrids and continuous backcrossing are likely responsible for the observed mitochondrial introgression. This case is unique in that it includes an ancient "forward" introgression and many recent "backward" introgressions, which re-constitutes the original nuclear and mitochondrial genomes of P. plancyi. This hybrid system provides an excellent opportunity to study cyto-nuclear interaction and co-adaptation.