Genome sequences and population genomics reveal climatic adaptation and genomic divergence between two closely related sweetgum species.

Understanding the genetic basis of population divergence and adaptation is an important goal in population genetics and evolutionary biology. However, the relative roles of demographic history, gene flow, and/or selective regime in driving genomic divergence, climatic adaptation, and speciation in non-model tree species are not yet fully understood. To address this issue, we generated whole-genome resequencing data of Liquidambar formosana and L. acalycina, which are broadly sympatric but altitudinally segregated in the Tertiary relict forests of subtropical China. We integrated genomic and environmental data to investigate the demographic history, genomic divergence, and climatic adaptation of these two sister species. We inferred a scenario of allopatric species divergence during the late Miocene, followed by secondary contact during the Holocene. We identified multiple genomic islands of elevated divergence that mainly evolved through divergence hitchhiking and recombination rate variation, likely fostered by long-term refugial isolation and recent differential introgression in low-recombination genomic regions. We also found some candidate genes with divergent selection signatures potentially involved in climatic adaptation and reproductive isolation. Our results contribute to a better understanding of how late Tertiary/Quaternary climatic change influenced speciation, genomic divergence, climatic adaptation, and introgressive hybridization in East Asia's Tertiary relict flora. In addition, they should facilitate future evolutionary, conservation genomics, and molecular breeding studies in Liquidambar, a genus of important medicinal and ornamental values.

Pangenome of water caltrop reveals structural variations and asymmetric subgenome divergence after allopolyploidization.

Water caltrop (Trapa spp., Lythraceae) is a traditional but currently underutilized non-cereal crop. Here, we generated chromosome-level genome assemblies for the two diploid progenitors of allotetraploid Trapa. natans (4x, AABB), i.e., diploid T. natans (2x, AA) and Trapa incisa (2x, BB). In conjunction with four published (sub)genomes of Trapa, we used gene-based and graph-based pangenomic approaches and a pangenomic transposable element (TE) library to develop Trapa genomic resources. The pangenome displayed substantial gene-content variation with dispensable and private gene clusters occupying a large proportion (51.95%) of the total cluster sets in the six (sub)genomes. Genotyping of presence-absence variation (PAVs) identified 40 453 PAVs associated with 2570 genes specific to A- or B-lineages, of which 1428 were differentially expressed, and were enriched in organ development process, organic substance metabolic process and response to stimulus. Comparative genome analyses showed that the allotetraploid T. natans underwent asymmetric subgenome divergence, with the B-subgenome being more dominant than the A-subgenome. Multiple factors, including PAVs, asymmetrical amplification of TEs, homeologous exchanges (HEs), and homeolog expression divergence, together affected genome evolution after polyploidization. Overall, this study sheds lights on the genome architecture and evolution of Trapa, and facilitates its functional genomic studies and breeding program.

In vitro anti-Toxoplasma gondii effects of a coccidiostat dinitolmide.

Coccidiosis, caused by Eimeria species, results in huge economic losses to the animal industry. Dinitolmide, a veterinary-approved coccidiostat, has a wide anticoccidial spectrum with no effect on host immunity. However, the mechanism of its anticoccidial effects remains unclear. Here, we used an in vitro culture system of T. gondii to explore the anti-Toxoplasma effect of dinitolmide and its underlying mechanism against coccidia. We show that dinitolmide has potent in vitro anti-Toxoplasma activity with the half-maximal effective concentration (EC50) of 3.625 µg/ml. Dinitolmide treatment significantly inhibited the viability, invasion and proliferation of T. gondii tachyzoites. The recovery experiment showed that dinitolmide can completely kill T. gondii tachyzoites after 24 h of treatment. Morphologically abnormal parasites were observed after dinitolmide exposure, including asynchronous development of daughter cells and deficiency of parasite inner and outer membrane. Further electron microscopy results showed that the drug could damage the membrane structure of T. gondii. By comparative transcriptomic analysis, we found that genes related to cell apoptosis and nitric-oxide synthase were up-regulated after dinitolmide treatment, which might be responsible for parasite cell death. Meanwhile, many Sag-related sequence (srs) genes were down-regulated after treatment, which could be closely associated with the reduction of parasite invasion and proliferation capacity. Our study indicates that the coccidiostat dinitolmide has a potent inhibitory effect on T. gondii in vitro and provides insight into the mode of action of the drug.

In Vitro and In Vivo Activities of Tilmicosin and Acetylisovaleryltylosin Tartrate against Toxoplasma gondii.

Toxoplasma gondii is a widespread intracellular pathogen that infects humans and a variety of animals. The current therapeutic strategy for human toxoplasmosis is a combination of sulphadiazine and pyrimethamine. However, this combination still has a high failure rate and is ineffective against chronic infections. Therefore, it is important to discover a new anti-T. gondii drug that is safer and more effective in both humans and animals. In this study, we describe the anti-T. gondii activities of the 16-membered macrolide tilmicosin and acetylisovaleryltylosin tartrate (ATLL). Both tilmicosin and ATLL potently inhibited T. gondii with a half-maximal effective concentration (EC50) of 17.96 µM and 10.67 µM, respectively. Interestingly, tilmicosin and ATLL had different effects on the parasites. ATLL exhibited a potent inhibitory effect on intracellular parasite growth, while tilmicosin suppressed parasites extracellularly. By studying the lytic cycle of T. gondii after treatment, we found that ATLL potently inhibited the intracellular proliferation of tachyzoites, while tilmicosin affected the invasion of tachyzoites. Immunofluorescence analysis using ATLL-treated T. gondii showed morphologically abnormal parasites, which may be due to the inhibition of tachyzoite proliferation and division. In addition, tilmicosin and ATLL significantly delayed the death of mice caused by acute toxoplasmosis. Our results suggest that ATLL has potent anti-Toxoplasma activity both in vitro and in vivo and may be an alternative to toxoplasmosis in the future.

Molecular signatures of parallel adaptive divergence causing reproductive isolation and speciation across two genera.

Parallel evolution of reproductive isolation (PERI) provides strong evidence for natural selection playing a fundamental role in the origin of species. However, PERI has been rarely demonstrated for well established species drawn from different genera. In particular, parallel molecular signatures for the same genes in response to similar habitat divergence in such different lineages is lacking. Here, based on whole-genome sequencing data, we first explore the speciation process in two sister species of Carpinus (Betulaceae) in response to divergence for temperature and soil-iron concentration in habitats they occupy in northern and southwestern China, respectively. We then determine whether parallel molecular mutations occur during speciation in this pair of species and also in another sister-species pair of the related genus, Ostryopsis, which occupy similarly divergent habitats in China. We show that gene flow occurred during the origin of both pairs of sister species since approximately 9.8 or approximately 2 million years ago, implying strong natural selection during divergence. Also, in both species pairs we detected concurrent positive selection in a gene (LHY) for flowering time and in two paralogous genes (FRO4 and FRO7) of a gene family known to be important for iron tolerance. These changes were in addition to changes in other major genes related to these two traits. The different alleles of these particular candidate genes possessed by the sister species of Carpinus were functionally tested and indicated likely to alter flowering time and iron tolerance as previously demonstrated in the pair of Ostryopsis sister species. Allelic changes in these genes may have effectively resulted in high levels of prezygotic reproductive isolation to evolve between sister species of each pair. Our results show that PERI can occur in different genera at different timescales and involve similar signatures of molecular evolution at genes or paralogues of the same gene family, causing reproductive isolation as a consequence of adaptation to similarly divergent habitats.

Pervasive hybridization during evolutionary radiation of Rhododendron subgenus Hymenanthes in mountains of southwest China.

Radiations are especially important for generating species biodiversity in mountainous ecosystems. The contribution of hybridization to such radiations has rarely been examined. Here, we use extensive genomic data to test whether hybridization was involved in evolutionary radiation within Rhododendron subgenus Hymenanthes, whose members show strong geographic isolation in the mountains of southwest China. We sequenced genomes for 143 species of this subgenus and 93 species of four other subgenera, and found that Hymenanthes was monophyletic and radiated during the late Oligocene to middle Miocene. Widespread hybridization events were inferred within and between the identified clades and subclades. This suggests that hybridization occurred both early and late during diversification of subgenus Hymenanthes, although the extent to which hybridization, speciation through mixing-isolation-mixing or hybrid speciation, accelerated the diversification needs further exploration. Cycles of isolation and contact in such and other montane ecosystems may have together promoted species radiation through hybridization between diverging populations and species. Similar radiation processes may apply to other montane floras in this region and elsewhere.

Evaluation of the Combined Effect of Artemisinin and Ferroptosis Inducer RSL3 against Toxoplasma gondii.

Toxoplasma gondii is a widespread intracellular pathogen that infects humans and a variety of animals. Dihydroartemisinin (DHA), an effective anti-malarial drug, has potential anti-T. gondii activity that induces ferroptosis in tumor cells, but the mechanism by which it kills T. gondii is not fully understood. In this study, the mechanism of DHA inhibiting T. gondii growth and its possible drug combinations are described. DHA potently inhibited T. gondii with a half-maximal effective concentration (EC50) of 0.22 µM. DHA significantly increased the ROS level of parasites and decreased the mitochondrial membrane potential, which could be reversed by ferroptosis inhibitors (DFO). Moreover, the ferroptosis inducer RSL3 inhibited T. gondii with an EC50 of 0.75 µM. In addition, RSL3 enhanced the DHA-induced ROS level, and the combination of DHA and RSL3 significantly increased the anti-Toxoplasma effect as compared to DHA alone. In summary, we found that DHA-induced ROS accumulation in tachyzoites may be an important cause of T. gondii growth inhibition. Furthermore, we found that the combination of DHA and RSL3 may be an alternative to toxoplasmosis. These results will provide a new strategy for anti-Toxoplasma drug screening and clinical medication guidance.

Complete chloroplast genome of Lamiophlomis rotata: comparative genome analysis and phylogenetic analysis.

Lamiophlomis rotata, the only species within the genus Lamiophlomis (family Labiatae), exhibits a broad geographical distribution in elevated highland areas in Qinghai-Tibetan Plateau and possesses significant therapeutic properties. Numerous chemical compositions and putative phylogenetic affiliations of this species have been documented in prior research. Nevertheless, there is a scarcity of accessible publications regarding the genomic data of L. rotata, particularly its chloroplast genome. This dearth of knowledge hampers the comprehensive investigation of its phylogenetic placement within the Labiatae family. In this study, we present a comprehensive analysis of the plastid genome of L. rotata. The plastid genome has a length of 151,837 base pairs (bp) and a GC content of 38.5%. Within this genome, a total of 135 genes were identified, including 90 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. By employing phylogenetic analysis, the taxonomic position of L. rotata within the family Labiatae is elucidated, highlighting a close relationship between the genus Lamiophlomis and the genus Phlomis. Notably, extensive genetic variations were uncovered between L. rotata and other Phlomis species. This study could provide significant insights for understanding the phylogenetic relationships of taxa within Labiatae.

Genome evolution of the psammophyte Pugionium for desert adaptation and further speciation.

Deserts exert strong selection pressures on plants, but the underlying genomic drivers of ecological adaptation and subsequent speciation remain largely unknown. Here, we generated de novo genome assemblies and conducted population genomic analyses of the psammophytic genus Pugionium (Brassicaceae). Our results indicated that this bispecific genus had undergone an allopolyploid event, and the two parental genomes were derived from two ancestral lineages with different chromosome numbers and structures. The postpolyploid expansion of gene families related to abiotic stress responses and lignin biosynthesis facilitated environmental adaptations of the genus to desert habitats. Population genomic analyses of both species further revealed their recent divergence with continuous gene flow, and the most divergent regions were found to be centered on three highly structurally reshuffled chromosomes. Genes under selection in these regions, which were mainly located in one of the two subgenomes, contributed greatly to the interspecific divergence in microhabitat adaptation.

Species Identification in the Rhododendron vernicosum-R. decorum Species Complex (Ericaceae).

Delimitating species boundaries is the primary aim of biological classification and could be critical for evaluating the evolving process of species and conserving biodiversity. Rhododendron is an iconic group with an extraordinary diversity in southwest China. However, it remains unknown whether the recorded species therein comprise independently evolving lineages or artificially delimitated morphological entities. In this study, we carried out species delimitation of four Rhododendron species in the R. vernicosum-R. decorum species complex based on morphological analyses and population genetic data from nuclear simple sequence repeats (SSR) markers. We randomly selected a total of 105 specimens of different individuals identified as four species across their distributional ranges to examine the statistically distinct phenotypic clusters based on 19 morphological traits. Similarly, we genotyped 55 individuals of four species from 21 populations using 15 SSR markers. The morphological analyses sorted R. decorum and the other three species into two different phenotypic clusters. The genetic clusters were consistent with the morphological clusters. However, we also recovered the third genetic cluster, comprising six R. vernicosum populations and containing the admixed genetic compositions of the other two distinct genetic clusters. This hybrid group was morphologically similar to the typical R. vernicosum (including the samples from its type specimen locality and both R. verruciferum and R. gonggashanense) but with more genetic ancestry from R. decorum. Based on our findings, we identify two distinct species and one putative hybrid group due to introgression in the R. vernicosum-R. decorum species complex. We propose to merge R. verruciferum and R. gonggashanense into R. vernicosum based on genetic compositions and our morphological analyses. The hybrid group inferred from our findings, however, needs further investigations.

Hybrid speciation via inheritance of alternate alleles of parental isolating genes.

It is increasingly realized that homoploid hybrid speciation (HHS), which involves no change in chromosome number, is an important mechanism of speciation. HHS will likely increase in frequency as ecological and geographical barriers between species are continuing to be disrupted by human activities. HHS requires the establishment of reproductive isolation between a hybrid and its parents, but the underlying genes and genetic mechanisms remain largely unknown. In this study, we reveal by integrated approaches that reproductive isolation originates in one homoploid hybrid plant species through the inheritance of alternate alleles at genes that determine parental premating isolation. The parent species of this hybrid species are reproductively isolated by differences in flowering time and survivorship on soils containing high concentrations of iron. We found that the hybrid species inherits alleles of parental isolating major genes related to flowering time from one parent and alleles of major genes related to iron tolerance from the other parent. In this way, it became reproductively isolated from one parent by the difference in flowering time and from the other by habitat adaptation (iron tolerance). These findings and further modeling results suggest that HHS may occur relatively easily via the inheritance of alternate parental premating isolating genes and barriers.

A chromosome-scale reference genome of Lobularia maritima, an ornamental plant with high stress tolerance.

Lobularia maritima (L.) Desv. is an ornamental plant cultivated across the world. It belongs to the family Brassicaceae and can tolerate dry, poor and contaminated habitats. Here, we present a chromosome-scale, high-quality genome assembly of L. maritima based on integrated approaches combining Illumina short reads and Hi-C chromosome conformation data. The genome was assembled into 12 pseudochromosomes with a 197.70 Mb length, and it includes 25,813 protein-coding genes. Approximately 41.94% of the genome consists of repetitive sequences, with abundant long terminal repeat transposable elements. Comparative genomic analysis confirmed that L. maritima underwent a species-specific whole-genome duplication (WGD) event ~22.99 million years ago. We identified ~1900 species-specific genes, 25 expanded gene families, and 50 positively selected genes in L. maritima. Functional annotations of these genes indicated that they are mainly related to stress tolerance. These results provide new insights into the stress tolerance of L. maritima, and this genomic resource will be valuable for further genetic improvement of this important ornamental plant.

Ocular biometric parameters are associated with non-contact tonometry measured intraocular pressure in non-pathologic myopic patients.

PURPOSES: This research aimed to investigate effects and risk factors on non-contact tonometer (NCT) readings in healthy myopic subjects by employing cross-sectional study design. METHODS: Totally, sixty otherwise healthy myopic volunteers (mean 28.4 years old) with 90% female were recruited in ophthalmic clinic. The routine ophthalmic tests, refractive evaluation, examination central corneal thickness (CCT), depth of anterior chamber, axial length, corneal curvature, white-to-white and NCT were assessed at baseline. The linear-mixed model was utilized to evaluate correlation between the readings and ocular biometric parameters. RESULTS: For population in this study, mean spherical equivalents were - 4.85 ± 1.79 diopters in right eyes and - 4.63 ± 1.95 diopters in left eyes. Meanwhile, 28.3% of the eyes had a refractive error exceeding - 6.0 diopters. The mean NCT reading was 15.02 ± 3.02 mmHg in left eyes and 15.33 ± 2.96 mmHg in right eyes. Among the factors analyzed, CCT was the most significant parameter associated with NCT readings. After adjusting for the other factors, per one standard deviation increase of central corneal thickness (36.11 µm) was associated a 1.14 (95% confidence interval 0.53-1.77) mmHg elevated NCT reading. The average central corneal curvature, age and spherical equivalence were also significantly and independently associated with NCT readings. CONCLUSIONS: Central corneal thickness, age, corneal curvature and degree of myopia were independently associated with NCT measured intraocular pressure. Central corneal thickness is one of the most influential factors.

Ancient introgression drives adaptation to cooler and drier mountain habitats in a cypress species complex.

Introgression may act as an important source of new genetic variation to facilitate the adaptation of organisms to new environments, yet how introgression might enable tree species to adapt to higher latitudes and elevations remains unclear. Applying whole-transcriptome sequencing and population genetic analyses, we present an example of ancient introgression from a cypress species (Cupressus gigantea) that occurs at higher latitude and elevation on the Qinghai-Tibet Plateau into a related species (C. duclouxiana), which has likely aided the latter species to extend its range by colonizing cooler and drier mountain habitats during postglacial periods. We show that 16 introgressed candidate adaptive loci could have played pivotal roles in response to diverse stresses experienced in a high-elevation environment. Our findings provide new insights into the evolutionary history of Qinghai-Tibet Plateau plants and the importance of introgression in the adaptation of species to climate change.

The complete chloroplast genome sequence of an Alpine flowering plant Kuepferia otophora (Gentianaceae).

The complete chloroplast genome sequence of Kuepferia otophora, a flowering plant occurring in Hengduan Mountains with high altitudes, is determined in this study. The plastome is 139,684 bp in length, with one large single-copy region of 76,787 bp, one small single-copy region of 16,635 bp, and two inverted repeat (IR) regions of 23,131 bp. It contains 128 genes, including 83 protein-coding, 8 ribosomal RNA, and 37 transfer RNA genes. Phylogenetic tree shows that this species is a sister to the clade of genus Gentiana. The first published plastome within Kuepferia provides significant insight for elucidating the phylogenetic relationship of taxa within tribe Gentianeae.

A transcriptome-based resolution for a key taxonomic controversy in Cupressaceae.

Background and Aims: Rapid evolutionary divergence and reticulate evolution may result in phylogenetic relationships that are difficult to resolve using small nucleotide sequence data sets. Next-generation sequencing methods can generate larger data sets that are better suited to solving these puzzles. One major and long-standing controversy in conifers concerns generic relationships within the subfamily Cupressoideae (105 species, approx. 1/6 of all conifers) of Cupressaceae, in particular the relationship between Juniperus, Cupressus and the Hesperocyparis-Callitropsis-Xanthocyparis (HCX) clade. Here we attempt to resolve this question using transcriptome-derived data. Methods: Transcriptome sequences of 20 species from Cupressoideae were collected. Using MarkerMiner, single-copy nuclear (SCN) genes were extracted. These were applied to estimate phylogenies based on concatenated data, species trees and a phylogenetic network. We further examined the effect of alternative backbone topologies on downstream analyses, including biogeographic inference and dating analysis. Results: Based on the 73 SCN genes (>200 000 bp total alignment length) we considered, all tree-building methods lent strong support for the relationship (HCX, (Juniperus, Cupressus)); however, strongly supported conflicts among individual gene trees were also detected. Molecular dating suggests that these three lineages shared a most recent common ancestor approx. 60 million years ago (Mya), and that Juniperus and Cupressus diverged about 56 Mya. Ancestral area reconstructions (AARs) suggest an Asian origin for the entire clade, with subsequent dispersal to North America, Europe and Africa. Conclusions: Our analysis of SCN genes resolves a controversial phylogenetic relationship in the Cupressoideae, a major clade of conifers, and suggests that rapid evolutionary divergence and incomplete lineage sorting probably acted together as the source for conflicting phylogenetic inferences between gene trees and between our robust results and recently published studies. Our updated backbone topology has not substantially altered molecular dating estimates relative to previous studies; however, application of the latest AAR approaches has yielded a clearer picture of the biogeographic history of Cupressoideae.

Single-Port Laparoscopic Assisted Transcrotal Orchidopexy for Palpable Inguinal Canalicular Cryptorchidism Accompany With Indirect Inguinal Hernia.

Purpose: To assess the outcomes of a novel laparoscopic assisted transcrotal orchidopexy (LATO) combined with percutaneous extraperitoneal closure (PEC) for palpable inguinal canalicular cryptorchidism accompany with indirect inguinal hernia, and evaluate its safety and efficiency. Materials and Methods: A retrospective cohort study for single-port LATO-PEC and traditional inguinal orchidopexy (TIO) was performed between 2011 and 2014. Totally 53 children with both palpable inguinal canalicular testes and indirect inguinal hernia were included. Median patient age was 15month (range, 6 months to 4 years). Of them, 35 patients underwent LATO-PEC procedure, utilizing an umbilical trocar for laparoscope, transcrotal dissection for orchidopexy, and an inner two-hooked cannula for ligation of the patent processus at the level of the internal ring. Three of them were bilateral, 12 on the left side and 20 on the right. Eighteen patients received TIO, seven of them on the left side and 11 on the right. Patient demographics, surgical technique, complications, and clinical outcomes were reviewed. Follow-up visits were performed to reassess position and size of the testes. Results: All 56 undescended testes were delivered into the scrotum successfully. In the LATO-PEC group, nine contralateral herniorrhaphy were accomplished simultaneously. Fifteen contralateral patent processus vaginalis (PPVs) in 32 unilateral undescended testis (UDT) were newly confirmed during the laparoscopy, while 6 of them received percutaneous extra-peritoneal herniorrhaphy for visible inguinal bubble in pneumoperitoneum condition. No additional port placement or conversion to open procedure was needed. Mean operative time for unilateral and bilateral LATO-PEC in this study was (37.81 ± 5.23) min and (53.33 ± 2.98) min, respectively. In TIO group, mean operative time was (41.11 ± 8.67) min. There was no statistical difference in operative time between the two approaches for unilateral UDTs (p = 0.098). Median follow-up interval was 24 months (range, 12-84 months). No operative complications were found in either group to date. Conclusions: Singe-port LATO-PEC is a safe, effective, and cosmetic choice for inguinal canalicular cryptorchidism accompany with indirect inguinal hernia, minimizing injuries to the vas deferens and testicular vessels. Laparoscopy can provide a diagnostic and therapeutic solution of contralateral PPV.

Laparoscopic Duhamel Procedure with Ex-Anal Rectal Transection for Right-Sided Hirschsprung's Disease.

BACKGROUND: Increased defecation frequency and soiling are common complications of surgery for right-sided Hirschsprung's disease (HD). Though the laparoscopic Duhamel procedure is a favorable option in right-sided HD, the conventional laparoscopic technique is time consuming and has complications that are associated with the reservoir. In this study, we described a modified laparoscopic Duhamel technique with ex-anal rectal transection combined with the Deloyer's procedure for right-sided HD. MATERIALS AND METHODS: Between March 2010 and January 2015, 18 right-sided HD underwent this modified laparoscopic Duhamel procedure (MLDP). The main modifications were to mobilize the colon and to dissect the retrorectal space under laparoscopy, pull out, and transect ex-anally by using a linear stapling device through a transverse incision on posterior wall of the rectum. Seven age-matched patients who underwent a conventional laparoscopic Duhamel procedure (CLDP) between March 2008 and December 2010 were included as the control group. Demographics, surgical procedures, operative data, postoperative complications, and clinical outcomes were compared between these two groups. RESULTS: Operative time was significantly shorter in the MLDP group (150 minutes versus 195 minutes; P = .03). There was no significant difference in mean timespan to obtain a normal defecation frequency (3.8 months versus 3.6 months), incidence of soiling (11.1% versus 14.3%; P = .34), and constipation (5.6% versus 14.3%; P = .69). Pouchitis were not found in the MLDP group, whereas 2 patients in the CLDP group (28.6%) presented with pouchitis and intractable diarrhea, requiring spur division. The incidence of postoperative enterocolitis was not significantly different in the two groups (11.1% in MLDP versus 14.3% in CLDP; P = .68). All 22 patients had a normal defecation over 4 years of age. CONCLUSIONS: Laparoscopic Duhamel with ex-anal rectal transection is a simple, easy-to-learn, and effective procedure for right-sided HD. It has low postoperative pouchitis because of a short pouch.

Hybrid Nuss Procedure for Pectus Excavatum With Severe Retrosternal Adhesions After Sternotomy.

BACKGROUND: The most striking feature of pectus excavatum (PE) after previous congenital heart disease (CHD) surgery through a median sternotomy is the postsurgical adhesions between the sternum and heart. For patients with severe adhesions, passing the introducer can be difficult and hazardous when performing a Nuss repair. We describe a hybrid Nuss procedure using a small subxiphoid incision for blunt and sharp anterior mediastinal dissection and using a thoracoscope to ensure the whole process of dissection is under direct visualization. METHODS: A retrospective case review was conducted of PE patients (previous CHD operation) who had undergone the hybrid Nuss procedure between January 2012 and June 2015. Demographic, treatment, and outcome variables were recorded. RESULTS: Eleven patients were included. The mean age was 4.7 ± 1.7 years (range, 3.2 to 8.9). The mean Haller index based on computed tomography was 4.15 ± 0.78 (range, 3.2 to 5.8). All cases were the symmetric type. Mean operating time was 95.5 ± 8.4 minutes (range, 80 to 110); mean blood loss was 15 ± 2.3 mL; and mean length of hospitalization was 6.0 ± 1.4 days (range, 4 to 8). There was no pneumothorax, cardiac injury, wound infection, discomfort requiring removal, or bar rotation at the average 27-month (range, 9 to 50) follow-up. Two patients had the bars removed. No reoperations were performed owing to recurrence. CONCLUSIONS: The hybrid Nuss procedure is a safe, simple modification of the Nuss repair for patients with severe retrosternal adhesions. This procedure achieves dissection with direct visualization. Hence, the risk of death due to cardiac perforation can be prevented.