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by claude@2026-07, 2026-07-03
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This study developed and validated a genus-specific qPCR toolkit using a hypervariable mitochondrial 16S rRNA target to detect and identify cavefish from the Sinocyclocheilus genus in karst habitats of southwestern China, where direct sampling is difficult. Using laboratory and field evaluations across 47 representative species, the authors reported high specificity and a detection limit of ~20 DNA copies per reaction, while controlled degradation experiments showed that temperature and pH strongly influence eDNA persistence. Field surveys across 47 karst sites detected Sinocyclocheilus eDNA in 33 caves only, and sequencing of qPCR amplicons enabled species-level identification and phylogenetic validation, including discovery of a new species; in a subset of sites, 16S metabarcoding was used for species discrimination. The work’s main caveats include that eDNA detection depends on environmental conditions affecting DNA persistence, which likely contributed to non-detection in some caves. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
Abstract
ABSTRACT Environmental DNA (eDNA) enables non-invasive detection of aquatic species through trace genetic material, but uncertainties regarding DNA persistence, detection accuracy, and the limited taxonomic resolution of stygian fauna have constrained its use in subterranean aquatic environments. The species-rich cavefish genus Sinocyclocheilus , endemic to the karst landscapes of southwestern China, offers an excellent model for understanding these issues. With over eighty species, many of which are endemics, and many still being described, this lineage is taxonomically rich, poorly understood, and highly threatened. However, access to these deep habitats remains challenging, making eDNA detection one of the most practical methods for documenting and monitoring them. We developed and validated a qPCR assay targeting a hypervariable region of the mitochondrial 16S rRNA gene, allowing for genus-specific detection and species-level identification. Laboratory and field evaluations involving 47 representative species confirmed high specificity and a detection limit of around 20 DNA copies per reaction (R² = 0.995). Controlled degradation studies indicated that temperature and pH strongly affect DNA persistence. Field surveys across 47 karst sites (37 caves and 10 surface sites) detected Sinocyclocheilus eDNA in only 33 caves. Dideoxy sequencing of qPCR amplicons provided species-level identification and phylogenetic validation, including detection of a new species. At three cave sites yielding multiple qPCR detections, 16S metabarcoding enabled species discrimination. By integrating qPCR assay validation, eDNA degradation modelling, hierarchical genetic identification via dideoxy sequencing, and selective NGS metabarcoding, this study presents a strategic framework for applying eDNA methods in subterranean environments.
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ABSTRACT
Environmental DNA (eDNA) enables non-invasive detection of aquatic species through trace genetic material, but uncertainties regarding DNA persistence, detection accuracy, and the limited taxonomic resolution of stygian fauna have constrained its use in subterranean aquatic environments. The species-rich cavefish genus Sinocyclocheilus, endemic to the karst landscapes of southwestern China, offers an excellent model for understanding these issues. With over eighty species, many of which are endemics, and many still being described, this lineage is taxonomically rich, poorly understood, and highly threatened. However, access to these deep habitats remains challenging, making eDNA detection one of the most practical methods for documenting and monitoring them. We developed and validated a qPCR assay targeting a hypervariable region of the mitochondrial 16S rRNA gene, allowing for genus-specific detection and species-level identification. Laboratory and field evaluations involving 47 representative species confirmed high specificity and a detection limit of around 20 DNA copies per reaction (R² = 0.995). Controlled degradation studies indicated that temperature and pH strongly affect DNA persistence. Field surveys across 47 karst sites (37 caves and 10 surface sites) detected Sinocyclocheilus eDNA in only 33 caves. Dideoxy sequencing of qPCR amplicons provided species-level identification and phylogenetic validation, including detection of a new species. At three cave sites yielding multiple qPCR detections, 16S metabarcoding enabled species discrimination. By integrating qPCR assay validation, eDNA degradation modelling, hierarchical genetic identification via dideoxy sequencing, and selective NGS metabarcoding, this study presents a strategic framework for applying eDNA methods in subterranean environments.
Competing Interest Statement
The authors have declared no competing interest.
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