The specimens were deposited at the Chengdu Institute of Biology, Chinese Academy of Sciences. The holotype was collected at 27.5065° N, 111.4597° E at approximately 350 metres above sea level. All three animals were found on plant leaves near mountain streams with relatively torrential flow. Animal procedures were conducted under a permit from the Chengdu Institute of Biology’s Animal Care Advisory Committee.

For the molecular analysis, researchers extracted DNA from muscle tissue and sequenced fragments of the mitochondrial 16S rRNA and cytochrome c oxidase subunit I, or COI, genes. They combined these new sequences with available GenBank data for related species, aligned them in BioEdit and analysed the concatenated dataset using maximum likelihood and Bayesian inference.

The concatenated dataset comprised 528 base pairs of 16S and 612 base pairs of COI. A GTR + G + I substitution model was selected using the Bayesian information criterion. Maximum-likelihood support was assessed with 1,000 bootstrap replicates. The Bayesian analysis used four Markov chains for 50 million generations, sampled every 1,000 generations, with the first 25% of sampled trees discarded as burn-in.

The maximum-likelihood and Bayesian trees had nearly consistent topologies, although relationships among many broader lineages remained unresolved. The Xinshao specimens formed a distinct lineage within Leptobrachella, with a Bayesian posterior probability of 1.00 and a maximum-likelihood bootstrap value of 100%. This lineage was sister to a clade containing L. wulingensis and L. yongshunensis.

Uncorrected sequence distances also distinguished the Xinshao population from its congeners. The smallest 16S divergence was 2.1% against L. wulingensis, while the minimum COI distance was 8.1% against L. yongshunensis. The authors reported that these values were equal to or greater than the differentiation observed between some recognized species pairs, including L. bijie and L. jinshaensis, and L. dong and L. dushanensis.

The diagnosis identifies L. xinshaoensis by a combination of characters. Males have a snout–vent length of 27.8–31.4 millimetres. Their toes have rudimentary webbing and narrow lateral fringes; the tympanum is distinct and surrounded by distinct tubercles; and the dorsal surface is shagreened with small, raised tubercles and longitudinal ridges. The ventral surface is creamy white with dark brown spots on the chest and margins, and black spots occur on the flanks. When the hindlimbs are flexed at right angles to the body axis, the heels do not meet.

Measurements and external features separate the new species from its closest relatives. Unlike L. wulingensis, whose heels meet when the hindlimbs are flexed, L. xinshaoensis has a tibia–tarsal articulation reaching between the eye and tympanum rather than the middle of the eye. In L. yongshunensis, the heels overlap, the tibia–tarsal articulation reaches the anterior edge of the eye, and the dorsal skin bears sparse large warts, dense small wart grains and, in some specimens, longitudinal ridges. Comparisons with 83 congeners north of the Isthmus of Kra and 27 species south of it also distinguished the new taxon by body size, glandular anatomy, coloration, toe webbing, limb proportions and skin texture.

The type specimens showed variation in preserved coloration. The holotype had a brown dorsum with an inverted triangular marking between the eyes and a W-shaped marking on the anterior mid-dorsum. Black flank spots occurred among two rows of large warts; the supra-axillary gland was orange; and the upper forelimbs were orange with black transverse bars below. Both paratypes were darker dorsally. One had a whiter abdomen, while the posterior abdominal margin of the other was pale purple. Adult males had subgular vocal sacs and femoral adipose glands, but no nuptial pads or spines.

The specific name xinshaoensis refers to Xinshao County, the type locality. The authors propose the English name “Xinshao Leaf-litter Toad” and the Chinese name “Xin Shao Zhang Tu Chan (新邵掌突蟾).” The species is currently known only from the type locality, and the survey found no females, eggs or tadpoles; late March is therefore described as a speculated breeding season rather than an observed one. Given its restricted known distribution and dependence on specific stream-side habitat, the authors recommend assessing its conservation status in future evaluations and advocate more intensive biodiversity surveys in central Hunan, where other cryptic species may occur.

Subject of Research: Description of Leptobrachella xinshaoensis from Xinshao County, Hunan, China, using integrative morphological and mitochondrial DNA analysis

Article Title: Morphological and molecular evidence support a new species of the genus Leptobrachella ( Anura , Megophryidae ) from central China

Article References: Jiang, L., Liu, J., Huang, Y., Zhang, H., & Wang, B. (2026). Morphological and molecular evidence support a new species of the genus Leptobrachella (Anura, Megophryidae) from central China. Zoosystematics and Evolution, 102(1), 253-269. https://doi.org/10.3897/zse.102.175462

Image Credits: AI Generated

DOI: 10.3897/zse.102.175462

Keywords: Leptobrachella xinshaoensis, new frog species, Hunan China, integrative taxonomy, morphological analysis, mitochondrial DNA, 16S rRNA, COI gene, cryptic species, amphibian diversity, Morphological, molecular

The researchers analyzed 16S rRNA gene amplicon sequences, shotgun metagenomes and metatranscriptomes generated from the same lakes. Sequencing depth was 3.31 Gbps for amplicons, 170.91 Gbps for metagenomes and 34.11 Gbps for metatranscriptomes. They tested three ways of making the datasets comparable: mapping shotgun reads to amplicon-derived operational taxonomic units, or OTUs; profiling each dataset with a method-specific taxonomic marker; and attempting to recover 16S rRNA genes from shotgun assemblies for a cross-method OTU library.

The assembly-based strategy was discontinued because recovered sequences had highly variable lengths and included chimeras longer than 1,600 base pairs. These sequences could not be compared meaningfully with the V9 region represented by the amplicon OTUs. The analysis therefore focused on the two remaining approaches, which converged on the finding that sequencing technology introduces systematic differences in the resulting community profiles.

In the OTU-based comparison, 2,535 representative amplicon OTUs passed a relative abundance threshold of 0.001% per sample. They recruited 9,356,192 amplicon reads, 154,996 metagenomic reads and 19,979,498 metatranscriptomic reads, with every OTU recruiting reads across all samples. Although the technologies represented the same 26 prokaryotic phyla, alpha-diversity estimates differed significantly. Amplicon data produced lower richness and Shannon diversity than both shotgun datasets, while metagenomes and metatranscriptomes did not differ significantly in those measures. Friedman tests gave p-values below 0.001, with Kendall’s W values of 0.76 for richness and 0.84 for Shannon diversity.

Marker-specific profiling produced a more pronounced ordering. The researchers used the single-copy rpS3 gene for metagenomes, rRNA reads for metatranscriptomes and amplicon OTUs as an independent reference. This analysis represented 23 prokaryotic phyla in metagenomes and 51 in metatranscriptomes. For every lake, estimated diversity ranked metatranscriptomes above metagenomes and metagenomes above amplicons. The consistent ranking across lakes indicates that these differences were systematic methodological effects rather than lake-specific patterns.

Community-level comparisons also depended on the analytical strategy. PERMANOVA attributed approximately 16% of variation in relative-abundance data and 13% of variation in presence-absence data to sequencing approach, with method effects reaching about 41% in the marker-based analysis. Metatranscriptomic profiles were the most homogeneous, while amplicon profiles were the most heterogeneous. Procrustes and Mantel tests showed greater concordance between metagenomes and metatranscriptomes than between either shotgun dataset and amplicons. Among the OTU-based comparisons, metagenomes and metatranscriptomes had the strongest congruence, with R² = 0.68 and P = 0.001.

Differential-abundance analysis identified method-specific taxonomic patterns. Relative to metagenomes, amplicon data contained more OTUs assigned to Alphaproteobacteria and Cyanobacteria and fewer assigned to Gammaproteobacteria, mainly Burkholderiales. Polynucleobacter and Limnohabitans were strongly underrepresented in the amplicon data, matching a pattern previously attributed to primer bias in the same lakes. Metagenomes had higher relative abundances of Cytophagales and Chitinophagales within Bacteroidetes, whereas amplicons overrepresented Flavobacteriales. Corrections based on 16S rRNA gene copy number and a weighted primer score did not materially alter community composition, with Spearman correlations of 0.9925 and 0.9968, respectively.

Metatranscriptomes and metagenomes differed in only six OTUs by differential abundance. However, the DNA dataset used as a reference affected conclusions about transcription. Relative to metagenomes, 22 taxonomic classes had similar proportions in the metatranscriptomic and metagenomic data, and Clostridia, including Clostridium species, appeared transcriptionally active. When amplicon data served as the baseline, a smaller proportion of that population appeared to be transcribing 16S rRNA genes. The comparison therefore depended on the reference method, not only on the metatranscriptomic measurements themselves.

Four analytical approaches—Mantel tests, Procrustes analyses, BioEnv and distance-based redundancy analysis with stepwise selection—were used to assess how well each dataset reflected environmental variation. Metatranscriptomes showed the strongest global community–environment associations in the Mantel and Procrustes analyses, while metagenomes produced the most stable explanatory OTUs in the BioEnv and redundancy analyses. The full metagenomic redundancy model explained about 20% of environmental variation, but forward selection retained one Cyanobacteria-affiliated OTU that explained 9.4% of the variation, with an adjusted R² of 0.094, F = 3.08 and p = 0.021. Neither amplicon nor metatranscriptomic datasets produced significant models after stepwise selection.

The findings identify trade-offs rather than a universally preferred method. Amplicon sequencing is comparatively rapid and inexpensive but is affected by primer and amplification biases. Metatranscriptomics captures transcriptional activity but is sensitive to RNA degradation and transcriptional dynamics. Metagenomics offers broad genomic context and stable explanatory features, but requires greater sequencing and computational resources and can have reduced sensitivity to rare organisms. The observational comparison was limited to 21 European lakes and does not establish which approach is best under all conditions; the authors noted that controlled experiments such as batch cultures would be required for that determination.

Subject of Research: Comparison of amplicon, metagenomic and metatranscriptomic methods for studying lake microbiomes and their relationships with environmental conditions

Article Title: Constraining lake ecology via metagenomics, metatranscriptomics, and amplicon sequencing

Article References: Nuy, J. K., Bornemann, T. L. V., Beisser, D., Probst, A. J., & Boenigk, J. (2025). Constraining lake ecology via metagenomics, metatranscriptomics, and amplicon sequencing. Metabarcoding and Metagenomics, 9, Article e156215. https://doi.org/10.3897/mbmg.9.156215

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.156215

Keywords: lake microbiomes, amplicon sequencing, metagenomics, metatranscriptomics, 16S rRNA, microbial diversity, environmental variation, European lakes, Constraining, lake, ecology, amplicon

The study tested whether DNA deposited or regurgitated by carrion flies could support surveys of terrestrial vertebrate diversity without directly observing or capturing animals. It also examined two ways to pool fly samples for sequencing and a field collection method designed to reduce the time required to survey large numbers of locations.

Sampling took place at Los Amigos Biological Station in the Madre de Dios region of the western Amazon, where the landscape includes old-growth terra firme forest, floodplain forest, dense stands of Guadua bamboo and Mauritia flexosa palm swamps. The researchers collected flies at 24 locations along six trail segments, three in floodplain habitat and three in terra firme habitat, over three days.

At each site, the team netted 10 flies directly from a white plastic grocery bag loosely tied around an open bag of rotting pork. Each fly was placed in a sterile 1.5- or 2-milliliter tube. After 3–5 hours at ambient temperature to allow flies to regurgitate or deposit vertebrate DNA, the flies were removed and preserved in 90% ethanol for morphological identification. The remaining tubes were dried with silica gel, stored at minus 20 degrees Celsius and later transferred to Utah State University, where they were held at minus 70 degrees Celsius. Because carrion flies were often difficult to identify beyond family level, the most common families were Calliphoridae, Sarcophagidae, Mesembrinellidae and Tachinidae.

In the laboratory, direct PCR lysis buffer and proteinase K were added to the tubes, followed by vortexing, incubation and heat treatment. The resulting solution was used directly as the PCR template, without macerating whole flies, dissecting their digestive tracts, or formally extracting and purifying DNA. The PCR targeted an approximately 138-base-pair fragment of the mitochondrial 16S rRNA gene and included blocking primers for human and domestic pig DNA. The authors noted that environmental DNA extraction kits typically cost US$3–8 per sample and that extracting 24 samples commonly takes 2–4 hours, although the study did not directly compare extraction-free PCR with conventional extraction.

Following quality filtering, denoising and chimera removal, the individual-fly and template-pool libraries retained 2,547,579 reads, while the PCR-product-pool library retained 3,086,005 reads. The researchers used BLAST and a naïve Bayes classifier to assign operational taxonomic units, supplementing uncertain assignments with local expertise and a published species checklist. After human and domestic-species sequences, the bobcat positive control and sequences that could not be assigned to family level were removed, 40 operational taxonomic units representing 31 wild vertebrate taxa remained. These included 27 mammalian taxa and four anurans, although 26 mammalian taxa were assigned to species known from the study area, three only to genus and two only to family. Two family-level assignments were designated presumed species based on local fauna knowledge and reference-sequence limitations.

Vertebrate DNA was recovered from 44.2% of individual flies, and some flies carried DNA from several vertebrates: 63 contained one taxon, 31 contained two, 10 contained three, one contained four and one contained five. Black spider monkey was detected in 40 of 240 flies, Bolivian squirrel monkey in 31 and lowland tapir in 14. The researchers described the eastern pygmy marmoset detection as uncertain because the species had not been observed at the study site, the top BLAST match was 93.4% to a related species and the naïve Bayes classifier supported only a genus-level assignment. They proposed DNA from a dead marmoset drifting downriver as one possible explanation for its detection in 10 flies, not a confirmed cause. A white-lipped peccary assignment was also inferred from local context after both classifiers produced a problematic match to Chacoan peccary, while a tayra assignment was inferred from a mustelid-level classification and local fauna knowledge.

The pooling comparison showed substantial differences between approaches. Template pooling, which combined lysis-buffer templates before the initial PCR, detected a mean of 2.25 vertebrate species per site, compared with 4.96 from individually sequenced flies, and reproduced the individual-fly species list at only one of 24 sites. PCR-product pooling, in which individual flies were first amplified and their products combined before indexing, detected a mean of 5.04 species per site and 29 species overall, the same total as the individual-fly analysis. Rarefying the PCR-product pools to the template-pool read depth changed only two site-level detections, indicating that sequencing depth alone did not explain the difference between the pooling methods.

The findings show that extraction-free carrion-fly iDNA can recover a broad range of rainforest vertebrates, including arboreal primates, at one Peruvian research site while reducing laboratory processing relative to maceration or digestive-tract extraction. Post-PCR pooling also closely reproduced individual-fly detections in this setting and may reduce sequencing costs. The study was limited to 240 flies from one site over three days, and capture rates may vary with location, weather, season and day. Taxonomic precision was constrained by the short 16S marker and incomplete reference sequences, while the absence of a direct comparison with formal DNA extraction prevents claims that the workflows are equivalent in every setting.

Subject of Research: Evaluation of extraction-free carrion-fly iDNA metabarcoding for surveying rainforest mammals

Article Title: An efficient iDNA method for surveying rainforest mammals with carrion flies

Article References: Rodgers, T. W., Basto, A., Castro, R., Groves, E. M. R., Xu, C. C. Y., Hernández, E., Mock, K. E., & Forsyth, A. (2026). An efficient iDNA method for surveying rainforest mammals with carrion flies. Metabarcoding and Metagenomics, 10, Article e174834. https://doi.org/10.3897/mbmg.10.174834

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.174834

Keywords: carrion flies, invertebrate-derived DNA, environmental DNA, mammal surveys, Amazon rainforest, direct PCR, DNA metabarcoding, sample pooling, biodiversity monitoring, 16S rRNA, efficient, iDNA

The study examined commercially available, primarily acidic concentrates purchased in Klaipėda, Lithuania, from seven local manufacturers, designated M1 through M7. Two production time points representing separate batches were sampled for each product, with three independently processed biological replicates. The beverages included drinks made from grains such as buckwheat, oats and rice, along with products formulated with lactic acid bacteria, Bifidobacterium cultures, honey or juices. Detailed pH, ethanol and residual-sugar measurements were unavailable because manufacturers did not provide them and the researchers did not measure them.

Researchers extracted DNA from homogenized beverages and used amplicon sequencing on an Illumina NextSeq 2000 platform. The V3–V4 region of the bacterial 16S ribosomal RNA gene was targeted to characterize bacteria, while the fungal internal transcribed spacer 2 region was used for fungi. DADA2 processing, taxonomic assignment against SILVA 138.2 and UNITE 10.0, and contamination filtering produced 6,352,987 bacterial reads representing 1,332 amplicon sequence variants and 1,295,070 fungal reads representing 133 variants across 42 samples. Following rarefaction and removal of rare variants, the retained datasets contained 375 bacterial and 65 fungal variants.

Lactic acid bacteria were prominent across many beverages, including Lacticaseibacillus, Limosilactobacillus, Lactobacillus, Streptococcus and Sporolactobacillus. Bifidobacterium was detected intermittently, while Acetobacter and other genera also occurred. Community structure differed markedly among manufacturers: Lactobacillus represented approximately 41–85.5% of detected bacterial genera in selected M3, M4, M5 and M6 samples, whereas Clostridium accounted for 72.1% of M2’s second-batch community. M7 had more than 50% relative abundance of Weizmannia in both batches. Fungal profiles included the fermentative yeast Saccharomyces, secondary yeasts such as Pichia, and numerous environmental or crop-associated genera.

Production-batch changes were also observed within manufacturers. In M3, Lactobacillus declined from 58.6% of bacterial reads in the first batch to 17.5% in the second, while Acetobacter rose from 0.78% to 56.1%. In M5, Lactobacillus fell from 85.5% to 13%, accompanied by an increase in Limosilactobacillus. Fungal dominance also shifted among batches. For example, Fusarium represented 96.1% of the fungal community in M4’s first batch but 31.0% in its second, while environmental yeasts changed in relative prominence in several other products. By contrast, M4 retained broadly consistent bacterial dominance by lactic acid bacteria across its two batches.

Statistical comparisons supported these observed differences. The number of observed bacterial variants differed significantly among manufacturers in a Kruskal–Wallis test (p = 7.14 × 10^-6), whereas fungal richness did not show a significant difference (p = 0.396). PERMANOVA identified significant effects of manufacturer and batch, as well as their interaction, for both bacterial and fungal communities; all three effects had p-values of 0.001 in the bacterial analysis, while batch and interaction had p-values of 0.023 and 0.024 in the fungal analysis. Manufacturer, batch and their interaction together accounted for 98.02% of bacterial variation and 55.53% of fungal variation. However, significant differences in within-group dispersion indicate that heterogeneity may also have influenced the PERMANOVA results.

Functional annotation assigned bacterial profiles through FAPROTAX and fungal profiles through FUNGuild, followed by literature checks and manual classification. Most resolved bacterial variants were categorized as beneficial under the study’s criteria, and most manufacturers had bacteria classified as potentially harmful below 1% relative abundance. Exceptions included samples from both M6 batches and M3’s first batch. In M6’s second batch, 84.3% of classified bacterial taxa were categorized as potentially harmful or undesirable, including taxa associated with spoilage or off-flavor formation such as Clostridium tyrobutyricum and Anoxybacillus flavithermus. Detection of Clostridium perfringens was treated as a signal requiring follow-up, not confirmation of pathogenicity.

Fungal functional assignments were less clear because many taxa had mixed, unclear or plant-associated ecological roles. M4, M5 and M6 had higher proportions of genera classified as potentially harmful, including Fusarium and Issatchenkia. The high Fusarium signal in M4’s first batch is relevant because some Fusarium species can produce mycotoxins under suitable conditions and cereal substrates are associated with these compounds. Nevertheless, the assay detected DNA rather than viability or active toxin production, so the signal could reflect fungal material carried through from malted barley, wheat, rye or another raw ingredient.

The study has important methodological limits. Amplicon sequencing does not establish that detected organisms are viable, metabolically active or present as pathogenic strains, and conserved markers often limit identification below genus level. PCR bias, gene copy-number variation, compositional data and gaps in reference databases can affect abundance estimates and classification. Several low-fungal-read samples were removed, including all fungal samples from M7’s second batch, further limiting comparisons. The authors therefore proposed a tiered monitoring system: retain standard culture assays for baseline compliance, add periodic 16S and ITS profiling to identify community drift and out-of-panel taxa, and use targeted strain-specific quantitative PCR, viability assays and rapid LC–MS/MS mycotoxin screening for verification. Within that framework, the study positions metabarcoding as a complementary first-pass method rather than a substitute for culture-based safety testing or targeted chemical analysis.

Subject of Research: Metabarcoding assessment of bacterial and fungal communities in fermented grain beverages across seven manufacturers and two production batches

Article Title: High-throughput microbiome profiling of fermented grain drinks: insights across manufacturers and batches

Article References: Scriver, M., Zaiko, A., Gudaitytė, R., & Samuilovienė, A. (2026). High-throughput microbiome profiling of fermented grain drinks: insights across manufacturers and batches. Metabarcoding and Metagenomics, 10, Article e191385. https://doi.org/10.3897/mbmg.10.191385

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.191385

Keywords: fermented beverages, metabarcoding, 16S rRNA, fungal ITS2, bacterial communities, batch consistency, food microbiology, microbial quality, probiotic bacteria, High-throughput, microbiome, profiling

Boulenophrys , a group of frogs within the subfamily Megophryinae, currently comprises 81 recognised species distributed from eastern and central China across south-eastern Asia to the Sunda Shelf islands, the Philippines, and westwards to Nepal and north-eastern India. Fifty-seven of those species have been described in the last decade alone, and as many as ten new species were published in 2025 by various research groups. The path to the new species began in June 2023, when the survey team collected seven adult males and one adult female from a mountain stream surrounded by evergreen broadleaved forest.

The specimens were euthanised using isoflurane, fixed in 10 percent buffered formalin, and later transferred to 75 percent ethanol for long-term preservation. Muscle tissue for molecular work was stored in 95 percent ethanol at minus 20 degrees Celsius. All voucher specimens were deposited at the Moutai Institute in Guizhou Province, and the survey and handling procedures were approved by the Forestry Bureau of Tianlin County and the Laboratory Animal Ethics Committee of the Moutai Institute.

To determine where the mystery toads sat on the amphibian family tree, the researchers extracted DNA from muscle tissue and amplified two mitochondrial markers: a 525 base pair fragment of the 16S rRNA gene and a 558 base pair fragment of the cytochrome c oxidase subunit I gene, or COI. These were concatenated into a single 1,083 base pair sequence for phylogenetic analysis. The team assembled and aligned sequences using the ClustalW module in BioEdit, selected the best partition scheme and evolutionary model with PARTITIONFINDER, which settled on the GTR plus G plus I model for each partition, and then reconstructed evolutionary relationships using both Maximum Likelihood methods in PhyML and Bayesian Inference in MrBayes.

All Boulenophrys samples formed a well-supported monophyletic clade, and the Tianlin lineage emerged as the sister group to B. anlongensis , a species described from Anlong County in neighbouring Guizhou Province, with maximal Bayesian posterior probability of 1.00 and a bootstrap support of 98 percent. Genetic distances told the same story: within the undescribed taxon, 16S divergence was zero and COI divergence stayed below 0.2 percent, but between the Tianlin toads and B. anlongensis the distance reached 1.4 percent for 16S and 8.2 percent for COI. Those figures are comparable to divergence levels considered valid for species delimitation in congeneric anurans, and they stand in contrast to some closely related species pairs in the genus, such as B. binlingensis , B. yezhongensis and B. binchuanensis , which differ by only 0.3 percent at 16S.

Morphology provided the second line of evidence. The researchers measured seventeen morphometric characters on the eight Tianlin specimens and on five male B. anlongensis , using dial calipers accurate to 0.1 millimetre. Mann-Whitney U tests on the raw measurements showed that B. anlongensis was significantly larger in snout-vent length and in most other traits, with p-values of 0.004 for many characters including head width, tympanum diameter, hind-limb length and tibia width.

Only interorbital space, eye diameter and thigh length failed to reach statistical significance, with thigh length approaching the threshold at p equals 0.051. Because much of this difference could simply reflect overall body size, the team corrected each character for allometry by dividing it by snout-vent length and log-transforming the result before running a principal component analysis. Even after removing the effect of body size, the two species remained clearly separated.

The first two principal components of the size-corrected male data accounted for 53.8 percent of total variance, with the first component, driven largely by interorbital space, hind-limb length and lower arm diameter, explaining 36.3 percent and the second, dominated by head width, explaining 17.5 percent. Specimens of the new species and B. anlongensis clustered apart along the first component axis, confirming that the two taxa differ not only in size but in body proportions as well. The diagnostic features of Boulenophrys tianlinensis are a distinctive combination.

It is a medium-sized frog, with males measuring 34.3 to 39.0 millimetres in snout-vent length and the single known female reaching 47.1 millimetres. Each upper eyelid margin bears one small horn-shaped tubercle. Vomerine ridges are present but vomerine teeth are absent, the tongue is not notched posteriorly, and the tympanum is rounded and clearly visible.

The toes carry only rudimentary webbing and lack lateral fringes, the heels overlap when the thighs are held perpendicular to the body axis, and the tibiotarsal articulation reaches the middle of the eye when the leg is stretched forwards. Males possess a single subgular vocal sac, and breeding males develop dark brown nuptial spines on the bases of the first and second fingers. Compared directly with B. anlongensis , the new species is significantly smaller in nearly every measured trait, lacks the narrow lateral fringes on its toes, and shows a different relative finger length order.

The holotype, an adult male with the catalogue number MT TL20230603004, was collected by Jing Liu on 3 June 2023 at the type locality. In life, the animal displays a dark brown dorsum with an inverted triangular brown speckle between the eyes, X-shaped ridges on the back, four transverse bands on the thighs and shanks, alternating dark and white bars along the lip margins, and a brown-and-white mottled belly. Individual variation among the paratypes is considerable, ranging from darker animals with indistinct striping to lighter ones with prominent brown patches, and the female carries dense black spines on the warty granules of her dorsum and flanks.

The species appears to favour stones in streams and gorges within evergreen broadleaved forest, where it lives alongside at least four other amphibian species, including Boulenophrys jingdongensis , Kurixalus odontotarsus , Amolops sinensis and Leptobrachella ventripunctata . The discovery also carries a broader message about how new species arise in this corner of Asia. Southwest China is a globally recognised biodiversity hotspot, and the authors argue that its karst canyon landscapes, with deep gorges and isolated peaks, act as robust geographic barriers that restrict gene flow, create microrefugia and drive allopatric speciation, a pattern particularly pronounced in groups like Boulenophrys that disperse poorly and occupy narrow ecological niches.

The straight-line distance between the type localities of the new species and B. anlongensis exceeds 100 kilometres, with the Nanpan River and intervening canyons forming effective dispersal barriers. Much remains unknown about the Tianlin Horned Toad: there are no data yet on its population size, breeding ecology or the threats it may face from climate change and human disturbance, and the authors stress that systematic surveys and long-term monitoring are needed to safeguard the species in these fragile karst ecosystems.

Subject of Research: Description of a new horned toad species, Boulenophrys tianlinensis, from western Guangxi, China

Article Title: A new species of the genus Boulenophrys ( Anura , Megophryidae ) from western Guangxi, China

Article References: Liu, J., Cheng, Y., Shen, T., Feng, C., Mu, L., Zhou, J., Wei, G., & Li, S. (2026). A new species of the genus Boulenophrys (Anura, Megophryidae) from western Guangxi, China. Zoosystematics and Evolution, 102(5), 1139-1157. https://doi.org/10.3897/zse.102.193995

Image Credits: AI Generated

DOI: 10.3897/zse.102.193995

Keywords: Boulenophrys tianlinensis, horned toad, new species, Guangxi, China, amphibian taxonomy, megophryid frogs, molecular phylogenetics, karst biodiversity, 16S rRNA, COI gene, cryptic species

For the research study, the authors combined direct RNA sequencing with complementary cDNA-based approaches and meta-analysis of publicly available RNA-seq datasets. The researchers assessed post-transcriptional modifications in the chloroplast 16S and 23S rRNAs of Arabidopsis thaliana. They also examined the plastid counterpart PFC1 of the bacterial dimethyltransferase KsgA and used single-molecule direct RNA sequencing to analyze defined 16S and 23S rRNA processing states. The reported detection of stage-specific modification-associated signatures was consistent with modifications being installed stepwise during plastid ribosome biogenesis.

Functional analysis of PFC1 indicated that loss of N6,N6-dimethyladenosine formation impaired 16S rRNA maturation, delayed chloroplast ribosome assembly and affected photosynthetic performance in young leaves. The study therefore associates this RNA modification with rRNA maturation and ribosome assembly while providing experimental evidence for a functional effect in young leaf tissue. The authors described the overall pattern as showing that plastid ribosomes retain a prokaryotic-type functional core, alongside the plant-lineage-associated C1940 modification that was not found in bacteria.

The work is a plant molecular-biology study centered on Arabidopsis, with comparative sequence evidence used to examine conservation across bacteria and plant lineages. Its functional experiments focused on one modification-related protein and the reported photosynthetic effect was specific to young leaves, so the abstract does not establish effects across all tissues, developmental stages or plant species. Only the supplied abstract and publisher metadata were available for this report; the source also notes that the accessible accepted version may undergo further edits before replacement by the final Version of Record.

Subject of Research: Mapping chloroplast rRNA modifications associated with ribosome maturation and assembly in Arabidopsis thaliana

Article Title: Integrative mapping of chloroplast rRNA modifications reveals conserved and lineage-specific features and stepwise modification during plastid ribosome biogenesis

Article References: Gołębiewska, K., Duszyn, M., Piątkowski, J., & Gawroński, P. (2026). Integrative mapping of chloroplast rRNA modifications reveals conserved and lineage-specific features and stepwise modification during plastid ribosome biogenesis. BMC Plant Biology. https://doi.org/10.1186/s12870-026-09896-7

Image Credits: AI Generated

DOI: 10.1186/s12870-026-09896-7

Keywords: Arabidopsis thaliana, chloroplasts, rRNA modifications, ribosome biogenesis, 16S rRNA, 23S rRNA, PFC1, KsgA, photosynthesis, RNA sequencing, Integrative, mapping

The specimens were collected during periods of maximum low tide in the upper intertidal and supralittoral zones. Two individuals were collected from Jindo on 11 July 2024, four from Suncheon on 23 August 2024 and three from Muan on 13 August 2025. They occurred within dense patches of saltmarsh vegetation on moist, muddy substrates that remained saturated when exposed by the retreating tide. No individuals were found in the middle or lower intertidal zones during the collections. The observations indicate that the species can occur in vegetation-covered upper-intertidal habitats, but they do not establish its abundance or distribution throughout the tidal flat.

Members of Onchidiidae are shell-less, air-breathing gastropods that inhabit intertidal environments. The family has particularly high species richness in coastal ecosystems of Southeast Asia, while records from Korea have been extremely limited. Species boundaries and nomenclatural assignments within the family have historically been difficult to resolve using morphology alone. Recent studies have consequently used integrative approaches that combine morphological observations with molecular data, allowing species delimitation to be assessed through more than one type of evidence.

Nine specimens were examined in the present study. One was a juvenile measuring 17 millimetres in body length; the other eight measured 43–74 millimetres in body length and 27–40 millimetres in width. The dorsal notum was grey to yellowish-grey and carried numerous fine papillae distributed across the surface. A distinctly enlarged central papilla, or peduncle, was present in the middle of the notum. The ventral foot was light yellow to beige, and the mantle was light grey with scattered black spots in some individuals, while some specimens lacked those spots. These features corresponded with descriptions of O. reevesii previously reported from China.

Radular morphology provided an additional morphological check on the identification. Researchers dissected the buccal mass from specimen MA2, collected at Muan, and isolated the radula after the surrounding tissues had been dissolved in sodium hydroxide. The structure was cleaned, mounted and coated with gold for scanning electron microscopy. The rachidian, innermost lateral and lateral teeth were consistent with published descriptions of O. reevesii from China. The study therefore based its identification on the combined external and radular characters of the Korean specimens rather than on coloration or body size alone.

Partial mitochondrial COI and 16S rRNA sequences were successfully obtained from specimens collected at Jindo, Suncheon and Muan. BLAST searches found that the Korean COI sequences were 99.64–99.83% similar to published O. reevesii reference sequences, using the five closest matches retrieved with specimen SC1. The Korean 16S sequences were 99.33–99.55% similar to the five closest matches. The sequences did not match other onchidiid genera such as Paromoionchis. All newly generated sequences were deposited in GenBank, providing public sequence records for the Korean material.

Maximum-likelihood phylogenetic analyses provided an additional test of the species assignment. The COI dataset included eight newly generated Korean sequences, five GenBank sequences identified as O. reevesii, six sequences representing the congeners O. stuxbergi and O. typhae, and Paromoionchis tumidus as the outgroup. The 14-sequence 16S dataset included nine newly generated sequences and five additional O. reevesii sequences from GenBank, with the same congeners and outgroup. In both trees, the Korean specimens clustered with Chinese O. reevesii sequences. No or only minimal sequence divergence was detected among the three Korean localities or between the Korean and Chinese samples in the COI dataset, and the 16S tree showed a nearly identical topology.

The new record also brings renewed attention to an earlier Korean report of an onchidiid slug. That record, from Muan, was identified as Onchidium hongkongensis on the basis of photographic evidence alone. The name is not recognized as valid in the World Register of Marine Species, and subsequent taxonomic revisions treated Onchidium hongkongense as a junior synonym of Paromoionchis tumidus. The earlier specimen’s identity therefore remains uncertain. Because it came from Muan, where the present study confirmed O. reevesii, the researchers said the possibility that the earlier record represented O. reevesii could not be ruled out. They also stated that the possible presence of P. tumidus in Korea merits further collection and examination.

The confirmed Korean occurrence adds a documented species to the biodiversity of the country’s tidal flats and provides a new biogeographic record for a genus whose Korean distribution has been poorly documented. The specimens were observed only in July and August, and the study did not include year-round sampling, so the seasonal pattern cannot be established from these observations alone. The limited sample also consists of nine animals from three localities, with the phylogenetic analyses based on partial mitochondrial sequences. Broader sampling across Korean and neighboring East Asian saltmarsh habitats, additional genetic markers and detailed anatomical work will be needed to clarify the species’ distribution, population variation and connectivity between Korean and Chinese populations, as well as the diversity of onchidiid slugs in the region.

Subject of Research: Integrative taxonomic confirmation of Onchidium reevesii in Korean tidal flats

Article Title: First record of Onchidium reevesii from Korean tidal flats ( Mollusca , Gastropoda , Onchidiidae )

Article References: Kim, B., Yang, I., Lee, S.-K., Kim, H., Khim, J. S., Ryu, J., & Park, J. (2026). First record of Onchidium reevesii from Korean tidal flats (Mollusca, Gastropoda, Onchidiidae). Biodiversity Data Journal, 14, Article e189286. https://doi.org/10.3897/bdj.14.e189286

Image Credits: AI Generated

DOI: 10.3897/BDJ.14.e189286

Keywords: Onchidium reevesii, Onchidiidae, air-breathing sea slugs, Korean tidal flats, saltmarsh biodiversity, integrative taxonomy, mitochondrial COI, 16S rRNA, maximum-likelihood phylogeny, Yellow Sea, First, record

The findings show that closely similar shrimp can conceal substantial species diversity, while also highlighting the vulnerability of the island’s isolated freshwater habitats. The research focused on a nine-member complex centered on Caridina clinata, a small freshwater shrimp originally described in 1999 from Cuc Phuong National Park in northern Vietnam. Its close relative C. haivanensis was described a decade later from the Hai Van Mountain Pass in central Vietnam.

Phylogenetic analyses based on the mitochondrial cytochrome c oxidase subunit I gene, or COI, and the 16S ribosomal RNA gene resolved the complex into nine distinct, well-supported clades. C. clinata was recorded from both Vietnam and China, C. haivanensis remained restricted to Vietnam, and the seven newly described members of the complex were endemic to Hainan. Hainan is a continental island of 33,900 square kilometres in the South China Sea and the second-largest island in China.

Its high central terrain and lower surrounding areas produce a radial river system, including the Nandu, Changhua and Wanquan rivers, which together drain more than 3,000 square kilometres. The island’s varied geology, climate and habitats spanning marine, brackish and freshwater environments have contributed to high endemism. The newly described shrimp are locally distributed: Caridina fontinalis was found at a spring outlet near Haikou, while C. wenchangensis, C. qiongzhongensis, C. huangi, C. hepingensis, C. tunchengensis and C. brevilineata were each recovered from streams associated with particular towns in Wenchang, Qiongzhong, Tunchang and Qionghai.

Fieldwork was conducted from 2023 to 2025 at 14 sites, comprising 12 locations on Hainan and two in Vietnam representing material of C. clinata and C. haivanensis from their type regions. Researchers used fine-mesh dip nets and baited traps, recorded GPS coordinates and photographed habitats and live animals, and measured site conditions including substrate, water current, pH, dissolved oxygen and ammonia nitrogen. Specimens were preserved in ethanol and examined with a stereomicroscope.

For molecular analysis, DNA was extracted from abdominal muscle, the COI and 16S loci were amplified and sequenced, and the aligned sequences were trimmed to 490 and 473 base pairs, respectively. A concatenated maximum-likelihood analysis used the GTR+G+I model, with branch support evaluated through 1,000 bootstrap replicates. The eight new species were named Caridina wenchangensis, C. qiongzhongensis, C. huangi, C. hepingensis, C. fontinalis, C. tunchengensis, C. brevilineata and C. caii.

Their recognition rested on combined evidence rather than a single diagnostic character. Relevant traits included the length and tooth armature of the rostrum, proportions of the walking legs, form of the male reproductive appendages, structure of the telson, uropodal spines and egg dimensions. For example, C. fontinalis, described from cool water with stable flow, has a shorter rostrum and fewer teeth than C. lanceifrons, with which it occurs at the same sampling site.

C. brevilineata, whose name refers to short body stripes, differs from C. clinata in its leaf-shaped male first pleopod endopod, reduced rostral armature and the absence of a finger-like projection on the first maxilliped palp. The Wuzhishan population supplied the clearest instance of a revised identification. It had been assigned to C. clinata by Cai in 2014 because of its resemblance to C. lanceifrons, but new specimens from the locality and topotypic C. clinata from Vietnam showed that it was distinct in both morphology and genetics.

The Wuzhishan shrimp differed from C. clinata by 25.4–29.3% in COI sequence divergence and 10.8–12.1% in 16S divergence. It also possessed a pointed, subtriangular male first pleopod endopod rather than the rounded form found in C. clinata, a relatively broad second walking-leg carpus, fewer spines on the fifth leg and smaller eggs. The researchers named the species C. caii in honour of carcinologist Cai Y.

X., who discovered the species and made substantial contributions to carcinology. Genetic evidence also supported retaining the Ledong population of Hainan within C. clinata. Shrimp from Ledong differed from Vietnamese populations by only 0.6–1.2% in COI sequences and 0.7–1.6% in 16S, and the two populations occupied the same well-supported phylogenetic clade.

Although some morphological measurements varied, the male first pleopod and second pleopod appendages were closely alike. By contrast, C. wenchangensis was genetically closest to C. tunchengensis and C. clinata but remained a separate supported lineage. C. huangi and C. hepingensis, collected from unconnected streams in the same county, were likewise distinguishable in both morphology and genetic distance.

Members of the C. clinata complex occupy the middle and upper reaches of mountain streams. They shelter beneath leaf litter, among marginal vegetation or on muddy, sandy, gravelly and rocky substrates, generally in slow- to moderately flowing water. The complex produces relatively few large eggs, a reproductive pattern associated with direct larval development into benthic juveniles and an exclusively freshwater life cycle.

Field observations recorded dense populations in several streams, including the Ledong, Wuzhishan, Qiongzhong, Heping and Tuncheng localities, but lower densities at the Wenchang, Haikou and Qionghai sites. The study sampled only a limited set of streams, so the geographic range and population trends of the newly described taxa remain incompletely characterized. The survey also documented pressures on the habitats of these isolated shrimp.

Rubber and areca-nut plantations surround many streams, and intensive irrigation has reduced flow in some locations. Temporary in-stream diversions installed during droughts have left sections of streams completely desiccated, while pipelines have been used to extract water for domestic use. Hydropower and irrigation infrastructure further fragments river corridors, and agricultural chemicals threaten water quality.

Their specific effects on native shrimp remain undocumented, although predation, resource competition and disease transmission are recognized pathways through which invasive species can reduce native biodiversity. Following the International Union for Conservation of Nature’s Red List Categories and Criteria, the researchers classified all eight newly described species as Data Deficient because they are known only from their type localities and broader distribution data are unavailable. C. clinata retained the Data Deficient assessment assigned in an earlier evaluation.

These classifications identify major gaps in knowledge rather than low or high extinction risk. The study supports regular monitoring of wild populations and public-awareness efforts emphasizing the ecological importance of freshwater shrimp, while its broader sampling and genetic records provide a baseline for future surveys of Hainan’s stream biodiversity and for reassessing the conservation status of the newly recognized species.

Subject of Research: Taxonomic and phylogenetic study of the Caridina clinata species complex in Hainan and Vietnam

Article Title: Integrative taxonomy reveals the Caridina clinata Cai, Nguyen & Ng, 1999 species complex ( Crustacea , Decapoda , Atyidae ), with descriptions of eight new species from Hainan Island, China

Article References: Hou, J., Zhang, J., Chen, B., Zhang, Y., Chen, W., & Guo, Z. (2025). Integrative taxonomy reveals the Caridina clinata Cai, Nguyen & Ng, 1999 species complex (Crustacea, Decapoda, Atyidae), with descriptions of eight new species from Hainan Island, China. Zoosystematics and Evolution, 101(4), 2295-2336. https://doi.org/10.3897/zse.101.172207

Image Credits: AI Generated

DOI: 10.3897/zse.101.172207

Keywords: Caridina shrimp, Hainan Island, integrative taxonomy, cryptic species, freshwater biodiversity, molecular phylogeny, COI sequencing, 16S rRNA, species delimitation, conservation assessment, Integrative, taxonomy

The new frog was found during two expeditions in October 2024 and April 2025 to the Reserva Natural Comunitaria Cerro El Inglés, a thousand-hectare cloud forest reserve in the municipality of El Cairo, Valle del Cauca, and from a second site at El Mirador in the municipality of Versalles. Ten researchers combed riparian zones, forest edges and interior leaf litter by day and night, shifting substrate by hand to flush out cryptic, ground-dwelling animals. The frogs were photographed alive, euthanized under approved protocols, and deposited in the amphibian collection of the Universidad del Quindío. The holotype, an adult male designated ARUQ-1885, was collected on 25 October 2024 at a site called Corazón, at 2,261 meters elevation, together with a type series of eleven additional specimens spanning adult males, females, subadults and juveniles.

Niceforonia tempestuosa is a small frog — adult males measure 21.5 to 25.1 millimeters from snout to vent, and the single adult female a heftier 31.1 millimeters — but it carries a distinctive combination of traits. It has dorsolateral folds and V-shaped scapular folds on its back, a visible tympanic membrane, a fleshy swelling of the upper lip in adult males, and small non-spinous nuptial pads on the thumbs. Circumferential grooves ring every finger, a feature absent from the first two fingers of several close relatives. In life the dorsum ranges from reddish-brown to grayish, marked by a dark facemask running from the snout through the lores and eyelids, while the belly, thighs and arms glow yellow and the groin and thigh surfaces blaze reddish-orange. Most tellingly, every specimen bears dark supra-inguinal spots — paired dark marks on the posterior flanks just above the groin — a character that turned out to be the key to the whole study.

Genetically, the new species is emphatically its own thing. The team sequenced fragments of two mitochondrial genes (12S and 16S ribosomal RNA, totaling 2,866 base pairs) and two nuclear genes (RAG-1 and tyrosinase, 631 and 531 base pairs respectively), comparing the new frog against every known Niceforonia species with available sequences, four candidate species, and a broad outgroup sampling of 198 terminals spanning all genera of Strabomantidae. Uncorrected p-distances at the 16S gene separate N. tempestuosa from all congeners by more than 7 percent — ranging from 7.44 percent against its closest relative, N. mantipus, up to 18.33 percent against N. philippi. In frog taxonomy, where new species are routinely recognized at 16S divergences of 3 to 5 percent, a gap that large is unambiguous.

The phylogenetic analyses, run under both maximum likelihood in IQ-TREE and parsimony in TNT with gaps treated as a fifth character state, recovered the new species as sister to N. mantipus. That relationship is biologically telling: N. mantipus is a widespread Colombian species, and the new frog had long been confused with it. By incorporating samples of N. mantipus from the Valle del Cauca and Quindío departments — including material from its type locality at Bosque de San Antonio, only about 15 kilometers west of Cali — the team confirmed that the two species are close but distinct, and hinted that further cryptic diversity may still lurk within N. mantipus across its range from Antioquia to Cauca.

But the study’s most consequential finding concerns those dark supra-inguinal spots. When the team mapped the presence or absence of this character across the family using the software YBYRÁ and TNT, the spots optimized unambiguously — with no homoplasy — as a shared derived character, or synapomorphy, for two separate clades within Strabomantidae. The first is a clade inside the genus Niceforonia comprising N. brunnea, N. dracula, N. elassodiscus, N. mantipus, N. nigrovittata, N. quilloturo, N. peraccai, four candidate species and the new species. The authors formally name this clade the Niceforonia nigrovittata species group, reviving a name proposed by William Lynch in 1989 and thereby preserving decades of established usage in the literature. Two additional species lacking molecular data, N. aderca and N. fallaciosa, show the spots and are tentatively added to the group pending genetic testing.

Remarkably, the same dark flank spots also diagnose a second, entirely unrelated clade: a strongly supported group (jackknife support of 99 percent) within the subfamily Holoadeninae, uniting the genera Bahius, Barycholos, Phyllonastes and Urkuphryne. This nested pattern dovetails with earlier anatomical work showing that the posterior origin of the iliacus externus muscle on the iliac shaft unites Phyllonastes and Barycholos. Taken together, muscle anatomy, DNA sequences and external coloration now independently corroborate the monophyly of the four-genus holoadenine group and of the Phyllonastes-plus-Barycholos subgroup — a textbook demonstration of how integrating molecular and phenotypic evidence can test and confirm evolutionary hypotheses in a group of frogs notorious for morphological conservatism.

The broader placement of Niceforonia within Brachycephaloidea, the superfamily of direct-developing frogs that skip the tadpole stage entirely, remains stubbornly unstable. Different studies have variously placed the genus in the subfamilies Strabomantinae, Holoadeninae, or Hypodactylinae, and the new analysis finds Niceforonia outside both Holoadeninae and Strabomantinae, though with weak support for its deeper affinities. The authors argue this reflects a genuine evolutionary or methodological conflict rather than mere lack of resolution, and they call for total-evidence analyses that fold phenotypic characters — carefully recoded, since traditional coding of digital discs conflates several independent traits — into molecular matrices. The genus itself, now 18 species strong, stretches from Colombia’s three Andean cordilleras south through Ecuador to central Peru and east into the Amazonian basin, and recent work in Ecuador suggests numerous cryptic species remain hidden under existing names.

Ecologically, N. tempestuosa is a creature of the forest floor. It inhabits the leaf litter of well-preserved forest interiors between 2,169 and 2,294 meters, showing a preference for sloping microhabitats, and has been observed sheltering in crevices formed by the buttress roots of large trees and among the stilt roots of Wettinia palms. It shares this litter layer with Strabomantis cerastes, another floor-dweller of the Serranía de los Paraguas. The region hosts more than 100 amphibian species, 32 of them threatened with extinction, yet parts of this hotspot have only recently begun to be explored — meaning both greater diversity and a wider range for the new frog may await discovery.

That discovery window is closing under pressure. The Serranía de los Paraguas faces habitat loss from illicit crop expansion, logging, agricultural frontier advance — including booming Hass avocado farming — and mounting large-scale mining interests promoted under the language of local development. Since 2019, the Fundación Calima and the community organization Corporación Serraniagua have run a joint conservation program, backed by the Rufford Foundation, the Prince Bernhard Nature Fund and the Critical Ecosystem Partnership Fund, combining acoustic and climatic monitoring, breeding-habitat protection, and community outreach. For the new frog specifically, zoning restricts foot traffic in dense leaf-litter zones where its populations peak, and fences are being installed with landowners to shield riparian forests from the agricultural frontier. A storm-named frog, protected by the people who live beneath those storms, is perhaps the most fitting emblem this biodiversity hotspot could have asked for.

Subject of Research: Description of a new Andean cloud-forest frog species and the phylogenetic significance of dark supra-inguinal spots in Strabomantidae

Article Title: A new species of Niceforonia ( Amphibia , Anura , Strabomantidae ) from the Serranía de los Paraguas, Colombia, with the description of a new phenotypic synapomorphy for the Niceforonia nigrovittata species group

Article References: Ospina-Sarria, J. J., Rodríguez-Betancourt, A. F., Gómez-Sepúlveda, X., Morales-Vertel, I. D., Vélez-Franco, V., Gallego-Posada, N., Herrera-Rengifo, J. D., Grajales Escobar, J. E., Ceballos-Castro, I., García-Gómez, I., Velásquez-Trujillo, D. A., Ortiz Baez, A. S., & Mendez Narvaez, J. (2026). A new species of Niceforonia (Amphibia, Anura, Strabomantidae) from the Serranía de los Paraguas, Colombia, with the description of a new phenotypic synapomorphy for the Niceforonia nigrovittata species group. ZooKeys, 1289, 159-184. https://doi.org/10.3897/zookeys.1289.198411

Image Credits: AI Generated

DOI: 10.3897/zookeys.1289.198411

Keywords: Niceforonia tempestuosa, Strabomantidae, new species, Colombia, cloud forest, phylogenetics, synapomorphy, 16S rRNA, Brachycephaloidea, Holoadeninae, cryptic species, conservation

The study, published in the journal Metabarcoding and Metagenomics, focused on bacterial communities living in sediments around two Atlantic salmon (Salmo salar) aquaculture installations in Scotland, one located near Oban and the other in Loch Linnhe. Fish farms deposit feed and faeces on the seabed, creating a gradient of organic enrichment that radiates outward from the cage edges. Bacteria respond quickly and predictably to this enrichment, making them powerful bioindicators of environmental impact. Regulators increasingly rely on DNA-based analysis of these communities to assess whether farms are operating within permitted environmental limits.

Traditionally, such monitoring has depended on Illumina sequencing, a second-generation technology prized for its accuracy, with a median error rate of roughly 0.5 percent. But Illumina’s short reads, capped at around 500 base pairs, capture only fragments of the bacterial 16S ribosomal RNA gene, the standard taxonomic marker. These short fragments typically cannot be assigned reliably to genus or species level, which limits the ecological information that can be extracted. In contrast, Nanopore sequencers can read entire genes in a single pass, but until recently their error rates, sometimes reaching 6 to 15 percent on older flow cells, made them unsuitable for the precision demands of compliance monitoring.

The breakthrough tested in this study is duplex sequencing, a strategy in which both strands of a DNA molecule are read sequentially through the same nanopore. Because errors in one strand can be cross-checked against its complementary partner, the approach dramatically improves base-calling accuracy. Oxford Nanopore reports accuracy above 99.9 percent (a Phred score of 30 or higher) when duplex reads are combined with super-accuracy base-calling models. The research team applied this chemistry, using the new R10.4.1 flow cell and the Dorado duplex base-caller, to full-length 16S rRNA gene amplicons generated from the same DNA extracts that had been sequenced on the Illumina platform.

The results were striking. Of the high-quality Nanopore amplicon reads retained for analysis, 36 percent were duplex reads with a Phred score of at least 30, a proportion notably higher than the duplex rates of roughly 6 to 7 percent reported in previous bacterial genome-sequencing studies using the same chemistry. The authors speculate that shorter DNA fragments, such as the roughly 1,500-base-pair 16S rRNA gene, may have a higher likelihood of forming duplex molecules at the pore than the much longer fragments typical of whole-genome sequencing, though they caution that this hypothesis requires further testing.

To compare the two platforms fairly, the researchers sampled sediment along transects running from each farm’s outer cage edge, through an intermediate Allowable Zone of Effect, to a distant reference site chosen to lie beyond the influence of farm deposits. Both sequencing approaches told the same ecological story. When diversity patterns were calculated from Illumina amplicon sequence variants (ASVs) and Nanopore operational taxonomic units (OTUs), both datasets split the samples into two clear clusters: a high-impact group comprising the cage-edge samples and the more affected Allowable Zone of Effect at one farm, and a low-impact group containing both reference sites and the less affected zone at the other farm. These patterns matched independent macroinvertebrate-based assessments, which rated the cage edge of one farm as heavily disturbed while the corresponding zone at the other farm remained in good ecological condition.

Notably, the Nanopore data appeared even more sensitive than the Illumina data in distinguishing between samples. Bray-Curtis distances between bacterial communities were larger in the Nanopore dataset, and differences in Shannon diversity between sites were more pronounced. For biomonitoring, where the goal is to detect subtle environmental change before it becomes severe, this heightened sensitivity could prove a genuine advantage.

The most consequential differences emerged at the taxonomic level. On average, 86 percent of high-quality Nanopore reads could be assigned to bacterial family and 82 percent to genus, compared with only 52 percent and 50 percent respectively for Illumina reads. At the species rank the gap widened dramatically: roughly 65 percent of Nanopore reads returned a species-level identification, against a mere 15 percent of Illumina reads, and more than 99 percent of the species-level Illumina assignments belonged to rare sequence variants. When the researchers collapsed their data to family level, the two platforms agreed well, with seven of the ten most abundant families shared between datasets, dominated by sulfur-cycling bacteria typical of organically enriched sediments. But at the genus level, agreement deteriorated for the Illumina data, and the taxonomy-based clustering of samples shifted slightly, a direct consequence of short reads lacking the sequence information needed to discriminate between closely related genera.

Some discrepancies between the platforms are attributable to the different PCR primer pairs used to amplify the short V3-V4 region for Illumina and the full-length gene for Nanopore sequencing. Both primer sets are known to miss certain bacterial groups, and the stochastic nature of PCR amplification can distort relative abundance estimates. The researchers acknowledge these limitations, noting that only PCR-independent metagenome sequencing could fully eliminate such biases, though that approach remains too expensive for routine monitoring.

The practical implications are considerable. Nanopore’s MinION device is compact and comparatively affordable, which would allow individual laboratories to run their own sequencing rather than outsourcing to commercial providers, shortening turnaround times and enabling protocol optimisation for specific sample types. The full-length 16S sequences also provide more material for designing indicator-specific primers for fast, sequencing-free quantitative PCR assays. The authors conclude that eDNA-based monitoring of aquaculture impacts could be conducted equally well with improved Nanopore duplex sequencing, and they propose the logical next step: a full validation against macroinvertebrate-derived biotic indices across numerous compliance-monitoring samples, mirroring the process that established Illumina sequencing as a trusted tool for regulators.

Subject of Research: Comparing Nanopore duplex sequencing and Illumina MiSeq for eDNA-based bacterial biomonitoring of salmon aquaculture impacts

Article Title: Nanopore duplex sequencing as an alternative to Illumina MiSeq sequencing for eDNA-based biomonitoring of coastal aquaculture impacts

Article References: Stoeck, T., Katzenmeier, S. N., Breiner, H.-W., & Rubel, V. (2024). Nanopore duplex sequencing as an alternative to Illumina MiSeq sequencing for eDNA-based biomonitoring of coastal aquaculture impacts. Metabarcoding and Metagenomics, 8, Article e121817. https://doi.org/10.3897/mbmg.8.121817

Image Credits: AI Generated

DOI: 10.3897/mbmg.8.121817

Keywords: environmental DNA, Nanopore sequencing, Illumina MiSeq, duplex sequencing, 16S rRNA, aquaculture, salmon farming, biomonitoring, bacterial communities, marine sediments, metabarcoding, Scotland