These plants are cultivated as medicinal and aromatic crops, but their classification has been impeded by ambiguous taxonomy, inconsistent nomenclature and genetic admixture, particularly between Z. bungeanum and Z. simulans. The research set out to clarify relationships among the sampled germplasm by integrating several classes of molecular marker rather than relying on a single identification system. Amplification with 36 selected primers produced 841 polymorphic bands across the assessed material.

The retained primers had a percentage of polymorphic bands, or PPB, of 100%. Among the three dominant marker systems, iPBS delivered the highest level of informativeness, exceeding CBDP and SCoT. Mantel tests also found significant pairwise correlations among all three systems, supporting the consistency of their combined genetic signals.

Cluster analysis and population-structure analysis divided the accessions into two principal genetic groups. Cluster I was dominated by Z. bungeanum, Z. simulans and Z. piasezkii, while Cluster II was dominated by Z. armatum, Z. schinifolium and Z. acanthopodium. These divisions were based on the 109 accessions included in the study and characterize the genetic patterns detected by the selected markers.

The two DNA barcodes supplied complementary taxonomic information. ITS2 supported Z. armatum as a monophyletic clade, indicating that the sampled accessions assigned to that species grouped into a distinct branch in the barcode analysis. In contrast, trnH-psbA exposed geographic substructure within Z. armatum corresponding to populations from Yunnan, the Shaanxi–Hubei region and Sichuan.

The most direct resolution of persistent classification uncertainty came from integrating the dominant-marker results with ITS2 data. Together, these datasets placed the local varieties “Choujiao,” “Qiujiao,” “Fuguhuajiao,” “Goujiao” and “Bayuejiao,” as well as the Shandong germplasm, closely related to Z. simulans. The study therefore establishes a coherent molecular system for identifying and classifying the Chinese prickly ash germplasm represented in the sample.

Its practical scope is resource standardization: the framework provides a way to distinguish accessions whose conventional names and classifications had been inconsistent. The results concern the relationships detected among the 109 evaluated accessions through the specified marker systems and do not by themselves establish the full taxonomic or genetic boundaries of every Zanthoxylum population. By demonstrating that iPBS, CBDP and SCoT markers capture related but complementary variation, and that DNA barcoding can resolve both broader relationships and geographic structure, the research provides an integrated molecular basis for Chinese prickly ash germplasm identification.

The principal reported outcome is a practical classification framework for the assessed material, with the closest affinities of five ambiguously named varieties and Shandong germplasm supported by combined marker and ITS2 evidence.

Subject of Research: Molecular classification and genetic relationships among 109 Chinese prickly ash germplasm accessions

Article Title: Integrated application of CBDP, iPBS, and SCoT markers with DNA barcoding enables effective classification of Chinese prickly ash (Huajiao)

Article References: Liang, S., Feng, Z., Yu, G., Li, Z., Liu, Y., & Liu, Y. (2026). Integrated application of CBDP, iPBS, and SCoT markers with DNA barcoding enables effective classification of Chinese prickly ash (Huajiao). BMC Plant Biology. https://doi.org/10.1186/s12870-026-09978-6

Image Credits: AI Generated

DOI: 10.1186/s12870-026-09978-6

Keywords: Chinese prickly ash, Zanthoxylum, DNA barcoding, iPBS markers, SCoT markers, CBDP markers, taxonomy, genetic diversity, germplasm identification, ITS2, Integrated, application

The site is smaller than 800 square metres, and all 21 matched pollen-and-tissue samples came from one collection event. Along a 500-metre transect running from northwest to southeast, the researchers recorded flowering plants within 30 metres of the route. Bumble bees were gently immobilised in a queen-marker cage fitted with coarse mesh and covered with sterile cling film.

A fine art brush was used to remove pollen from each bee’s corbicula through the mesh. The researchers then clipped the tarsus from one middle leg with cuticle scissors before releasing the insect. The study cited previous work indicating that clipping a middle-leg tarsus does not affect bumble bee productivity.

The authors also observed bees gathering pollen while lacking a mid-tarsus, which they interpreted as consistent with minimal impairment of foraging. Nevertheless, the method removed a tissue sample, and the study did not establish that sampled bees were literally uninjured or demonstrate population-level effects across sites and seasons. After each bee was released, the collected material was transferred to a 2 ml centrifuge tube.

Equipment was cleaned with a 10% bleach solution, rinsed with 70% ethanol and dried before reuse to reduce cross-contamination. In total, the field procedure produced 21 samples containing both pollen and tarsal tissue from the same insect. For plant analysis, pollen was processed in a controlled clean room, homogenised by bead milling for 2.5 minutes at 30 Hz and subjected to DNA extraction.

The researchers amplified the ITS2 region of nuclear ribosomal DNA, included extraction and PCR controls, and sequenced the products with Illumina MiSeq V3 chemistry using paired-end 300-base reads. Exact sequence variants were matched against a custom database of 583,533 vouchered plant and fungal ITS2 sequences downloaded from GenBank. Negative controls produced only low read totals and were discarded, while positive controls recovered the expected plant species and were also excluded from the final analysis.

Plant identification depended on the completeness of the public reference database. The authors stated that this approach might have contributed to some plants being identified only to genus level and that a database containing regional plants could improve resolution. Insect identification combined field morphology with sequencing of the mitochondrial COI barcode region.

Twelve of the 21 bees were assigned unambiguously by morphology: one Bombus hypnorum , eight B. lapidarius and three B. pascuorum . Nine had ambiguous morphological characteristics, and COI barcoding resolved those specimens within the complete sample as two B. cryptarum , four B. pascuorum and three B. terrestris . Across all 21 individuals, the researchers identified five species: two B. cryptarum , one B. hypnorum , eight B. lapidarius , seven B. pascuorum and three B. terrestris .

Pollen metabarcoding generated 642,151 raw sequences and yielded unambiguous species-level assignments for 18 plants. Four sequence variants were identifiable only to genus: Lotus , Ranunculus , Rosa and Trifolium . Trifolium repens was detected on all 21 bees.

Jasione montana , Lotus , Prunus persica , Ranunculus , Ranunculus sardous , Rubus caesius and Rumex acetosella were each detected on one bee. Individual bees carried pollen from two to seven plant taxa, producing a network of 28 plant-pollinator interactions. The vegetation survey recorded 17 flowering plant species, 13 of which were also detected in the pollen analysis.

Achillea millefolium , Galium saxatile , Hieracium pilosella and Stellaria graminea were flowering but absent from the metabarcoding results. Conversely, Prunus persica , Prunus serotina , Rosa and Rubus caesius appeared in the pollen analysis but not in the on-site flowering survey. The authors related this discrepancy to the small protected area and bumble bees’ ability to forage beyond the surveyed route; they also noted that P. persica is a common ornamental plant that may have been available in nearby gardens.

Some measured pollen patterns differed among bumble bee species. Pollen from Vicia and Trifolium pratense occurred exclusively on B. pascuorum , whereas Jasione montana , Lotus and Hypochaeris radicata occurred only on B. lapidarius . The authors said the workflow could potentially be adapted to smaller pollinators, but that extension was not tested.

Overall, the case study supports the method as a means of studying bumble bee foraging while releasing sampled insects, while its single-site, single-event design leaves broader ecological validation outside the scope of the study.

Subject of Research: A non-lethal DNA sampling case study of plant-pollinator interactions among 21 bumble bees in northern Germany

Article Title: Case study of non-lethal sampling for plant-pollinator networks via barcoding and metabarcoding on bumble bees in Germany

Article References: Edwards, A., & Gemeinholzer, B. (2025). Case study of non-lethal sampling for plant-pollinator networks via barcoding and metabarcoding on bumble bees in Germany. Metabarcoding and Metagenomics, 9, Article e141904. https://doi.org/10.3897/mbmg.9.141904

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.141904

Keywords: bumble bees, non-lethal sampling, pollen metabarcoding, DNA barcoding, plant-pollinator networks, Bombus, pollination ecology, Germany, ITS2, COI sequencing, Case, non-lethal

Each survey zone was five metres wide and included both riverbanks and the riverbed. Specimens were collected for microscopic identification, while DNA barcoding was used to verify morphologically difficult taxa. The molecular analyses reused river-water environmental DNA samples collected in October 2020.

For PCR metabarcoding, the researchers evaluated 17 rbcL primer pairs and 21 ITS2 primer pairs in silico, allowing up to three mismatches between primers and binding sites. Because the literature did not provide a satisfactory bryophyte-specific ITS2 pair, they designed BraF and BraR by examining available sequences in NCBI and identifying a conserved region among mosses that differed from vascular plants. They also modified the rbcL265 and rbcL556 primers for the study.

Visual inventories identified 68 species, including 29 found only through field sampling; eight of those species lacked reference sequences in the database. PCR metabarcoding recovered 37 of the 68 field-recorded species, more than half of the observed flora, and detected 57 additional species that were not recovered by the other methods. The study also detected Buxbaumia viridis, a species listed in Annex II of the European Habitats Directive and strictly protected in France, at read abundances comparable to those of abundant taxa despite its absence from the transect survey.

The species can persist as microscopic protonema that is not visible to the naked eye. Hybridisation capture detected 27 bryophyte species, including 11 of the field species, or 16% of the field inventory, with an average of five species per site. Nine species were found only by hybridisation capture.

Two were described as very rare in the study region—Dicranella cerviculata and Riccia huebeneriana—and three as quite rare—Aneura pinguis, Hookeria lucens and Trichocolea tomentella. The remaining four were described as fairly common or common. These findings show that capture recovered some taxa not found by the other approaches, but fewer species overall than PCR metabarcoding.

The molecular methods also differed in their recovery of individual taxa. In the combined eDNA marker comparison, ITS2 identified three times as many species as rbcL, and only two species were detected by both markers: Fontinalis antipyretica and Ptychostomum capillare. Within the PCR data specifically, rbcL detected 67 species and ITS2 detected 59.

Approximately 40% of species in the PCR comparison were recovered by only one marker, while 20% were shared. ITS2 also had finer taxonomic resolution: 21 of 31 unique sequences were assigned to species, compared with six of 12 for rbcL, despite both markers having the same amplicon size in the comparison. Community composition differed significantly among the three methods.

A principal coordinate analysis based on Sørensen dissimilarity showed distinct clusters according to method, with the first two coordinates explaining 32.3% and 15.9% of total variation. A PERMANOVA confirmed the separation, with method explaining a substantial share of variation in the model (R² = 0.898, F = 70.81, p = 0.001). Tests did not support the hypotheses that detection differences were associated with preferences for moist habitats or dispersal through sporophytes and vegetative propagules, although available trait data for bryophytes were limited.

Indicator-species analysis nevertheless identified taxa more likely to be detected by particular methods. River eDNA may include signals transported from beyond the local survey area. Leptobryum pyriforme was detected at one site by PCR although it was not observed there and was considered unlikely to occur naturally in the region; the authors noted that it occurs in synanthropic habitats found upstream.

Fissidens fontanus may likewise have arrived through runoff from nearby villages. Conversely, Kindbergia praelonga, observed at every site, and the widespread riparian species Plagiomnium undulatum were not detected by either eDNA approach despite having reference barcodes for at least one marker. The study reported that 57% of eDNA-detected species were absent from field inventories, while 42% of field-identified species went undetected by eDNA.

The analysis had several bounded limitations: it covered seven sites in one river system, used water samples collected at one time for the molecular comparison, relied on incomplete reference databases and limited trait data, and did not yet provide the capture baits and full raw hybridisation-capture dataset. Marker specificity and reference-database composition were important determinants of detection. The researchers associated some differences between capture markers with short, randomly recovered rbcL fragments of 150–220 base pairs, which have lower taxonomic resolution than sequences spanning positions 265–500, and suggested 200–250-base-pair paired-end sequencing to improve rbcL detection.

Overall, the results support eDNA as an effective means of detecting bryophytes from river water while showing that PCR, hybridisation capture and expert visual surveys recover different subsets of the community and provide complementary information.

Subject of Research: Comparative study of field surveys, PCR metabarcoding and hybridisation capture for monitoring riparian bryophytes

Article Title: A comparative analysis of hybridisation capture and PCR-based eDNA metabarcoding for monitoring bryophytes in riparian ecosystems

Article References: Reinhart, T., Espinosa Prieto, A., Begoc, T., Tinguy, H., Bick, F., Chanez, E., Beisel, J.-N., & Hardion, L. (2025). A comparative analysis of hybridisation capture and PCR-based eDNA metabarcoding for monitoring bryophytes in riparian ecosystems. Metabarcoding and Metagenomics, 9, Article e154548. https://doi.org/10.3897/mbmg.9.154548

Image Credits: AI Generated

DOI: 10.3897/mbmg.9.154548

Keywords: bryophytes, environmental DNA, PCR metabarcoding, hybridisation capture, riparian ecosystems, river monitoring, ITS2, rbcL, species detection, biodiversity, comparative, analysis

The work was a sequence-collection and sequence-comparison study, not a field validation of environmental DNA monitoring. The target list was based primarily on a comprehensive inventory of fish inhabiting the Lake Biwa basin. Mugil cephalus and Lateolabrax japonicus, distributed in the downstream Yodo River, were also included. Specimens came mainly from the Lake Biwa and Yodo River basins, although some species, including non-native species, were collected in Gifu and Kanagawa prefectures. Other specimens were obtained from the Lake Biwa Museum, freshwater fish researchers, aquarium shops and a fishery cooperative in Shiga Prefecture.

The forward primer, Fish_ITS2_F, was placed in the conserved 5.8S region, and the reverse primer, Fish_ITS2_R, was placed in the 28S region, allowing amplification of the intervening ITS2 sequence. The primers were designed manually from NCBI sequences aligned with MUSCLE, with target melting temperatures of approximately 60 °C. Forward-primer design used sequences from 113 species in 64 genera, 36 families and 12 orders, while reverse-primer design used sequences from 96 species in 58 genera, 34 families and 11 orders.

The researchers tested the primer pair with tissue-derived DNA from 12 fish species inhabiting Lake Biwa, and PCR products of the expected size were obtained for every tested species. Reported amplified lengths among taxa in the study area ranged from 298 base pairs for Channa argus to 481 base pairs for Micropterus nigricans. This laboratory test showed that the primers could amplify tissue DNA, but it did not assess their detection performance in environmental water samples.

For reference-library construction, genomic DNA was extracted from tissue or pelvic-fin samples representing the 84 target species. Amplicons were prepared for Illumina MiSeq sequencing, which produced 12,651,181 reads across two runs. The researchers obtained new ITS2 sequences for 61 species, represented by 291,855 reads. Together with previously available sequences for Cyprinus carpio and Plecoglossus altivelis altivelis, the collection covered 63 species, or 75% of the targets. Sequences were unavailable for 21 species because samples were unavailable for three species or sequencing quality was poor for 18 species.

Species distinguishability was assessed using BLAST percent identities and maximum-likelihood phylogenetic trees. The study used the MiFish identity threshold of 98.5% to define four categories. Identities at or below 98.5% and lower than the MiFish value were considered easily distinguishable; identities at or below the threshold but comparable to MiFish were considered distinguishable. Identities above 98.5% or substantially overlapping within-species values were treated as difficult to distinguish, and identical sequences were classified as indistinguishable.

Results differed among taxonomic groups. Oncorhynchus biwaensis had ITS2 identities 1.9–2.8 percentage points lower than its MiFish identities when compared with O. masou ishikawae and O. mykiss, allowing the study to classify O. biwaensis as easily distinguishable from them. The other two Oncorhynchus species were classified as distinguishable because their interspecific identities were no higher than 98.5% and were comparable to MiFish. In Rhinogobius, R. flumineus had ITS2 identities 1.0–3.0 percentage points lower than MiFish when compared with Rhinogobius sp. OM and R. biwaensis, making it easily distinguishable by the study’s criteria. However, Rhinogobius sp. OM and R. biwaensis exceeded 99.5% identity and overlapped with their within-species values, so they remained difficult to distinguish.

ITS2 also failed to resolve every species pair. Cobitis magnostriata and C. minamorii oumiensis shared identical ITS2 sequences and were classified as indistinguishable. Carassius sp. and C. buergeri grandoculis likewise shared identical sequences. The two BIWAE types of Cobitis exceeded 99.5% identity and overlapped with within-species values, while Carassius cuvieri had identities above 98.5% relative to Carassius sp. and C. buergeri grandoculis. These findings show that the marker’s discriminatory value was taxon-dependent rather than uniformly superior to MiFish.

The study has several bounded limitations. Reference coverage was incomplete, with sequences recovered for 63 of 84 targets, and only a small number of individuals contributed to some species comparisons. The analysis used tissue-derived reference DNA rather than a controlled comparison of environmental DNA detection, so it did not establish field sensitivity, false-detection rates or performance across habitats. The authors concluded that ITS2 can be effective for some species that are difficult to distinguish with MiFish and recommended combining both markers for broader biodiversity monitoring. They also called for field validation in the Lake Biwa basin and expansion of the reference database with additional species and individuals. The generated sequences were deposited in DDBJ, EMBL and GenBank under accession numbers LC876517–LC876631.

Subject of Research: ITS2 reference sequences for freshwater fish in the Lake Biwa basin

Article Title: Collection of ITS2 region reference sequences for freshwater fish in the Japanese archipelago for environmental DNA metabarcoding applications

Article References: Ito, G., Sasaki, D., Sakata, M. K., Kawase, S., Minamoto, T., & Yamanaka, H. (2026). Collection of ITS2 region reference sequences for freshwater fish in the Japanese archipelago for environmental DNA metabarcoding applications. Metabarcoding and Metagenomics, 10, Article e162895. https://doi.org/10.3897/mbmg.10.162895

Image Credits: AI Generated

DOI: 10.3897/mbmg.10.162895

Keywords: environmental DNA, metabarcoding, ITS2, Lake Biwa, freshwater fish, MiFish, reference library, species discrimination, biodiversity monitoring, Collection, region, reference

The research, led by Gonçalo Alves of The MENTOR Initiative and the Instituto de Higiene e Medicina Tropical at Universidade NOVA de Lisboa, together with Cátia Marques of Population Services International and an international team spanning the US Centers for Disease Control and Prevention, the US President’s Malaria Initiative, Angola’s National Malaria Control Program and provincial health authorities, drew on entomological surveillance carried out between 2015 and 2022. Field teams worked in four provinces that trace the Atlantic coastline of Angola: Luanda, Benguela, Cuanza Sul and Namibe. The work was approved by Angola’s National Institute for Health Research and funded through a constellation of international donors, including USAID’s President’s Malaria Initiative and The Global Fund, reflecting the practical stakes of the question at hand.

Correct species identification sits at the heart of malaria control. Public health campaigns spray walls with residual insecticides, distribute bed nets and deploy larval-source management, all calibrated against the known habits of the mosquito species that transmit Plasmodium parasites. When a species is routinely misidentified, those calibrations go wrong. An. azevedoi has long been one of the casualties of this confusion, its adults difficult to distinguish morphologically from related anophelines and its biology so poorly documented that surveillance programs had little reason to pay it attention. The new study set out to correct the record by combining traditional morphology with two molecular markers: the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA and the mitochondrial cytochrome c oxidase subunit I (COI) gene, both workhorses of insect barcoding.

The results confirmed that An. azevedoi occupies a surprisingly broad and varied range. The species was found in multiple habitats along the Atlantic coast of Angola, with new geographical records in Luanda and Benguela provinces expanding its known distribution. Perhaps more striking was the setting: the mosquito turned up frequently in urban and peri-urban environments, a habitat association that matters enormously in a country where cities such as the capital Luanda have grown explosively and where urban malaria transmission has become an increasing concern for control programs. A mosquito that thrives near human settlements is, by definition, a mosquito that control measures must account for.

To understand whether the species actually feeds on people, the researchers screened a subset of wild-caught adults for blood-meal sources. Human blood was identified in three field-collected specimens, offering the first direct evidence that An. azevedoi bites humans in the study area. The team also tested the mosquitoes for Plasmodium infection, and importantly, none of the screened adults carried the malaria parasite. That absence does not declare the species a non-vector; it simply means that, in this sample, no infections were detected. Determining whether An. azevedoi contributes to malaria transmission will require larger screens, but the demonstration of human blood feeding alone is enough to justify closer attention.

The most consequential findings, however, concern insecticides. Using World Health Organization tube bioassays and bottle bioassays on live mosquitoes reared from field-collected larvae, the team measured the susceptibility of An. azevedoi populations to the major classes of public health insecticides. The outcome was sobering: the species exhibited resistance to pyrethroids, the chemical class that impregnates the vast majority of insecticide-treated bed nets, and to DDT, an organochlorine still used in some indoor residual spraying programs. Resistance to two different insecticide classes signals that the standard toolkit of vector control is losing traction against this mosquito wherever it occurs.

Yet the bioassays also contained a note of hope. Two populations from Luanda remained fully susceptible to chlorfenapyr, a pyrrole insecticide with a distinct mode of action that disrupts energy production in mosquito cells and is increasingly deployed in new-generation treated nets and indoor residual formulations precisely because it can overcome pyrethroid resistance. That chlorfenapyr retains activity against these An. azevedoi populations gives Angolan control programs a concrete, evidence-based option should the species ever prove to be a significant player in transmission, and it illustrates a broader principle: knowing the susceptibility profile of every locally relevant species, not just the classic primary vectors, is essential when resistance reshapes the landscape.

Methodologically, the study offers a template for rescuing other neglected species from taxonomic obscurity. Larvae were collected in the field and reared to adulthood so that live specimens of known provenance could be used in the bioassays, avoiding the ambiguity that comes with testing damaged or variable wild adults. Adults were first identified morphologically and then confirmed by sequencing ITS2 and COI, and the authors have made these markers publicly available for the first time, meaning that any laboratory in the region can now verify an identification of An. azevedoi with a simple PCR and sequencing workflow. In a genus where cryptic species complexes routinely confound surveillance, the publication of validated reference sequences is a quiet but genuinely transformative contribution.

The collaboration behind the work is itself noteworthy, uniting Angola’s National Malaria Control Program and provincial health directorates in Benguela, Cuanza Sul and Sumbe with international partners, and crediting specialists such as John Gimnig at CDC Atlanta for taxonomic determination and Adeline Chan, the PMI entomologist in Angola during 2015 and 2016, for coordinating surveillance and preserving field specimens. The funding came from USAID through the President’s Malaria Initiative via projects including AIRS, VectorLink and Health for All, and from The Global Fund through the NMF3 Angola grant, with additional support from the Portuguese Foundation for Science and Technology. The authors stress that the funders played no role in the design, analysis or publication of the study, and that the findings do not necessarily represent the official positions of USAID, PMI or the CDC.

What emerges is a call to widen the surveillance lens. Malaria control in sub-Saharan Africa has understandably focused on the dominant vectors, chiefly members of the Anopheles gambiae complex and the Anopheles funestus group, whose efficiency as parasite carriers has shaped a half-century of strategy. But as primary vectors come under increasing pressure from insecticides and as urban transmission complicates the epidemiological picture, secondary and previously ignored species may occupy ecological space left vacant by successful interventions. An. azevedoi, feeding on humans in cities and peri-urban districts, already resistant to pyrethroids and DDT yet vulnerable to newer chemistries, exemplifies exactly the kind of species that deserves routine monitoring rather than perpetual misidentification. The authors argue for including it in future entomological surveillance and for research into its potential public health relevance, and the tools to do so now exist. After 57 years in the taxonomic shadows, one of Angola’s overlooked mosquitoes finally has a name that can be checked, a map that can be read and a susceptibility profile that can inform decisions, and the study stands as a reminder that in the fight against malaria, the mosquitoes you do not know may matter as much as the ones you do.

Subject of Research: Distribution, molecular identification and insecticide resistance of the malaria mosquito Anopheles azevedoi in Angola

Article Title: Anopheles azevedoi (Ribeiro, 1969) in Angola: new geographical records, molecular characterization, and insecticide susceptibility profile of a commonly misidentified species

Article References: Alves, G., Marques, C., Marcet, P., Chipepa, V., Fedorova, A., Sutcliffe, A., do Rosario, J., Calles, D., Troco, A. D., Chissanga, M. S., Espalhado, F., Nóbrega, T., Sousa, C., Pinto, J., Jorge, C. P., Martins, J. F., Yoshimizu, M., & Gutierrez, C. T. (2026). Anopheles azevedoi (Ribeiro, 1969) in Angola: new geographical records, molecular characterization, and insecticide susceptibility profile of a commonly misidentified species. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07706-5

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07706-5

Keywords: Anopheles azevedoi, Angola, malaria, mosquitoes, insecticide resistance, pyrethroids, DDT, chlorfenapyr, COI barcoding, ITS2, vector surveillance, urban malaria

A team of mycologists and microbial ecologists from Rovira i Virgili University in Reus, Spain, led by Alan Omar Granados-Casas, Alberto Miguel Stchigel, Ana Fernández-Bravo and José Francisco Cano-Lira, compared sediment communities from two strikingly different neighbouring water bodies. Salada Grande de Chiprana is a hypersaline lagoon whose waters are far saltier than the ocean, while the nearby La Estanca is an oligohaline lagoon with only low levels of dissolved salts. By sampling both, the researchers could ask a deceptively simple question: does salinity structure the invisible communities of fungi and prokaryotes that live in lagoon sediments, and how reliable are the molecular tools used to detect them?

The team used DNA metabarcoding, a technique that extracts environmental DNA from sediment, amplifies a specific genetic marker with the polymerase chain reaction, and then sequences the resulting mixture to identify which organisms are present. For fungi, the researchers targeted the internal transcribed spacer region of the nuclear ribosomal DNA, the standard barcode for fungal identification. Crucially, they ran two versions of this marker, ITS1 and ITS2, which flank the 5.8S ribosomal RNA gene and are often treated as interchangeable in the literature. For prokaryotes, meaning bacteria and archaea, they sequenced the 16S ribosomal RNA gene, the long-standing workhorse of bacterial diversity surveys.

When the two fungal marker datasets were analysed together, the fungal communities in both lagoons were dominated by three major lineages: the phylum Ascomycota, which contains most described fungal species; the Rozellomycota, a mysterious group of obligate parasites of other microorganisms; and the Basidiomycota, which includes mushrooms and their relatives. The prokaryotic assemblages, by contrast, were composed mainly of Pseudomonadota, a hugely diverse bacterial phylum, along with Cyanobacteriota, the photosynthetic bacteria that often form conspicuous microbial mats in saline environments, plus Bacteroidota and Chloroflexota, both common in sediments rich in organic matter.

The first surprise came from the diversity estimates themselves. When the researchers counted the number of distinct amplicon sequence variants, or ASVs, in each sample, they found no significant difference in fungal alpha diversity between the hypersaline and oligohaline lagoons. In other words, the saltier environment did not appear to host a depauperate fungal community compared with its brackish neighbour. However, the choice of marker mattered: the ITS2 primer pair recovered a significantly higher number of observed ASVs than ITS1, a difference the authors report as statistically significant with a p-value of 0.016. Two markers from the same genomic region, amplified from the same sediments, delivered measurably different inventories of fungal life.

The deeper analysis sharpened that warning. Using Bray-Curtis dissimilarity, a measure of how different community compositions are between samples, combined with PERMANOVA, a permutation-based statistical test for community data, the team found that fungal community composition was significantly associated with two factors: the identity of the lagoon and the molecular marker used. Remarkably, the marker choice explained more of the variation in the fungal data than the lagoon identity itself, and there was also a significant interaction between the two, meaning the apparent effect of the lagoon depended on which marker was being examined. For a field that often draws ecological conclusions from a single marker, that is a sobering result.

Salinity, the environmental variable most obviously differentiating the two lagoons, turned out to be a weaker predictor than expected. The researchers used electrical conductivity as a proxy for salinity and found that it explained only a limited and statistically non-significant fraction of the variation in fungal community composition. The authors are careful to note an important caveat: because the two lagoons differ in more ways than one, conductivity’s independent contribution could not be cleanly separated from other environmental differences between the sites. The hypersaline lagoon is not simply a saltier version of La Estanca; it is a distinct ecosystem with its own chemistry, hydrology and history, and any of those factors could be shaping its fungal residents.

The prokaryotic side of the story unfolded differently. While the bacterial and archaeal communities showed clear taxonomic differences between the two lagoons, the beta-diversity analysis found no statistically significant separation between them at the community level, largely because of substantial variability among samples taken within each lagoon. This contrast between fungi and prokaryotes is one of the study’s most intriguing findings. Two major branches of life, sampled from the same handful of sediments with standard molecular methods, responded in visibly different ways to the same environmental gradient. It suggests that fungal and prokaryotic communities are governed by different ecological rules in these systems, perhaps reflecting differences in dispersal ability, dormancy strategies, or the way each group partitions the sediment environment.

Perhaps the most tantalising result is what the researchers could not identify. A large fraction of the fungal ASVs recovered from the sediments remained taxonomically or functionally unclassified, meaning they did not match any known reference sequence well enough to assign a name or a role. These sediments, it appears, harbour poorly characterised fungal diversity, possibly including lineages new to science or at least new to databases. Inland hypersaline environments are understudied compared with marine and terrestrial habitats, and European examples in particular have rarely been surveyed for fungi with modern molecular methods. Chiprana’s sediments may therefore represent a reservoir of unknown fungal life adapted to some of the saltiest non-marine conditions on the continent.

The broader lesson of the study extends well into the booming world of environmental DNA research. Metabarcoding studies now underpin everything from biodiversity monitoring to climate impact assessments, and most rely on a single marker per organismal group. This work shows that for fungi, ITS1 and ITS2 are not interchangeable windows onto the same community: they recover complementary, partially overlapping views, and conclusions drawn from one may not survive scrutiny with the other. The authors argue that marker choice must be explicitly considered when interpreting metabarcoding-based ecological patterns, and that combining markers, as they did here, offers a more complete picture. For the microbial ecologists charting life in Earth’s extreme environments, the message is clear: before declaring what lives in a hypersaline lagoon, it pays to check which lens you are looking through.

Subject of Research: Fungal and prokaryotic sediment communities in hypersaline and oligohaline lagoons assessed by ITS and 16S rRNA metabarcoding

Article Title: Marker-Dependent Biases and Contrasting Fungal and Prokaryotic Community Patterns in Hypersaline and Oligohaline Lagoons

Article References: Granados-Casas, A. O., Stchigel, A. M., Fernández-Bravo, A., & Cano-Lira, J. F. (2026). Marker-Dependent Biases and Contrasting Fungal and Prokaryotic Community Patterns in Hypersaline and Oligohaline Lagoons. Microbial Ecology. https://doi.org/10.1007/s00248-026-02900-5

Image Credits: AI Generated

DOI: 10.1007/s00248-026-02900-5

Keywords: hypersaline lagoons, fungal ecology, metabarcoding, ITS1, ITS2, 16S rRNA, prokaryotes, salinity, Chiprana, microbial diversity, extremophiles, sediment microbiology

The investigation was led by Márcio Galvão Pavan of the Oswaldo Cruz Institute (Fiocruz) in Rio de Janeiro, together with Rodrigo Gurgel-Gonçalves of the University of Brasília and colleagues, including Fernando Abad-Franch and Fernando Araujo Monteiro, who contributed equally to the work. The team set out to answer a deceptively simple question: when light-colored bugs turn up inside carnaúba palms, are they R. nasutus, the species whose primary habitat these palms represent in northeastern Brazil, or could they actually be R. neglectus wearing, in effect, a different species’ colors? The answer, according to the combined genetic and morphological evidence, is frequently the latter.

The study’s foundation lies in an ecological and biogeographical puzzle. Rhodnius nasutus is typical of the Caatinga, Brazil’s seasonally dry, thorny scrubland biome, where it dwells chiefly in the crowns of Copernicia prunifera palms and is characteristically light brown in color. Rhodnius neglectus, by contrast, is a core-Cerrado species associated with Mauritia flexuosa palms and typically shows a much darker brown body. Because the Caatinga and Cerrado intergrade across a broad transition zone, the two palm species, and the bugs that inhabit them, occasionally come into geographic contact. That overlap set the stage for the color confusion the researchers document.

To untangle the situation, the team sampled triatomines from C. prunifera palms across the semiarid Caatinga and the Caatinga-Cerrado transition, comparing them with specimens collected from core-Cerrado M. flexuosa palms. Every insect was first identified by phenotype, meaning its visible color pattern and morphology. The researchers then turned to genetics, sequencing clones of the mitochondrial cytochrome b gene, a standard barcoding marker, and the nuclear ribosomal DNA internal transcribed spacer 2, or ITS2. This two-marker strategy allowed the team to compare signals from the maternally inherited mitochondrial genome with those from the nuclear genome, a contrast that proved decisive.

The morphological results were clear at the extremes but murky in the middle. Adult specimens from core-Cerrado Mauritia palms consistently displayed the dark brown coloration typical of R. neglectus, while adults from core-Caatinga localities showed the light brown hue typical of R. nasutus. But in bugs captured in carnaúba palms from the Caatinga-Cerrado transition zone and parts of the central-western Caatinga, color patterns were dubious, matching neither type reliably. Specimens that looked like typical light brown R. nasutus could not confidently be assigned on appearance alone, raising the possibility that field identifications based on color might have been systematically wrong in exactly these regions.

Genetics began to resolve the ambiguity. Bayesian genealogies built from cytochrome b sequences placed the dubious-phenotype specimens that resembled typical R. nasutus in a sub-clade that is sister to typical R. neglectus from Mauritia flexuosa palms, with roughly 2% mean cytb divergence separating the two sub-clades. In other words, the bugs that looked like R. nasutus carried mitochondrial genomes pointing to R. neglectus ancestry. This mitochondrial signal effectively dissolved the taxonomic uncertainty created by the shared coloration, demonstrating that insects occupying carnaúba palms in the transition zone belong, at least by maternal lineage, with R. neglectus.

The nuclear ITS2 marker told a less tidy story. ITS2 genealogies recovered both R. neglectus and R. nasutus as paraphyletic, meaning individuals of each nominal species were scattered across the gene tree rather than clustering neatly by species. Such patterns often arise when recently diverged species still share ancestral genetic variation. When the researchers applied a multispecies-coalescent analysis, a statistical framework designed to account for this shared ancestry, the dubious-phenotype bugs from C. prunifera clustered with typical R. neglectus. The analysis suggested that the messy ITS2 picture reflects incomplete lineage sorting, the retention of ancestral polymorphisms, rather than hybridization or misassigned species limits. The mito-nuclear discordance observed in the study is thus itself scientifically informative, illustrating how different genomes can carry different historical signals during the early stages of species divergence.

Why would two species converge on the same color? The authors propose that predator-driven natural selection is the most plausible mechanism. Triatomine bugs live in palm crowns where the substrate color is set by the palm’s dried leaf bases and fibers. In the core Cerrado, dark Mauritia crowns favor dark R. neglectus; in the core Caatinga, the lighter carnaúba substrate favors pale R. nasutus. But when R. neglectus colonizes carnaúba palms, individuals whose coloration better matches the light palm-crown substrate presumably enjoy better camouflage against visual predators such as birds and lizards. Over time, this selection pressure could produce R. neglectus populations whose light brown bodies are nearly indistinguishable from typical R. nasutus. This is a textbook scenario of adaptive phenotypic convergence, conceptually related to classic camouflage and mimicry systems in evolutionary biology, but documented here in disease vectors, where species identity has public health consequences.

The practical implications are significant. Chagas disease remains a major neglected tropical disease in Latin America, and surveillance programs depend on correctly identifying which vector species are present in a given area, because species differ in habitat preferences, domestic invasion behavior, and epidemiological relevance. If R. neglectus can masquerade as R. nasutus, field records based on color alone may misattribute vectors to the wrong species and thereby misguide control strategies. The study’s findings, the authors argue, call into question the widespread use of overall body color as a key phenotypic character in triatomine taxonomy. As molecular tools become more accessible, integrating DNA-based confirmation into routine vector surveillance may be essential wherever palm-dwelling Rhodnius species co-occur or share similar microhabitats.

Beyond the immediate applied concerns, the work contributes to a broader understanding of how color variation evolves in triatomines and how convergence can erode the morphological signals taxonomists depend on. The researchers received support from Brazil’s CAPES and CNPq funding agencies, and sampling was conducted under permit from the country’s biodiversity authority, ICMBio. By combining careful field collection across two biomes with mitochondrial and nuclear sequence analysis and modern coalescent modeling, the team has shown that even a character as apparently straightforward as body color can be shaped powerfully by ecology, and that species identities written in DNA may diverge sharply from those written on the insect’s back. For the bugs of the carnaúba palms, looking like the neighbor may be good for survival, but it makes life considerably harder for the scientists trying to tell them apart.

Subject of Research: Phenotypic convergence in the Chagas disease vector bugs Rhodnius neglectus and Rhodnius nasutus inhabiting Copernicia prunifera palms in Brazil

Article Title: Phenotypic convergence in Chagas disease vectors: Rhodnius neglectus from Copernicia prunifera palms may express the typical Rhodnius nasutus color phenotype

Article References: Pavan, M. G., Gurgel-Gonçalves, R., Corrêa-Antônio, J., Morelli, K. A., Bahia, A. C., Abad-Franch, F., & Monteiro, F. A. (2026). Phenotypic convergence in Chagas disease vectors: Rhodnius neglectus from Copernicia prunifera palms may express the typical Rhodnius nasutus color phenotype. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07651-3

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07651-3

Keywords: Triatominae, Rhodnius, Chagas disease, phenotypic convergence, convergent evolution, camouflage, Copernicia prunifera, cytochrome b, ITS2, incomplete lineage sorting, mito-nuclear discordance, Brazil