Seven-site river study finds bryophyte-enhanced PCR detects more species than hybridisation capture

A comparative study at seven sites along the Falkensteinerbach River found that PCR metabarcoding with bryophyte-enhanced primers detected 101 bryophyte species, compared with 68 in expert field surveys and 27 through hybridisation capture. Across all three approaches, researchers recorded 145 species, but only nine were recovered by every method, indicating that visual and molecular surveys captured substantially different parts of the riparian bryophyte community. The research was conducted in the Vosges du Nord Regional Nature Park and compared three monitoring approaches: visual transect surveys based on macro- and micromorphology, PCR metabarcoding of the chloroplast rbcL gene and nuclear ribosomal ITS2 region, and hybridisation capture using the same two markers.

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