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Frontiers in ZoologyZoologyPeer-reviewed journal article

An integrative taxonomic approach reveals unexplored diversity in Croatian planarians

Miquel Vila‐FarréiD, Jeremias N. BrandiD, Tobias BootheiD, Maren Brockmeyer, Fruzsina Ficze-Schmidt, Markus A. GrohmeiD, Uri WeilliD, Kasper H. Kluiver, Ludwik GąsiorowskiiD, Lucija Kauf, Yuliia KananaiD, Helena BilandžijaiD, Marta RiutortiD, Jochen Christian RinkiD

PDFDOIPreprint

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (16 Apr 2025) · CC BY 4.0

Degenerate COI primers combined with histology, karyology and transcriptomics raise the known Croatian freshwater planarian species from eight to sixteen and describe Dendrocoelum pigmentatum sp. nov.

Species-level DNA barcoding of freshwater planarians is held back by COI primers that work poorly on planariids and dendrocoelids and by a GenBank reference set skewed toward Dugesiidae, so the fauna of species-rich regions such as karstic Croatia remains poorly documented. The work adds optimised degenerate primers, curated barcodes and transcriptomes for undersampled families, and pairs them with histology and karyology to settle species identities.

Key results

  1. ShownA large pigmented dendrocoelid found at three karst sites in Croatia is described as Dendrocoelum pigmentatum sp. nov. Its closest relatives are pigmented Dendrocoelum species otherwise confined to the Ohrid-Prespa lakes region.

    The transcriptome IQ-TREE (55 transcriptomes) places it in a maximum-support clade with two pigmented Ohrid species. COI barcodes separate it from D. cf. sanctinaumi and D. maculatum. The sites lie about 600 km north of Ohrid-Prespa and fall in two drainages (Adriatic and Black Sea). · Croatia field sampling campaign and preliminary…; Phylogenetic relationships of the Croatian…; Habitat; Taxonomic discussion

  2. ShownSurveys of karst surface waters lifted Croatia's documented freshwater planarian tally from eight to sixteen species. They also recovered Polycladodes alba, which may have gone unrecorded in the country for over 100 years.

    About 35 new planarian records. Barcodes identified Girardia tigrina, Dugesia sicula, Schmidtea polychroa, S. lugubris, Dendrocoelum lacteum, Planaria torva and Polycelis felina; histology confirmed P. alba and Crenobia montenigrina. The over-100-year gap for P. alba holds only if Sorocelopsis decemoculata is a synonym. · DNA barcoding of Croatian planarians; Taxonomic discussion; Taxonomic discussion; General discussion

  3. ShownA new degenerate COI primer pair with inosine bases (MVCOI900) amplifies an ~880 bp fragment in dugesiids, planariids and dendrocoelids. It substantially expands curated barcodes for the two families that were poorly represented.

    PCR success was 87.5 % for dendrocoelids, 71.4 % for dugesiids and 68.18 % for planariids (Phagocata and Crenobia were difficult). Dataset Barcoding holds 124 sequences, 77 of them new (two made with another primer pair). New amplicons were 734 to 890 bp (mean 844 bp) vs 302 to 1755 bp (mean 620 bp) for 47 GenBank records. Includes the first COI record for Planaria torva. · Optimisation of COI primer pair for European…; DNA barcoding of Croatian planarians; Taxonomic discussion

  4. SuggestedSchmidtea lugubris splits into a Central and an Adriatic COI haplotype clade. Low transcriptomic divergence, a shared 2n = 8 karyotype and similar genital anatomy point to lineages within one species rather than cryptic species.

    Branch length between the two clades was 0.0121, vs 0.1293 for S. mediterranea vs S. polychroa and 0.1359 for S. nova vs British S. lugubris. Dataset 1 (308 bp) had 10 haplotypes and Dataset 2 (702 bp) had 8. Of 54 metaphase plates from 9 individuals, most showed eight chromosomes; five plates had 7, 9 or 12. · Metaphasic plate production and analysis; Phylogenetic relationships of the Croatian…; Karyological analysis

  5. ShownFlatworm COI references in GenBank are skewed. Parasitic Neodermata dominate. Among freshwater triclads, Dugesiidae (especially Dugesia and Schmidtea) is overrepresented while Planariidae and Dendrocoelidae are sparse, and two records appear assigned to the wrong genus.

    Of 41743 platyhelminth COI entries, 87.6 % are Neodermata. Share of triclad accessions vs share of known species: Dugesiidae 37.8 % vs 13.1 %, Planariidae 6 % vs 10.3 %, Dendrocoelidae 0.6 % vs 14.5 %. Kenkiidae (24 species) has none. DQ666053 (Phagocata) groups with Girardia, and AF178312 (D. lacteum) groups with Bdellocephala punctata. · DNA barcoding analysis reveals taxon biases in…; DNA barcoding of Croatian planarians

Methods, data and reproducibility

Field-collected planarians were sorted by live morphology and barcoded with new degenerate COI primers, then placed in IQ-TREE COI trees. Identities were then resolved with RNA-seq phylotranscriptomics (1701 orthologous protein alignments), COI haplotype networks, histological reconstruction of copulatory anatomy and DAPI-stained metaphase karyology.

  • Planarians collected from rivers, lakes and springs in the Croatian karst and on other European expeditions, mostly between 2013 and 2023 · freshwater planarians (Tricladida), multiple species
  • Dataset Barcoding: COI sequences of European freshwater planarians, new and from GenBank · Tricladida, multiple species · n = 124 sequences (77 new, 47 GenBank) · New accessions referenced to Additional File 2
  • Flatworm COI entries from the NCBI nucleotide archive, retrieved 2024-04-11, for the taxon-coverage analysis · Platyhelminthes · n = 41743 entries
  • Phylotranscriptomic set: new transcriptomes (P. alba, D. pigmentatum, D. cf. sanctinaumi, Dugesia sp., Croatian S. lugubris) plus published ones · 51 planarians plus four outgroups · n = 55 transcriptomes
  • S. lugubris COI haplotype datasets: Dataset 1 (308 bp) and Dataset 2 (702 bp) · Schmidtea lugubris · n = 16 and 12 sequences
  • S. lugubris metaphase plates from the Nottingham, Black Drim and Croatian populations · Schmidtea lugubris · n = 54 plates from 9 individuals
  • Serial histological sections of S. lugubris, P. alba, D. pigmentatum, P. torva and C. montenigrina · Tricladida, five species · Holotype H0366; slides to be deposited at ZMUG
Methods & tools
Degenerate COI primer pair MVCOI900 (inosine-containing), Geneious and PerlPrimer (alignment, primer design), MAFFT (v7.490, v7.525), IQ-TREE with ModelFinder, 1000 ultrafast bootstraps, TaxonKit (v0.18.0), DnaSP6 and Network v10.2 (median-joining haplotype networks), fastp (v0.23.4), Kraken 2 (v2.1.3), KrakenTools, Trinity (v2.15.2) and TransDecoder (v5.7.1), OrthoFinder (v2.5.4), FastTree (v2.1.10), PhyloPyPruner, MMseqs2 (contaminant filtering, homology search), Mallory-Cason/Heidenhain histology; Olympus VS200 slide scanner, Nocodazole metaphase arrest, DAPI staining, FIJI deconvolution
Reagents & environment
Stated versions: TaxonKit v0.18.0, MAFFT v7.490 and v7.525, fastp v0.23.4, Kraken 2 v2.1.3, Trinity v2.15.2, TransDecoder v5.7.1, OrthoFinder v2.5.4, FastTree v2.1.10, Network v10.2. No container or environment file is given.
Reproducibility gaps
COI sequences, raw RNA-seq, transcriptome assemblies and slides are promised for future public deposition only. New GenBank IDs are deferred to Additional File 2, and no analysis code repository is given.
Limitations (6)
  • Sampling in Croatia was limited in scope and covered only surface waters. The authors expect further surface and cave sampling to raise the regional species count. · stated by the authors
  • MVCOI900 is not yet a universal planarian primer. Amplification was hard for Phagocata and Crenobia, possibly because of primer mismatch or PCR inhibitors, and further optimisation is needed. · stated by the authors
  • COI barcoding was inconclusive for P. alba, Croatian Phagocata, several Polycelis samples and the pigmented dendrocoelids. The authors note that COI alone is limited when the reference library is taxonomically biased, so histology was still needed. · stated by the authors
  • Sparse sampling in the Western and Southern Balkans leaves the extent of the Adriatic S. lugubris clade uncertain. A human introduction of D. pigmentatum cannot be fully ruled out. · stated by the authors
  • The D. pigmentatum species description rests on one barcoded individual per locality and a few sectioned specimens, and traits such as pharynx length and adenodactyl papilla shape differ between populations. · noted in this summary
  • The intraspecific reading of the S. lugubris clades rests on comparing branch lengths with sister-species pairs. The Croatian karyotype comes from a single individual, and no nuclear population-genetic or crossing data are given. · noted in this summary

Read this if you work on freshwater invertebrate barcoding, planarian taxonomy or Balkan biogeography and need COI primers, curated reference barcodes, or the description of a new pigmented Dendrocoelum species.

Citations
3
FWCI
15.19
Field percentile
top 3%

Abstract

BackgroundFreshwater ecosystems are among the most endangered habitats on Earth, with approximately one-fourth of aquatic species at risk of extinction. Effective conservation efforts require comprehensive monitoring and accurate species identification, including often overlooked groups. DNA barcoding promises rapid and accessible species identification but requires the availability of "universal" primer pairs and robust, taxonomically curated reference libraries. Planarian flatworms are one such group for which these resources are currently lacking, even though they are common constituents of freshwater ecosystems worldwide. As a result, the true extent of planarian diversity remains underappreciated in many areas. ResultsMotivated by the highly skewed representation of planarian diversity in current GenBank records of the barcoding gene mitochondrial cytochrome c oxidase subunit I (COI), we optimised existing barcoding primers and tested them on planarians collected in a species-rich but, so far, poorly surveyed region of Croatia. Using an integrative approach combining the new primers, traditional taxonomy and karyology, we generated new taxonomically curated COI barcode sequences for European dugesiids, dendrocoelids and planariids, which, in the case of the latter two groups, represent a substantial increase of the number of entries for those families. Our efforts resulted in several significant findings, including the description of a new pigmented Dendrocoelum species, Dendrocoelum pigmentatum sp. nov, the discovery of two highly differentiated haplotypic clades in Schmidtea lugubris, and the rediscovery of Polycladodes alba in Croatia after a century. ConclusionsOverall, our work extends the utility of DNA barcoding for species-level identification of planarians to previously inaccessible groups. Additionally, it integrates Croatia as an underexplored but species-rich region into the endeavour to systematically describe the planarian fauna of Europe. The expansion of a known number of Croatian planarian species from eight to sixteen and the discovery of a new, large planarian species in continental Europe, Dendrocoelum pigmentatum, demonstrate the effectiveness of our integrative approach. Overall, our work highlights the underappreciated diversity of planarians, even in continental Europe and supports practical conservation efforts to preserve aquatic biodiversity.

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