# Molecular characterisation of the invasive terrestrial nemertean Geonemertes pelaensis: long and complex mitogenome and presence of NUMTs

Romain Gastineau, Mathieu Coulis, Christian Otis, Brian Boyle, Claude Lemieux, Monique Turmel, Sima Mohammadi, Roger Lévesque, David Herbert, Barna Páll-Gergely, et al.

► To cite this version:

Romain Gastineau, Mathieu Coulis, Christian Otis, Brian Boyle, Claude Lemieux, et al. Molecular characterisation of the invasive terrestrial nemertean Geonemertes pelaensis: long and complex mitogenome and presence of NUMTs. Scientific Reports, 2026, 16 (1), pp.3312. 10.1038/s41598-025-33230-0. hal-05518437

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# Molecular characterisation of the invasive terrestrial nemertean *Geonemertes pelaensis*: long and complex mitogenome and presence of NUMTs

Romain Gastineau<fnref n="1" />✉, Mathieu Coulis²,³, Christian Otis⁴, Brian Boyle⁴, Claude Lemieux<fnref n="5" />, Monique Turmel<fnref n="5" />, Sima Mohammadi<fnref n="5" />, Roger C. Lévesque<fnref n="5" />, David G. Herbert⁶, Barna Páll-Gergely⁷,⁸, Ira Richling<fnref n="9" />, Leigh Winsor¹⁰ & Jean-Lou Justine⁶

The complete mitochondrial genome of the invasive terrestrial nemertean *Geonemertes pelaensis* Semper, 1863 (Nemertea: Prosorhochmidae) was sequenced from two specimens collected in geographically distant French overseas territories—Martinique in the Caribbean and New Caledonia in the South-West Pacific. In both specimens, the mitogenome contained 13 protein-coding genes, two rRNA genes, and 21 tRNA genes, and was unusually large, approaching 32 kb. The two genomes differed by only four single nucleotide polymorphisms and one indel. A comparison with 22 *cox1* sequences available in GenBank confirmed this high level of genetic conservation, suggesting a recent introduction from related source populations. The extraordinary length of the mitogenome was largely attributable to two extended regions comprising only tRNA genes and long intergenic sequences. These results were contrasted with data from an unpublished SRA sequencing project (SRS20559370) of an unlocalized specimen identified as *G. pelaensis*; its reconstructed mitogenome was only 18 kb in length (14 kb shorter) and showed extensive sequence divergence. Phylogenetic analyses placed this specimen as the sister lineage to *G. pelaensis*, highlighting the need for further investigation of this taxon. In the Martinique specimen, several NUMTs (nuclear mitochondrial pseudogenes) were also detected, which could complicate future studies relying solely on Sanger sequencing. Sequencing additionally revealed prey DNA from the gut contents of both worms: the New Caledonian specimen had consumed an unidentified noctuid moth, while the Martinique specimen had likely fed on the invasive cockroach *Periplaneta australasiae* (Fabricius, 1775), itself an introduced species.

Nemertea constitutes a phylum of invertebrates also known as “ribbon worms”. The vast majority of its representatives are marine organisms, which includes *Lineus longissimus* (Gunnerus, 1770), the longest known animal. Among Nemertea, the suborder Monostilifera singularises itself by containing terrestrial species among marine ones. These terrestrial species are distributed among two families, the Acteonemertidae and the Prosorhochmidae, the later one also encompassing parasitic species.

Parallels can be drawn between terrestrial Nemertea and terrestrial flatworms (phylum Platyhelminthes: family Geoplanidae), with which they are easily and frequently misidentified. Indeed, both represent terrestrial

<footnote n="1">¹Institute of Marine and Environmental Sciences, University of Szczecin, Szczecin, Poland. ²CIRAD, UPR GECO, 97285 Le Lamentin, Martinique, France. ³GECO, CIRAD, University Montpellier, Montpellier, France. ⁴Plateforme d'Analyse Génomique, Institut de Biologie Intégrative et des Systèmes, Université Laval, Quebec, QC, Canada.</footnote>

<footnote n="5">⁵Département de Biochimie, de Microbiologie et de Bio-Informatique, Institut de Biologie Intégrative et des Systèmes, Université Laval, Quebec, QC, Canada. ⁶ISYEB, Institut de Systématique, Évolution, Biodiversité (UMR7205 CNRS, EPHE, MNHN, UPMC, Université des Antilles), Muséum National d'Histoire Naturelle, CP 51, 55 Rue Buffon, 75231 Paris Cedex 05, France. ⁷HUN-REN Centre for Agricultural Research, Plant Protection Institute, Brunszvik u. 2, Martonvásár 2462, Hungary. ⁸Department of Soil, Water and Natural Sciences, Albert Kázmér Faculty of Agricultural and Food Sciences of Széchenyi István University, Vár 2., Mosonmagyaróvár 9200, Hungary.</footnote>

<footnote n="9">⁹Stuttgart State Museum of Natural History, Rosenberg Gewann 1, 70191 Stuttgart, Germany. ¹⁰College of Science and Engineering, James Cook University, Townsville, QLD, Australia. ✉email: romain.gastineau@usz.edu.pl</footnote>
exceptions in predominantly marine phyla, and both are now known for the invasive character of some species, whether potential or assessed<sup>1-5</sup>. Although there is an increasing number of invasive land flatworms recorded, only two species of Nemertea, *Argonemertes dendyi* Dakin, 1915, and *Geonemertes pelaensis* Semper, 1863, both considered endemic to the Australasian region, are now widely dispersed through human agency. The latter species is now invasive in most tropical regions of the world<sup>6-8</sup> and also in greenhouses in other regions; see map in GBIF ([https://www.gbif.org/species/6786919](https://www.gbif.org/species/6786919)). The success in dispersal of these two species can be attributed to both being hermaphroditic: *A. dendyi* protandrous, and *G. pelaensis* a cyclic hermaphrodite with subsequent multiple sex reversals<sup>7,9</sup>. With the rise of citizen science and the development of internet resources such as iNaturalist, an increasing number of observations have been done worldwide, although many of which are waiting to be confirmed.

Two specimens of *G. pelaensis* were collected in two very distant islands, Martinique in the Caribbean and New Caledonia in the South-West Pacific (Fig. 1). In the context of our genomic investigations of invasive invertebrates<sup>10-13</sup>, these samples were subjected to short and long reads sequencing. The study aimed at obtaining their mitogenomes, which were quickly proven to be large and complex, and also to gather information regarding their diet. As an unexpected outcome, when these results were compared to an other data from a non-curated SRA BioProject conducted on a specimen ascribed to *G. pelaensis* (BioSample SAMN40098146), more questions arose.

# Results

## The extra-large mitogenome

For unknown reasons, the first attempts to assemble the mitochondrial genome of specimen MNHN JL402 using short-reads failed, with several high coverage contigs retrieved after data-mining that Consed<sup>14</sup> failed to

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** (a) *Geonemertes pelaensis* MNHN JL402. Martinique. A typical specimen, exhibiting faint paired lateral stripes obvious anteriorly, in addition to the usual brown median dorsal stripe. Unscaled. Photo by Mathieu Coulis. (b) *Geonemertes pelaensis* MNHN JL632. New Caledonia. The four groups of eyes and the common opening for the mouth and proboscis are clearly evident. Unscaled. Photo by David G. Herbert. (c) *Geonemertes pelaensis* MNHN JL632. New Caledonia. The preserved specimen with the proboscis everted (arrow). The beginnings of the paired lateral stripes are obvious anteriorly. Photo by Jean-Lou Justine.</figcaption></figure>
merge. The possible explanations of these difficulties could be the distribution of the reads and/or the presence of NUMTs within this sample, as described and discussed below. These issues did not occur with specimen MNHN JL632, but it should be underlined that its sequencing returned three time fewer short reads. Long-reads assemblies both concurred to retrieve the mitogenome as circular contigs (as detected by the assembler) of more than 31 kb with high coverages of 527× and 106× for MNHN JL402 and MNHN JL632, respectively.

After polishing with short-reads, the final length of the mitogenome is 31,738 bp long for MNHN JL402 (GenBank: PQ336987) and 31,739 bp for MNHN JL632 (GenBank: PX238424). It codes for 13 protein-coding genes (PCG), two rRNA and 21 tRNA. All genes are on the same strand, except for two tRNA (*tRNA-Pro* and *tRNA-Thr*).

The short-reads derived mitogenome obtained on MNHN JL632 is available as Supplementary file S1. It came as a 31,870 bp contig with a 89× coverage. It had redundant endings of 125 bp, congruent with the k-mer used for assembly. After circularization, the genome is 31,745 bp long, thus a 6 bp difference of length with the long-reads derived mitogenome. Except for these indels, no differences could be found between the two versions of the mitogenome. For questions of consistency however, this is the long-reads derived mitogenome that for both samples was later kept for subsequent analysis.

Large portions of non-coding sequences are interspersed along the mitogenome. There is a 14,129 bp region between *ND4* and *ATP8* that contains a single PCG (*cox3*) and 11 tRNA. Queries for microsatellites or tandem repeats did not return significant results, neither for this region nor for the whole genome. Surprisingly, the large intergene between *tRNA-Glu* and *tRNA-Lys* was shown to align with *ND1* on its whole CDS, although with only 72.92% identity. This alignment can be found as Supplementary file S2. Since they are colinear and conserved, only the map of the mitogenome of MNHN JL402 is shown (Fig. 2). The two genomes differed only by four single nucleotide polymorphisms and one indel. The indel was found in the *rrnL* gene. Two of the polymorphisms concern intergenic sequences located in the aforementioned large area (*cox3/tRNA-Gly*, *tRNA-Val/tRNA-Ala*) but the two others were found in *ND5*, one of which being a silent mutation.

The mitogenome retrieved for BioSample SAMN40098146 from the unpublished BioProject PRJNA1080026 is only 18,540 bp long (Fig. 3). It is not colinear with those of specimens MNHN JL402 and MNHN JL632 but also displays only *tRNA-Pro* and *tRNA-Thr* on its negative strand (Supplementary file S3). All its coding content presents massive differences when compared to *G. pelaensis* MNHN JL402 and MNHN JL632. For example, the *cox1* gene, whose length is identical (1,536 bp), is only 91.21% conserved (protein sequences conserved at 96.87%).

## The rRNA cluster

For MNHN JL402, the rRNA cluster was retrieved as a 7,719 bp long contig (GenBank: PV031326) distributed as 1,832 bp (18S), 1,284 bp (*ITS1*), 153 bp (5.8S), 480 bp (*ITS2*) and 3,970 bp (28S). Blastn query of the complete cluster returned 100% identity with sequences EU255578 (18S part)¹⁵ and HQ856887 (28S part)¹⁶. The rRNA cluster of MNHN JL632 was 7,711 bp long (GenBank: PX232434) and was completely conserved with previous sequence, except for an 8 bp sequence located in the ITS1. It is to note that these 8 bp are a dimer of an 'ATGC' motif. This motif is copied 8 times in the ITS1 of MNHN JL402, and 6 times in MNHN JL632.

For BioSample SAMN40098146 an 8,082 bp rRNA cluster could be retrieved, distributed 1,832 bp (18S), 1,337 bp (*ITS1*), 153 bp (5.8S), 652 bp (*ITS2*) and 4,108 bp (28S) (Supplementary file S4). The 18S displays six polymorphisms when compared to MNHN JL402/MNHN JL632.

## Phylogeny

The multiprotein tree distinguishes four groups (Fig. 4, excluding the outgroups). Two correspond to the class Palaeonemertea, one to the class Pilidiophora and the last one to the class Hoplonemertea. *Geonemertes pelaensis* MNHN JL402 and MNHN JL632 are contained among Hoplonemertea with the longest branch from the common node. They cluster together and are sister to BioSample SAMN40098146 with maximum support at the nodes. Then, the closest relative is *Prosadenoporus spectacula* (Yamaoka, 1940)¹⁷ but with a limited support of 44%.

The two-genes *16S-cox1* tree (Fig. 5) clearly distinguishes *G. pelaensis* from *Prosadenoporus* spp. It associates *G. pelaensis* MNHN JL402 and MNHN JL632 to voucher specimens of *G. pelaensis* (MCZ DNA102574 and MCZ DNA102574.1 from St Davis, Bermuda) in a single clade. The closest relative is once more BioSample SAMN40098146, with maximum support. This cluster of terrestrial species is sister to the marine *Malacobdella grossa* (Müller, 1776), with a support of 91%.

The *cox1*-inferred tree (subtree shown as Fig. 6) associates in a single clade all specimens of *G. pelaensis* for which a *cox1* gene is available, minus BioSample SAMN40098146 which is outside this clade, yet sister to it with maximum support. *Malacobdella grossa* is again sister to this clade with a support of 97%.

## Nuclear mitochondrial pseudogenes (NUMTs)

While data-mining the short-read derived contigs file obtained from MNHN JL402, three large contigs with lower coverage returned positive results (Supplementary file S5). The first one was 6,655 bp long with a coverage of 17.05x. No large open-reading frame (ORF) could be evidenced using neither genetic code 1 nor 5, but a blastx query on the NCBI database with code 5 suggested the presence of a pseudogene version of *cox3* containing internal stop codons. The largest ORF that could be identified within this fragment was a 156 AA fragment that returned 92.05% of identity with the Cox3 protein of *G. pelaensis* and an e-value of 3e-84. A megablast query of this contig also showed that two portions of it could align with the mitogenome of *G. pelaensis*, one on a large span of 1,224 bp on 1,360 bp, a smallest one of 216 bp on 236 bp. Both aligning fragments are separated by ca. 3,500 bp which did not return a megablast result once trimmed. The second contig was 4,210 bp long with a coverage of 22.94x, aligned on a far large span with the mitogenome (3,885 bp identical on 4,215) and contained
<figure><img src="image_4.png" /><figcaption>Fig. 2. Map of the mitochondrial genome of *Geonemertes pelaensis* MNHN JL402.</figcaption></figure>

pseudogenized versions of *cox1*, *cox2*, *ATP6* and *ND2*, in identical order as in the mitogenome. A third one was 3,250 bp long with a coverage of 23.70× and contained pseudogenized versions of *cob* and *ND1*. The same analysis was performed on the contigs file of MNHN JL632 but returned no comparable results.

## Alien DNA and prey

Data-mining of the contigs file from MNHN JL402 allowed retrieval of both a complete mitogenome (Supplementary file S6) and a complete cluster of nuclear rRNA (Supplementary file S7) of prey DNA. The cluster of rRNA was found in a 14,068 bp long contig. The mitogenome was found in a 15,674 bp contig showing redundant ending. After trimming, the rRNA is 6,777 bp long. Megablast query returned as best result the 1,808 bp long partial *18S* gene of *Periplaneta australasiae* (Fabricius, 1775) (DQ874169) with e-value 0.0 and 100% of identity¹⁸. After trimming, the mitogenome is 15,604 bp long. Megablast query returned as best result the 15,605 bp long mitogenome of *P. australasiae* (NC_034841) with e-value 0.0 and 99.70% of identity¹⁹.

For MNHN JL632, another alien cluster of nuclear rRNA was found as an 8,791 bp contig with a 5.18× coverage (Supplementary file S8). When querying the *18S* region on NBCI BLAST, the best result was the moth *Anticarsia gemmatalis* Hübner, 1818 (XR_012960467) (Lepidoptera, Erebidae), with e-value 0.0 and 99.79% of identity. Reads were assembled a second time with a k-mer of 85, and it was then possible to extract from the contigs file a
<figure><img src="image_5.png" /><figcaption>Fig. 3. Map of the mitochondrial genome of *Geonemertes pelaensis* BioSample SAMN40098146.</figcaption></figure>

5,568 bp long contig with a 12.31 × coverage that represented the partial mitogenome of a moth (Supplementary file S9). Its *cox1* gene was extracted, and the best Megablast result was a partial 658 bp *cox1* gene ascribed to and unidentified *Avitta* sp. ANIC1 (HQ949103) (Lepidoptera, Noctuidae) from Queensland (Australia) with e-value 0.0 and 98.63% of identity.

Discussion

A long and complex mitochondrial genome

The mitochondrial genome of *G. pelaensis* is for now the largest among Nemertea. Complex regions in the mitogenome of invertebrates that could only be resolved by long-read sequencing were observed on Platyhelminthes such as the terrestrial flatworm *Arthurdendyus triangulatus* (Dendy, 1895)¹⁰ or the parasite *Schistosoma haematobium* (Bilharz, 1852)²⁰. These two examples are however very different compared to *G. pelaensis*. For example, in *A. triangulatus* and *S. haematobium*, there is a single region that contains several tandem repeats motifs in many copies, making it impossible to resolve this area by short-reads. In *G. pelaensis*, no noticeable tandem-repeats or microsatellites were detected, and the number of non-coding regions, or at least large region depleted of PCG, is higher. It is possible that the extra-length results from the duplication of genes,
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Maximum likelihood phylogenetic tree inferred from a concatenated alignment of 13 mitochondrial protein sequences. The subtree shown excludes the outgroups.</figcaption></figure>

followed by pseudogenization and accumulation of mutations, as suggested by the similarities between one of the large intergene and *ND1*. For now however, it would be safer to refrain from drawing conclusions regarding the mechanism behind this extension.

The second longest mitogenome found so far in the Nemertea is that derived from BioSample SAMN40098146 (18,540 bp) (assigned by its author to *G. pelaensis*), followed, among the available and verified mitogenomes from GenBank, by *Micrura bella* (Stimpson, 1857) (KR703629)²¹, with 16,847 bp. This result suggests that the size extension of the mitogenome of *G. pelaensis* occurred after the shared ancestor adapted to terrestrial life. This could represent a promising model to study the dynamic of organellar genomes and the accumulation of non-coding sequences among them, provided that the specific identity of BioSample SAMN40098146 can be verified.

**Geonemertes pelaensis: genetically conserved populations vs. cryptic diversity?**

To date, of the 14 known species of terrestrial nemerteans there are only three species of *Geonemertes*: *G. pelaensis*, *Geonemertes rodericana* Gulliver, 1879⁷,²² and *Geonemertes philippinensis* Gibson & Moore, 1998²³. Once native to the Island of Rodrigues in the Indian Ocean, *G. rodericana* is feared to be extinct since the end of the 20th century²⁴. *Geonemertes philippinensis* is known only from a single specimen from the Philippines found in a cave on the Island of Luzon. As a consequence, genetic studies of the genus *Geonemertes* are only available for *G. pelaensis*. Consequently, the evolutive history of this genus remains to be untangled. Based on
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Maximum likelihood phylogenetic tree inferred from a concatenated alignment of *cox1* and *16S* genes.</figcaption></figure>

the partial *cox1* phylogeny, there was an almost complete, if not complete, identity between populations of *G. pelaensis* from Martinique, Bermuda and Cuba, which can make sense considering the proximity of all these islands in the Caribbean and suggests common source of introduction. But the identity was also complete with the specimen from New Caledonia and several specimens from Japan, with sampling locations described as the Ryukyu (Okinawa) and Daito (Minamidaitojima) islands. It was thus surprising when the data derived from BioSample SAMN40098146 showed such important differences with our own results. Unfortunately, the available information concerning this specimen are scarce. There was no picture provided, and the BioSample description does neither include locality name nor coordinates. So far, our investigations could not provide more clues concerning the provenance of this organism. There are massive differences in terms of colour between the two known remaining species of *Geonemertes*, *G. pelaensis* and *G. philippinensis*. This, plus the limited known distribution of *G. philippinensis*, suggest that misidentification with *G. pelaensis* is unlikely. As for now, we might suppose that BioSample SAMN40098146 belongs to a species morphologically close to *G. pelaensis*, possibly a cryptic species that could not be distinguished from *G. pelaensis* based on its external appearance. We regard this hypothesis as worthy further investigations, especially in the frame of the worldwide monitoring of the spread of *G. pelaensis*. We emphasize the use of molecular methods whenever possible, when a specimen similar to *G. pelaensis* is found. To a broader extent, the species richness of the genus *Geonemertes* might be re-evaluated in the future, if more sampling campaigns are being conducted in the Australasian area.
<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Maximum likelihood phylogenetic tree inferred from an alignment of *cox1* gene. The subtree containing all sequences ascribed to *Geonemertes pelaensis* is shown.</figcaption></figure>

## Phylogenetic position

In the multiprotein tree (Fig. 4), the sister-group to the cluster formed by all specimens ascribed to *G. pelaensis* is *Prosadenoporus spectaculum*, opening a path of reflexion. Some species of the genus *Prosadenoporus* Bürger, 1890 are semi-terrestrial and as such, their phylogenetic proximity to terrestrial specimens could make sense. The large comprehensive phylogeny published by Andrade et al.¹⁶, although including *G. pelaensis*, did not comprise taxa of the genus *Prosadenoporus*, and this was among the rationales for performing the 2-genes phylogeny presented here. The tree derived did not enforce this supposed proximity between *G. pelaensis* and *Prosadenoporus* spp., nor associated *G. pelaensis* with *Tetras temma laminariae* Ushakov, 1928; instead, it corroborated the topology obtained by Andrade et al.¹⁶ that found *G. pelaensis* as sister to *M. grossa*. The proximity between *G. pelaensis* and *Malacobdella* spp. has also been highlighted in Morffe et al.⁸. This result is surprising, as *M. grossa* is not known for any terrestrial or semi-terrestrial lifestyle. Instead, species of *Malacobdella* spp. are usually found living inside marine bivalve shells, in a relationship with the molluscs that can be seen as either commensal or kleptivore²⁵. So far there is no available mitogenome of *Malacobdella* spp., so they could unfortunately not be included in the multiprotein phylogeny.

## The challenge of NUMTs

Regarding the results obtained on specimen MNHN JL402, it seems that complete portions of the mitogenome have been copied in the nuclear genome. This can represent an additional challenge when performing molecular barcoding by PCR and Sanger sequencing²⁶ with the risk also of overestimating cryptic diversity²⁷. The fact that a pseudogenized copy of *cox1* has been found in *G. pelaensis* MNHN JL402 illustrates this challenge, considering how often this gene is being used for molecular barcoding. Although likely present in a lesser number of copies
in each cells than the mitochondrial *cox1* gene, there is still a probability that the NUMT might be amplified by PCR. At the very least, such non-specific amplification could impair a Sanger sequencing performed directly on PCR product. A protocol that includes high-throughput sequencing, such as the one employed here, can help avoiding this issue. In case of serious doubts concerning the results of a *cox1* molecular barcoding performed on a specimen regarded as *G. pelaensis*, we would strongly advocate the community members involved in the monitoring of invasive species to consider a NGS approach.

## Invasive vs. invasive: the diet of Geonemertes pelaensis in Martinique

*Geonemertes pelaensis* is a rather non-specialized predator, which is known to feed on a wide variety of invertebrates. The list includes gastropods<sup>28</sup> and various arthropods<sup>5,8</sup>, but we are not aware of any report of *G. pelaensis* feeding on cockroaches and the current study might represent the first one. Although less reliable than direct observations, gut-DNA sequencing can provide information on diets<sup>1,2,9</sup>. The high identity found between the two sequences extracted from the contigs file with those ascribed to *P. australasiae* makes us rather confident that *G. pelaensis* had been indeed feeding on it. *Periplaneta australasiae* is mistakenly named the 'Australian cockroach', but its origin might actually be Africa. It is known since at least the end of the 1960s in Martinique<sup>30</sup>, where it was found to host the introduced parasitoid 'Blue-eyed Ensign wasp' *Evania appendigaster* (Linnaeus, 1758)<sup>31</sup>.

Concerning MNHN JL632 from New Caledonia, it was harder to pinpoint which prey it might have consumed. The use of the *18S* gene was not accurate enough, which might apply to a broad spectrum of organisms for which *18S* cannot be used below the generic level. *Anticarsia gemmatalis*, which represented the best BLASTn result, is an American species of moth, where it is regarded as a pest that is damaging soybean plantations. But so far, it does not seem to have been observed often outside of America, and never in the Australasian area. The use of the *cox1* gene was possibly more efficient in determining the origin of prey DNA, although in the current case, no species name could be linked to this sequence. The fact that the reference, noted as *Avitta* sp., originates from Australia seems compatible with the presence of this species, or a closely related one, in New Caledonia. Whether *G. pelaensis* is capable of preying on flying adults, or is restricted to feeding on caterpillars and pupae, remains to be determined through direct observation.

## Material and methods

### Sampling

The specimen of *G. pelaensis* from Martinique (Fig. 1a) was collected on 31 January 2018 by Mathieu Coulis in Fort de France (14.62493 N, 61.05320 W). The specimen from New Caledonia (Fig. 1b, c) was collected on 6 April 2024 in Yaté, South Province (22.05053 S, 166.85344 E) by David G. Herbert, Barna Páll-Gergely and Ira Richling during a land snail campaign. Both were stored in ethanol 99% and transferred to the collections of the National Museum of Natural History in Paris, France (MNHN), where they were identified by Jean-Lou Justine before being registered within the collections with accession number MNHN JL402 (Martinique) and MNHN JL632 (New Caledonia).

### DNA extraction and sequencing

DNA extraction and sequencing were performed at the Genomic Analysis Platform (PAG) of the Institute of Integrative Biology and Systems at the Laval University (Québec, Canada) (https://www.ibis.ulaval.ca/en/services-2/genomic-analysis-platform). Extraction was done using the NucleoMag HMW DNA extraction kit by Macherey-Nagel (Düren, Germany).

The paired-end Illumina library was produced by shearing 500 ng of DNA with a Covaris M220 apparatus (Covaris, Woburn MA, United States) and using the NEBNext Ultra II DNA Library Prep Kit Illumina from New England Biolab (Ipswich, MA, United States). The library was sequenced using an AVITI system and AVITI 2×150 sequencing kit cloudbreak FS High Output (Element Biosciences, San Diego, CA).

The Oxford Nanopore libraries were produced after checking the quality and purity of DNA using a Femto Pulse System with the genomic DNA 16S-kb kit (Agilent, Santa Clara, CA, USA). Small fragments were removed on a 3 µg DNA sample using the Short Read Eliminator XS kit (PacBio, Menlo Park, CA, USA). The LSK-114 kit (Oxford Nanopore, Littlemore, UK) was then used to prepare the long fragments library from a 2 µg DNA sample. Finally, 700 ng of the resulting DNA library were loaded on a R10 flow cell and sequenced on a PromethION 2 Solo device (Oxford Nanopore, Littlemore, UK) for 72 h.

### Assembly, data-mining and annotation

Short reads were filtered using fastp, with a minimum size threshold of 125 bp<sup>32</sup>. A total of ca. 133 M and ca. 45 M clean reads remained for MNHN JL402 and MNHN JL632 respectively. Reads were assembled using SPAdes 4.0<sup>33</sup> and a k-mer of 125. The resulting contigs file was subjected to a blastx query<sup>34</sup> using the proteins of *Amphiporus formidabilis* Griffin, 1898 (NC_023929) and *Gononemertes parasita* Bergendal, 1900 (NC_024822)<sup>17,35</sup> as database, using genetic code 5 and an e-value filter of 1e<sup>-40</sup> and positive matches were extracted from the contigs file. The 12,233,132 (for MNHN JL402) and 115,912 (for MNHN JL632) long reads were assembled using Flye 2.9.1 with the -meta option and an overlap parameter of 2000<sup>36</sup>. The contig identified as the mitogenome was polished with 3 iterations of Pilon 1.24<sup>37</sup>. Annotations were done with the help of MITOS<sup>38</sup> and tRNA were verified using ARWEN v.1.2<sup>39</sup>. The presence of repetitions in the mitogenome was investigated using Tandem repeats finder<sup>40</sup>. Boundaries of the nuclear rRNA clusters retrieved from short-reads assemblies were found with the help of Rfam 15<sup>41</sup>. Traces of alien DNA corresponding to food were first investigated as detailed in Justine et al.<sup>12</sup>. In the case of the gut content of MNHN JL402, once a positive matches had been found, a second blastn query was done using the mitogenome of *P. australasiae* (KX640825)<sup>19</sup> as reference. For what
concerns the BioSample SAMN40098146 ascribed to *G. pelaensis*, short reads data were downloaded from SRA (SRS20559370) and processed the same way as explained above (quality trimming and assembly).

## Phylogenies

For the multiprotein phylogeny, amino-acid sequences of the mitochondrial proteins were extracted from 42 available and verified mitogenomes of Nemertea obtained from GenBank, to which the two specimens of *G. pelaensis* and the mitogenome retrieved from SRA BioProject PRJNA1080026 were added. The outgroups were chosen in accordance to the previous phylogeny published by Thol lesson and Norenburg⁴², and consisted in the Brachiopoda *Terebratalia transversa* (Sowerby, 1846) (AF331161)⁴³ the Mollusca *Aplysia californica* J. G. Cooper, 1863 (AY569552)⁴⁴, the Annelida *Urechis caupo* Fisher & MacGinitie, 1928 (AY619711)⁴⁵ and *Phascolosoma similis* (Chen & Yeh, 1958), currently designated as *Antillesoma antillarum* (Grube, 1858) (MN813482)⁴⁶. Proteins were aligned independently using MAFFT 7⁴⁷ then trimmed with *trimAl* and the -automated1 option⁴⁸. Trimmed alignments were concatenated using Phyutility 2.7.1⁵⁰ (Supplementary file S10). The best model of evolution was selected using *ModelTest-NG* v0.1.7⁵⁰ with default options and was found to be the MTZOA + I + G4 + F. Maximum likelihood phylogeny was conducted using IQ-TREE 2.2.0⁵¹ with 1000 ultrafast bootstrap replicates.

A second phylogeny was obtained by appending 16S and *cox1* data from the works of Andrade et al.¹⁶ with those of Maslakova and Norenburg⁵² and also the *cox1* sequence (AY791979) of *T. laminariae*¹⁵ plus those from the current study (Supplementary file S11). The same software and options were used for alignment and trimming but the models of evolution were verified on each gene separately, and the phylogeny was partitioned using these results, which according to the Bayesian inference criterion, were TPM3uf + I + G4 for 16S and TPM1uf + I + G4 for *cox1*.

Finally, the *cox1* only phylogeny was done by completing the previous dataset with all the remaining *cox1* sequences available on GenBank for *G. pelaensis*, with TPM1uf + I + G4 as model and 10,000 ultrafast bootstrap replicates (Supplementary file S12).

## Data availability

Reads are available on the Sequence Reads Archive (SRA) under BioProject PRJNA1223316, BioSamples SAMN46813856 (for MNHN JL402) and SAMN50810529 (for MNHN JL632), SRA accession numbers SRR32329171, SRR32329172, SRR35157170 and SRR35157171.

Received: 20 October 2025; Accepted: 17 December 2025

Published online: 22 January 2026

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## Author contributions

Collection of the samples by M.C., D.G.H., B.P.L., I.R. Taxonomic identifications by J.L.J., L.W. Sequencing by C.O., B.B., R.G.L., S.M. Bioinformatic analyses by R.G., C.L. and M.T. First draft written by R.G. Draft edited by L.W., I.R., B.P.L., C.L., J.L.J. All authors read and approved the final draft.
**Funding**

This work was co-financed by the Minister of Science under the “Regional Excellence Initiative” Program for 2024–2027 (RID/SP/0045/2024/01). Claude Lemieux and Monique Turmel were supported by grant RG-PIN-2017-04506 from the Natural Sciences and Engineering Research Council of Canada (NSERC). Brian Boyle and Christian Otis were supported by the “Programme d’appui aux plateformes technologiques stratégiques” from the Ministère de l’Économie, de l’Innovation et de l’Énergie Québec.

**Competing interests**

The authors declare no competing interests.

**Additional information**

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1038/s41598-025-33230-0.

**Correspondence** and requests for materials should be addressed to R.G.

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