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Post mortem damage by predators varies with geography, climate, and location of cadavers. Frequently encountered facultatively parasitic terrestrial organisms include fly larvae (maggots), ants and beetles. This report describes for the first time opportunistic post mortem damage caused by the terrestrial flatworm Platydemus manokwari de Beauchamp,1963 (New Guinea Flatworm) (phylum Platyhelminthes: order Tricladida: suborder Continenticola: family Geoplanidae) to exposed skin of a body located in a tropical urban location. Several flatworms were attached to the body associated with areas of skin loss. Microscopy showed epidermal damage with no vital reaction and the presence of PAS-positive granules consistent with flatworm secretions. No human DNA profile was recovered with genetic testing of two of the flatworms. This case extends the range of potential post mortem predators to include flatworms, albeit opportunistically., Competing Interests: Declarations Ethical approval No ethical approval required for a case report. Consent from the Northern Territory Coroner has been obtained to report the de-identified case information. Competing interests None to declare., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Specimens of a flat and dark brown land planarian were found in a plant nursery in North Carolina, USA in 2020. On the basis of examination of photographs of the live specimens only, the specimens were considered as belonging to Obama nungara , a species originally from South America, which has now invaded a large part of Europe. Unexpectedly, a molecular analysis revealed that the specimens did not belong to this species, neither to the genus Obama . We then undertook its histological study, which finally confirmed that the species is a member of the genus Amaga : the species is herein described as a new species, Amaga pseudobama n. sp. The species has been found in three locations in North Carolina and some infested plants were from Georgia. We reinvestigated specimens collected in Florida in 2015 and found that they also belong to this species. Citizen science observations suggest its presence in other states. Therefore, it is likely that A. pseudobama has already invaded a part of south-east USA and that the invasion took place more than ten years ago. The complete 14,909 bp long mitochondrial genome was obtained. The mitogenome is colinear with those of other Geoplanidae and it was possible to find and annotate a tRNA-Thr, which has been reported missing in several geoplanids. Amaga pseudobama shares with other Geoplaninae the presence of alternative start codons in three protein-coding genes of its mitogenome. The availability of this new genome helped us to improve our annotations of the ND3 gene, for which an ATT start codon is now suggested. Also, the sequence of the ATP6 gene raised questions concerning the use of genetic code 9 to translate the protein-coding genes of Geoplanidae, as the whole translated protein would not contain a single methionine residue when using this code. Two maximum likelihood phylogenies were obtained from genomic data. The first one was based on concatenated alignments of the partial 28S , Elongation Factor 1-alpha ( EF1) and cox1 genes. The second was obtained from a concatenated alignment of the mitochondrial proteins. Both strictly discriminate A. pseudobama from O. nungara and instead associate it with Amaga expatria . We note that the nine species currently accepted within Amaga can be separated into two groups, one with extrabulbar prostatic apparatus, including the type species A. amagensis , and one with intrabulbar prostatic apparatus, including the new species A. pseudobama . This suggests that species of the latter group should be separated from Amaga and constitute a new genus. This finding again illustrates the possible emergence of new invasive species in regions naturally devoid of large land planarians, such as North America. Amaga pseudobama thus deserves to be monitored in the USA, although its superficial resemblance to O. nungara and Geoplana arkalabamensis will complicate the use of photographs obtained from citizen science. Our molecular information provides tools for this monitoring., Competing Interests: Jean-Lou Justine is an Academic Editor for PeerJ., (©2024 Justine et al.)

Using a combination of short- and long-reads sequencing, we were able to sequence the complete mitochondrial genome of the invasive 'New Zealand flatworm' Arthurdendyus triangulatus (Geoplanidae, Rhynchodeminae, Caenoplanini) and its two complete paralogous nuclear rRNA gene clusters. The mitogenome has a total length of 20,309 bp and contains repetitions that includes two types of tandem-repeats that could not be solved by short-reads sequencing. We also sequenced for the first time the mitogenomes of four species of Caenoplana (Caenoplanini). A maximum likelihood phylogeny associated A. triangulatus with the other Caenoplanini but Parakontikia ventrolineata and Australopacifica atrata were rejected from the Caenoplanini and associated instead with the Rhynchodemini, with Platydemus manokwari. It was found that the mitogenomes of all species of the subfamily Rhynchodeminae share several unusual structural features, including a very long cox2 gene. This is the first time that the complete paralogous rRNA clusters, which differ in length, sequence and seemingly number of copies, were obtained for a Geoplanidae., (© 2024. The Author(s).)

We present here the first observation of Bipalium admarginatum de Beauchamp, 1933 since its original description 90 years ago. Three specimens were found on Perhentian Kecil Island, off Terengganu State, Malaysia and photographed in the field, and two were collected. This report thus includes the first colour photographs published for this species, from a locality close to the type-locality, Tioman Island (which is ca. 200 km south of the locality in this study, on the east coast of Peninsula Malaysia). We describe the external morphology and colour pattern of the species, which correspond well to the original description, itself based only on two preserved specimens. We performed an in-depth molecular characterisation of the species, including its complete mitochondrial genome, the 18S sequence and elongation 1-alpha (EF1-α) sequence. In addition, EF1-α sequences were also retrieved for 5 additional geoplanid species. No tRNA-Thr could be detected in the mitogenome of B. admarginatum, a lack already reported in several species of geoplanids, but we found a 13 bp sequence that contains the anticodon loop and seems to be conserved among geoplanids and might thus possibly represent a non-canonical undetected tRNA. We discuss the difficulties encountered in trying to reconstruct the cluster of nuclear ribosomal genes, a problem already mentioned for other Triclads. Three phylogenies, based respectively on all mitochondrial proteins, 18S, and EF1-α, were computed; the position of B. admarginatum within the Bipaliinae was confirmed in each tree, as sister-group to various bipaliine species according to the sequences available for each tree. In the mitochondrial proteins tree, which had high support, B. admarginatum was sister to Bipalium kewense and Diversibipalium multilineatum.

We sequenced the complete mitochondrial genome of the flatworm Australopacificaatrata . The species, originally described from New South Wales, Australia, has been found in various locations in the British Isles, New Zealand and in the United States of America; it is thus potentially invasive. The genome is 16513 bp long, encodes for 12 protein coding genes, two ribosomal RNA genes and 20 tRNA genes, and is completely colinear with the other two available Rhynchodeminae. In addition, it shares with them some unusual characters discriminating them from members of the other subfamilies of Geoplanidae, the most noticeable being the extra length of its cox2 gene. The data allow a reliable multigene phylogeny to be derived, and also provide a means of accurate biomonitoring of possible invasiveness by A.atrata ., (Romain Gastineau, Leigh Winsor, Jean-Lou Justine.)

The land flatworm Obama nungara, a species originating from South America and already invasive in many European countries, is recorded from La Runion, a French island in the Indian Ocean. This is the first record of O. nungara from this locality and also the first record of the species for Africa. Three specimens were collected in 2021 and 2022, in the communes of Saint Paul, Saint Joseph and Le Tampon, respectively; the three localities are widely separated, with two in the Western part and one in the South-eastern part of the island. This suggests that the species is already present in several locations in La Runion, and it is likely that the species is already present since 2020. The specimen from Saint Paul had the same cox1 haplotype as specimens previously recorded from several countries of Europe; it is hypothesized that the species was imported from Europe, probably from France. We mapped climatic suitability of the species in La Runion and found that O. nungara could potentially invade a large part of the island. One record was apparently associated with the transport of plates of travertine, a construction material which has numerous cavities thus suitable for the transport and survival of adult or cocoons of land flatworms.

Background: New records of alien land planarians are regularly reported worldwide, and some correspond to undescribed species of unknown geographic origin. The description of new species of land planarians (Geoplanidae) should classically be based on both external morphology and histology of anatomical structures, especially the copulatory organs, ideally with the addition of molecular data., Methods: Here, we describe the morphology and reproductive anatomy of a species previously reported as Diversibipalium "black", and the morphology of a species previously reported as Diversibipalium "blue". Based on next generation sequencing, we obtained the complete mitogenome of five species of Bipaliinae, including these two species., Results: The new species Humbertium covidum n. sp. (syn: Diversibipalium "black" of Justine et al., 2018) is formally described on the basis of morphology, histology and mitogenome, and is assigned to Humbertium on the basis of its reproductive anatomy. The type-locality is Casier, Italy, and other localities are in the Department of Pyrénées-Atlantiques, France; some published or unpublished records suggest that this species might also be present in Russia, China, and Japan. The mitogenomic polymorphism of two geographically distinct specimens (Italy vs France) is described; the cox1 gene displayed 2.25% difference. The new species Diversibipalium mayottensis n. sp. (syn: Diversibipalium "blue" of Justine et al., 2018) is formally described on the basis of external morphology and complete mitogenome and is assigned to Diversibipalium on the basis of an absence of information on its reproductive anatomy. The type- and only known locality is the island of Mayotte in the Mozambique Channel off Africa. Phylogenies of bipaliine geoplanids were constructed on the basis of SSU, LSU, mitochondrial proteins and concatenated sequences of cox1 , SSU and LSU. In all four phylogenies, D. mayottensis was the sister-group to all the other bipaliines. With the exception of D. multilineatum which could not be circularised, the complete mitogenomes of B. kewense , B. vagum , B. adventitium , H. covidum and D. mayottensis were colinear. The 16S gene in all bipaliine species was problematic because usual tools were unable to locate its exact position., Conclusion: Next generation sequencing, which can provide complete mitochondrial genomes as well as traditionally used genes such as SSU, LSU and cox1 , is a powerful tool for delineating and describing species of Bipaliinae when the reproductive structure cannot be studied, which is sometimes the case of asexually reproducing invasive species. The unexpected position of the new species D. mayottensis as sister-group to all other Bipaliinae in all phylogenetic analyses suggests that the species could belong to a new genus, yet to be described., Competing Interests: Jean-Lou Justine is an Academic Editor of PeerJ., (© 2022 Justine et al.)

The land flatworm Platydemus manokwari (Platyhelminthes, Geoplanidae) is recorded from the islands of Guadeloupe, Martinique, and Saint Martin in the Caribbean arc. Photographs and records were obtained mainly from citizen science and ranged from the end of 2018 to February 2021; several specimens were deposited in the collections of the Muséum National d'Histoire Naturelle in Paris, France. Thirty records were from Guadeloupe, but only one from Martinique and from Saint Martin, respectively. The COI sequences of 3 specimens from Guadeloupe show that they belong to the World haplotype also found in many countries. We also report P. manokwari from Fort Myers, Florida, USA, with molecular characterization, which was also the World haplotype. This is the first published record of P. manokwari for Guadeloupe, Martinique and Saint Martin and the second for islands in the Caribbean, after Puerto Rico.

Background: Obama nungara is a species of land flatworm originating from South America; the species was recently described and distinguished from a similar species, Obama marmorata . Obama nungara has invaded several countries of Europe, but the extent of the invasion has not been thoroughly mapped., Methods: In this article, based on a five and a half-year survey undertaken by citizen science, which yielded 530 records from 2013 to 2018, we analysed information about the invasion of Metropolitan France by O. nungara . We also investigated the variability of newly obtained cytochrome c oxidase 1 (COI) sequences of specimens from France, Italy and Switzerland., Results: Obama nungara was recorded from 72 of the 96 Departments of Metropolitan France. The species is especially abundant along the Atlantic coast, from the Spanish border to Brittany, and along the Mediterranean coast, from the Spanish border to the Italian border. More than half of the records were from an altitude below 50 m, and no record was from above 500 m; mountainous regions such as the Alps, Pyrenees and Massif Central are not invaded. Local abundance can be impressive, with 100 of specimens found in a small garden. An analysis of our new COI sequences, combined with published sequences of specimens from several countries, confirmed that three clades comprise the species. The first clade, 'Brazil', is currently confined to this country in South America; the second clade, 'Argentina 2', was found in Argentina and in Europe, only in Spain; and the third, 'Argentina 1', was found in Argentina and in Europe, in Spain, Portugal, France, UK, Italy, Belgium, and Switzerland. This suggests that two clades of O. nungara from Argentina have invaded Europe, with one widely spread., Discussion: The present findings strongly suggest that O. nungara is a highly invasive species and that the population which has invaded several countries in Europe comes from Argentina. The wide dispersion of the species and its reported local abundance, combined with the predatory character of the species, make O. nungara a potential threat to the biodiversity and ecology of the native soil fauna in Europe, and probably the most threatening species of all invasive land planarians present in Europe., Competing Interests: Jean-Lou Justine is an Academic Editor for PeerJ., (© 2020 Justine et al.)

Specimens of Bipalium adventitium (Platyhelminthes, Geoplanidae) were found in Montréal, Québec, Canada. The specimens showed the typical colour pattern of the species and barcoding (Cytochrome Oxidase I) demonstrated near-identity with a sequence of the same species from the USA. This is the first record of the species in Canada.

We report the presence of the land planarian Endeavouria septemlineata from Tahiti, French Polynesia, on the basis of a single specimen collected in 2017. Identification of the species was ascertained by external and internal morphology and DNA COI sequence. The finding is of importance for conservation, since this species is a predator of soil animals.

Background: Species of the genera Bipalium and Diversibipalium , or bipaliines, are giants among land planarians (family Geoplanidae), reaching length of 1 m; they are also easily distinguished from other land flatworms by the characteristic hammer shape of their head. Bipaliines, which have their origin in warm parts of Asia, are invasive species, now widespread worldwide. However, the scientific literature is very scarce about the widespread repartition of these species, and their invasion in European countries has not been studied., Methods: In this paper, on the basis of a four year survey based on citizen science, which yielded observations from 1999 to 2017 and a total of 111 records, we provide information about the five species present in Metropolitan France and French overseas territories. We also investigated the molecular variability of cytochrome-oxidase 1 (COI) sequences of specimens., Results: Three species are reported from Metropolitan France: Bipalium kewense , Diversibipalium multilineatum , and an unnamed Diversibipalium 'black' species. We also report the presence of B. kewense from overseas territories, such as French Polynesia (Oceania), French Guiana (South America), the Caribbean French islands of Martinique, Guadeloupe, Saint Martin and Saint Barthélemy, and Montserrat (Central America), and La Réunion island (off South-East Africa). For B. vagum , observations include French Guiana, Guadeloupe, Martinique, Saint Barthélemy, Saint Martin, Montserrat, La Réunion, and Florida (USA). A probable new species, Diversibipalium sp. 'blue,' is reported from Mayotte Island (off South-East Africa). B. kewense , B. vagum and D. multilineatum each showed 0% variability in their COI sequences, whatever their origin, suggesting that the specimens are clonal, and that sexual reproduction is probably absent. COI barcoding was efficient in identifying species, with differences over 10% between species; this suggests that barcoding can be used in the future for identifying these invasive species. In Metropolitan south-west France, a small area located in the Department of Pyrénées-Atlantiques was found to be a hot-spot of bipaliine biodiversity and abundance for more than 20 years, probably because of the local mild weather., Discussion: The present findings strongly suggest that the species present in Metropolitan France and overseas territories should be considered invasive alien species. Our numerous records in the open in Metropolitan France raise questions: as scientists, we were amazed that these long and brightly coloured worms could escape the attention of scientists and authorities in a European developed country for such a long time; improved awareness about land planarians is certainly necessary., Competing Interests: Jean-Lou Justine is an Academic Editor for PeerJ.

The chapter provides a concise account of collecting techniques, and basic histological techniques for investigating planarians, intended for use by practicing scientists and postgraduate students new to the field. Selected hands-on methods are described that will lead to well-stained preparations suitable for light microscopic examination for subsequent taxonomic identification or general investigations into planarian functional cytology and histology. Major equipment items that are minimal essential requirements of a functional histological laboratory are indicated. The chapter includes sectioning and staining troubleshooting guides that will be very helpful for those who must work in the laboratory without the opportunity to consult qualified and experienced technicians or microscopists.