Delphine Gey

Use of RAD sequencing for delimiting species

RAD-tag sequencing is a promising method for conducting genome-wide evolutionary studies. However, to date, only a handful of studies empirically tested its applicability above the species level. In this communication, we use RAD tags to contribute to the delimitation of species within a diverse genus of deep-sea octocorals, Chrysogorgia, for which few classical genetic markers have proved informative. Previous studies have hypothesized that single mitochondrial haplotypes can be used to delimit Chrysogorgia species. On the basis of two lanes of Illumina sequencing, we inferred phylogenetic relationships among 12 putative species that were delimited using mitochondrial data, comparing two RAD analysis pipelines (Stacks and PyRAD). The number of homologous RAD loci decreased dramatically with increasing divergence, as >70% of loci are lost when comparing specimens separated by two mutations on the 700-nt long mitochondrial phylogeny. Species delimitation hypotheses based on the mitochondrial mtMutS gene are largely supported, as six out of nine putative species represented by more than one colony were recovered as discrete, well-supported clades. Significant genetic structure (correlating with geography) was detected within one putative species, suggesting that individuals characterized by the same mtMutS haplotype may belong to distinct species. Conversely, three mtMutS haplotypes formed one well-supported clade within which no population structure was detected, also suggesting that intraspecific variation exists at mtMutS in Chrysogorgia. Despite an impressive decrease in the number of homologous loci across clades, RAD data helped us to fine-tune our interpretations of classical mitochondrial markers used in octocoral species delimitation, and discover previously undetected diversity.

The invasive New Guinea flatworm Platydemus manokwari in France, the first record for Europe: time for action is now

Non-indigenous terrestrial flatworms (Platyhelminthes) have been recorded in thirteen European countries. They include Bipalium kewense and Dolichoplana striata that are largely restricted to hothouses and may be regarded as non-invasive species. In addition there are species from the southern hemisphere such as the invasive New Zealand flatworm Arthurdendyus triangulatus in the United Kingdom, Eire and the Faroe Islands, the Australian flatworm Australoplana sanguinea alba in Eire and the United Kingdom, and the Australian Blue Garden flatworm Caenoplana coerulea in France, Menorca and the United Kingdom. The United Kingdom has some twelve or more non-indigenous species most of which are Australian and New Zealand species. These species may move to an invasive stage when optimum environmental and other conditions occur, and the flatworms then have the potential to cause economic or environmental harm. In this paper, we report the identification (from morphology and molecular analysis of COI sequences) of non-indigenous terrestrial flatworms found in a hothouse in Caen (France) as the New Guinea flatworm Platydemus manokwari de Beauchamp, 1963 (Platyhelminthes, Continenticola, Geoplanidae, Rhynchodeminae). Platydemus manokwari is among the “100 World’s Worst Invader Alien Species”. Lists of World geographic records, prey in the field and prey in laboratories of P. manokwari are provided. This species is considered a threat to native snails wherever it is introduced. The recent discovery of P. manokwari in France represents a significant extension of distribution of this Invasive Alien Species from the Indo-Pacific region to Europe. If it escaped the hothouse, the flatworm might survive winters and become established in temperate countries. The existence of this species in France requires an early warning of this incursion to State and European Union authorities, followed by the eradication of the flatworm in its locality, tightening of internal quarantine measures to prevent further spread of the flatworm to and from this site, identifying if possible the likely primary source of the flatworm, and tracing other possible incursions that may have resulted from accidental dispersal of plants and soil from the site.

The invasive land planarian Platydemus manokwari (Platyhelminthes, Geoplanidae): records from six new localities, including the first in the USA

The land planarian Platydemus manokwari de Beauchamp, 1963 or “New Guinea flatworm” is a highly invasive species, mainly in the Pacific area, and recently in Europe (France). We report specimens from six additional countries and territories: New Caledonia (including mainland and two of the Loyalty Islands, Lifou and Maré), Wallis and Futuna Islands, Singapore, Solomon Islands, Puerto Rico, and Florida, USA. We analysed the COI gene (barcoding) in these specimens with two sets of primers and obtained 909 bp long sequences. In addition, specimens collected in Townsville (Australia) were also sequenced. Two haplotypes of the COI sequence, differing by 3.7%, were detected: the “World haplotype” found in France, New Caledonia, French Polynesia, Singapore, Florida and Puerto Rico; and the “Australian haplotype” found in Australia. The only locality with both haplotypes was in the Solomon Islands. The country of origin of Platydemus manokwari is New Guinea, and Australia and the Solomon Islands are the countries closest to New Guinea from which we had specimens. These results suggest that two haplotypes exist in the area of origin of the species, but that only one of the two haplotypes (the “World haplotype”) has, through human agency, been widely dispersed. However, since P. manokwari is now recorded from 22 countries in the world and we have genetic information from only 8 of these, with none from New Guinea, this analysis provides only partial knowledge of the genetic structure of the invasive species. Morphological analysis of specimens from both haplotypes has shown some differences in ratio of the genital structures but did not allow us to interpret the haplotypes as different species. The new reports from Florida and Puerto Rico are firsts for the USA, for the American continent, and the Caribbean. P. manokwari is a known threat for endemic terrestrial molluscs and its presence is a matter of concern. While most of the infected territories reported until now were islands, the newly reported presence of the species in mainland US in Florida should be considered a potential major threat to the whole US and even the Americas.

The monogenean which lost its clamps.

Ectoparasites face a daily challenge: to remain attached to their hosts. Polyopisthocotylean monogeneans usually attach to the surface of fish gills using highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 60 years ago, the clamps were considered to be absent but few specimens were available and this observation was later questioned. In addition, genera within the family Protomicrocotylidae have either clamps of the “gastrocotylid” or the “microcotylid” types; this puzzled systematists because these clamp types are characteristic of distinct, major groups. Discovery of another, new, species of the genus Lethacotyle, has allowed us to explore the nature of the attachment structures in protomicrocotylids. Lethacotyle vera n. sp. is described from the gills of the carangid Caranx papuensis off New Caledonia. It is distinguished from Lethacotyle fijiensis, the only other species of the genus, by the length of the male copulatory spines. Sequences of 28S rDNA were used to build a tree, in which Lethacotyle vera grouped with other protomicrocotylids. The identity of the host fish was confirmed with COI barcodes. We observed that protomicrocotylids have specialized structures associated with their attachment organ, such as lateral flaps and transverse striations, which are not known in other monogeneans. We thus hypothesized that the clamps in protomicrocotylids were sequentially lost during evolution, coinciding with the development of other attachment structures. To test the hypothesis, we calculated the surfaces of clamps and body in 120 species of gastrocotylinean monogeneans, based on published descriptions. The ratio of clamp surface: body surface was the lowest in protomicrocotylids. We conclude that clamps in protomicrocotylids are vestigial organs, and that occurrence of “gastrocotylid” and simpler “microcotylid” clamps within the same family are steps in an evolutionary sequence, leading to the absence of these attributes in species of Lethacotyle.

Pseudorhabdosynochus sulamericanus (Monogenea, Diplectanidae), a parasite of deep-sea groupers (Serranidae) occurs transatlantically on three congeneric hosts (Hyporthodus spp.), one from the Mediterranean Sea and two from the western Atlantic

Little is known of the diversity of the monogenean parasites infesting deep-sea groupers, and there is even less information available about their geographic distributions within the ranges of their hosts. To improve our understanding of these host-parasite relationships we conducted parasitological evaluations of the deep-water Haifa grouper Hyporthodus haifensis from the southern Mediterranean off Tunisia and Libya. We collected more than one species of diplectanid monogeneans from this host, but among these only one dominant species was abundant. This proved to be morphologically very similar to Pseudorhabdosynochus sulamericanus Santos, Buchmann & Gibson, 2000, a species originally described from the congeneric host H. niveatus off Brazil and also recorded from H. niveatus and H. nigritus off Florida. Here, we conducted a morphological comparison between newly collected specimens and those previously deposited in museum collections by other authors. Further, we used COI barcoding to ascertain the specific identity of the three host species to better elucidate the circumstances that might explain the unexpectedly broad distribution of P. sulamericanus. We assigned our specimens from H. haifensis to P. sulamericanus primarily on the basis of morphological characteristics of the sclerotized vagina. We also noted morphological characteristics of eastern and western Atlantic specimens that are not clearly described or not given in previous descriptions and so prepared a redescription of the species. We confirmed, by COI barcoding, that no sister-species relationships were evident among the three hosts of P. sulamericanus. Our observation that P. sulamericanus infects unrelated host species with putatively allopatric distributions was unexpected given the very limited dispersive capabilities and the high degree of host specificity common to members of Pseudorhabdosynochus. This transatlantic distribution raises questions with regard to phylogeography and assumptions about the allopatry of Atlantic grouper species from the Americas and Afro-Eurasia. Here, we propose some hypothetical explanations for our findings.

A new species of Microcotyle (Monogenea: Microcotylidae) from Scorpaena notata (Teleostei: Scorpaenidae) in the Mediterranean Sea

We collected specimens of Microcotyle spp. from two species of scorpaeniform fishes off Algeria, namely Scorpaena notata and Helicolenus dactylopterus. The identification of both fishes was confirmed by molecular barcoding of the COI gene. Sequences of COI gene were also obtained for both parasite species. The species from S. notata is described as Microcotyle algeriensis n. sp., on the basis of morphological differences from other species (number of clamps, number of spines in genital atrium, number of testes). Its COI sequence differs from M. sebastis Goto, 1894 (from Sebastes schlegeli from a fish farm in South Korea) by 14.6%. The species from H. dactylopterus is distinct from M. algeriensis on the basis of morphology (number of clamps, number of spines in genital atrium) and COI sequence (4.5% divergence) and is also distinct from M. sebastis in its COI sequence (12.3%). We refrained from describing it as new because M. sebastis, a species originally described from scorpaeniform fishes off Japan, has been recorded in various hosts in the North and South Pacific, Atlantic and Mediterranean (for the latter, in the same host, H. dactylopterus). We believe that correct specific assignment of species of Microcotyle from scorpaeniform fishes needs a detailed morphological and molecular study of representatives from various locations and hosts.

Species of Pseudorhabdosynochus (Monogenea, Diplectanidae) from Groupers (Mycteroperca spp., Epinephelidae) in the Mediterranean and Eastern Atlantic Ocean, with Special Reference to the ‘Beverleyburtonae Group’ and Description of Two New Species

Pseudorhabdosynochus Yamaguti, 1958 is a species-rich diplectanid genus, mainly restricted to the gills of groupers (Epinephelidae) and especially abundant in warm seas. Species from the Mediterranean are not fully documented. Two new and two previously known species from the gills of Mycteroperca spp. (M. costae, M. rubra, and M. marginata) in the Mediterranean and Eastern Atlantic Ocean are described here from new material and slides kept in collections. Identifications of newly collected fish were ascertained by barcoding of cytochrome c oxidase subunit I (COI) sequences. Pseudorhabdosynochus beverleyburtonae (Oliver, 1984) Kritsky & Beverley-Burton, 1986 and P. sosia Neifar & Euzet 2007 are redescribed from type-specimens and new specimens collected off Tunisia and Libya from M. marginata and M. costae, respectively. Pseudorhabdosynochus oliveri n. sp., from M. marginata (type-host) off the Mediterranean coast of France (type-locality), is described from specimens found among voucher specimens of P. beverleyburtonae deposited by Guy Oliver in the collection of the Muséum National d’Histoire Naturelle, Paris. Pseudorhabdosynochus oliveri is distinguished by the shape of its sclerotised vagina; it was not found in the other localities investigated. Pseudorhabdosynochus hayet n. sp. is described from M. rubra (type host) off Senegal (type-locality) and Tunisia. Pseudorhabdosynochus hayet is morphologically similar to P. sosia (type-host: M. costae) but was distinguished by differences in measurements of the vagina and male copulatory organ, different host, and divergent COI sequences. The four species (P. beverleyburtonae, P. sosia, P. oliveri, and P. hayet) share common characteristics such as squamodiscs with 2 innermost circular rows of rodlets and a similar general structure of the sclerotised vagina; we propose to group them into a ‘beverleyburtonae group’ within Pseudorhabdosynochus.

Nematode parasites of four species of Carangoides (Osteichthyes: Carangidae) in New Caledonian waters, with a description of Philometra dispar n. sp. (Philometridae)

Parasitological examination of marine perciform fishes belonging to four species of Carangoides, i.e. C. chrysophrys, C. dinema, C. fulvoguttatus and C. hedlandensis (Carangidae), from off New Caledonia revealed the presence of nematodes. The identification of carangids was confirmed by barcoding of the COI gene. The eight nematode species found were: Capillariidae gen. sp. (females), Cucullanus bulbosus (Lane, 1916) (male and females), Hysterothylacium sp. third-stage larvae, Raphidascaris (Ichthyascaris) sp. (female and larvae), Terranova sp. third-stage larvae, Philometra dispar n. sp. (male), Camallanus carangis Olsen, 1954 (females) and Johnstonmawsonia sp. (female). The new species P. dispar from the abdominal cavity of C. dinema is mainly characterised by the body length (5.14 mm), the lengths of markedly unequal spicules (163 and 96 μm) and gubernaculum (102 μm long) provided with a dorsal protuberance and a small, reflexed dorsal barb on its posterior portion. The finding of C. bulbosus represents the first record of this parasite a century after its discovery; the first study of this species by scanning electron microscopy (SEM) enabled detailed redescription. The finding of Johnstonmawsonia sp. in C. fulvoguttatus is the first record of a rhabdochonid nematode from a host belonging to the Carangidae family. Johnstonmawsonia africana Moravec & Puylaert, 1970 and J. campanae Puylaert, 1973 are transferred to Prosungulonema Roytman, 1963 as P. africanum (Moravec & Puylaert, 1970) comb. n. and P. campanae (Puylaert, 1973) n. comb.

Species of Philometra (Nematoda, Philometridae) from fishes off the Mediterranean coast of Africa, with a description of Philometra rara n. sp. from Hyporthodus haifensis and a molecular analysis of Philometra saltatrix from Pomatomus saltatrix

Two gonad-infecting species of Philometra Costa, 1845 (Nematoda, Philometridae) were recorded for the first time from marine perciform fishes off Tunisia and Libya: Philometra rara n. sp. from the rare, deep-water Haifa grouper Hyporthodus haifensis (Serranidae) off Libya and Philometra saltatrix Ramachandran, 1973 from the bluefish Pomatomus saltatrix (Pomatomidae) off Tunisia. Identification of both fish species was confirmed by molecular barcoding. Light and scanning electron microscope studies of Ph. rara n. sp. showed that it is characterized by the length of spicules (216–219 μm) and the gubernaculum (90–93 μm), the gubernaculum/spicules length ratio (1:2.32–2.43), and mainly by the shape and structure of the distal end of the gubernaculum (shovel-shaped with a wide median smooth field in dorsal view), appearing as having a dorsal protuberance in lateral view, and by the structure of the male caudal mound (dorsally interrupted); large subgravid females (70–137 mm long) are characterized by the presence of four oval submedian cephalic elevations, each of them bearing a pair of cephalic papillae of the outer circle. The finding of Ph. saltatrix off Tunisia confirms that this species is widespread throughout the Mediterranean region. A molecular analysis of our Ph. saltatrix specimens and other available philometrid cytochrome c oxidase 1 (COI) sequences showed that most species have robust clades. Sequences of Ph. saltatrix from Tunisia diverge from Ph. saltatrix from Brazil and the USA, suggesting that speciation is currently occurring between populations from both sides of the Atlantic Ocean.

Giant worms chez moi! Hammerhead flatworms (Platyhelminthes, Geoplanidae, Bipalium spp., Diversibipalium spp.) in metropolitan France and overseas French territories

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.