Morphological, acoustic and genetic identification of a reproducing population of the invasive African clawed frog Xenopuslaevis (Anura, Pipidae) recently discovered in Belgium.

Using external morphology of adults and tadpoles, osteology from high-resolution microcomputed tomography, vocalization analysis, and DNA sequence data, the identity of a reproducing Belgian population of invasive Xenopus at the current northernmost edge of the distribution of the genus in Europe was assessed. All data concur to an identification as Xenopus (Xenopus) laevis (Daudin, 1802). Genetically it is most closely related to populations of the Cape region in South Africa. No studies on the natural history of the Belgian Xenopus population and its impact on the local environment have been made to date.

A new jewel-like species of the pill-millipede genus Sphaerobelum Verhoeff, 1924 (Diplopoda, Sphaerotheriida, Zephroniidae) from Thailand.

A new species of the giant pill millipede genus Sphaerobelum is described: Sphaerobelumturcosasp. nov. from the northeastern part of Thailand. Species delimitation is based on morphological characters and COI sequence data. The new species can be clearly discriminated from congeners by its greenish-blue body color, the face mask-like appearance of the thoracic and anal shields jointly when rolled up, and the combination of the following four characters: (1) the coxa of the second leg laterally with a sharp and long process, (2) the tarsi of legs 4-21 with 6-7 ventral spines, (3) the anterior telopods consisting of four conspicuous telopoditomeres, and (4) the immovable, slender (not strongly humped) and distally curved finger of the posterior telopods without a membranous spot. The interspecific COI sequence divergence between the new species and other Sphaerobelum species ranges from 17% to 23% (mean 20%). The intergeneric COI sequence divergence between the new species and Zephronia species ranges from 18% to 21% (mean 20%). The relationships among Sphaerobelum and Zephronia species based on the COI sequence data were not resolved in this study. Sphaerobelumturcosasp. nov. is restricted to limestone habitat in Loei province and is probably endemic for the Thai fauna.

The systematics and evolution of the Sri Lankan rainforest land snail Corilla: New insights from RADseq-based phylogenetics.

The stylommatophoran land-snail genus Corilla is endemic to Sri Lanka and India's Western Ghats. On the basis of habitat distribution and shell morphology, the 10 extant Sri Lankan species fall into two distinct groups, lowland and montane. Here, we use phylogenetic analyses of restriction-site-associated DNA sequencing (RADseq) data and ancestral-state reconstructions of habitat association and shell morphology to clarify the systematics and evolution of Sri Lankan Corilla. Our dataset consists of 9 species of Corilla. Phylogenetic analyses were based on 88 assemblies (9,604-4,132,850 bp) generated by the RADseq assembler ipyrad, using four parameter combinations and different levels of missing data. Trees were inferred using a maximum likelihood (ML) approach. Ancestral states were reconstructed using maximum parsimony (MP) and ML approaches, with 1 binary state character analysed for habitat association (lowland vs montane) and 6 binary state characters analysed for shell morphology (shape, colour, lip width, length of upper palatal folds, orientation of upper palatal folds and collabral sculpture). Over a wide range of missing data (40-87 % missing individuals per locus) and assembly sizes (62,279-4,132,850 bp), nearly all trees conformed to one of two topologies (A and B), most relationships were strongly supported and total branch support approached the maximal value. Apart from the position of Corilla odontophora 'south', topologies A and B showed similar, well-resolved relationships at and above the species level. Our study agrees with the shell-based taxonomy of C. adamsi, C. beddomeae, C. carabinata, C. colletti and C. humberti (all maximally supported as monophyletic species). It shows that C. erronea and C. fryae constitute a single relatively widespread species (for which the valid name is C. erronea) and that the names C. gudei and C. odontophora each apply to at least two distinct, yet conchologically-cryptic species. The MP and ML ancestral-state reconstructions yielded broadly similar results and provide firm evidence that diversification in Sri Lankan Corilla has involved evolutionary convergence in the shell morphology of lowland lineages, with a pale shell and wide lip having evolved on at least two separate occasions (in C. carabinata and C. colletti) from montane ancestors having a dark, narrow-lipped shell.

130 years of heavy metal pollution archived in the shell of the intertidal dog whelk, Nucella lapillus (Gastropoda, Muricidae).

Heavy metals in coastal waters are a great environmental concern in the North Sea since the middle of the 20th century. Regulatory efforts have led to a significant reduction in atmospheric and water-transported heavy metals. Still, high concentrations of these in sediments remain a risk for ecosystems, requiring close monitoring. Here, we investigated the applicability of Nucella lapillus museum collections as a tool for targeted tracking of chronic anthropogenic heavy metal pollution. We analysed the concentration ratios of the common heavy metals Cu, Cd, Pb, and Zn in relation to Ca in N. lapillus shells collected from the Dutch and Belgian intertidal zone over the last 130 years. We found that shell Cu/Ca and Zn/Ca concentration ratios remained remarkably constant, whereas Pb/Ca concentration trends were closely aligned with emissions of leaded petrol in Europe. Our results suggest that N. lapillus provides a suitable Pb pollution archive of the intertidal zone.

Eating eggplants as a cucurbit feeder: Dietary shifts affect the gut microbiome of the melon fly Zeugodacus cucurbitae (Diptera, Tephritidae).

While contemporary changes in feeding preferences have been documented in phytophagous insects, the mechanisms behind these processes remain to be fully clarified. In this context, the insect gut microbiome plays a central role in adaptation to novel host plants. The cucurbit frugivorous fruit fly Zeugodacus cucurbitae (Diptera, Tephritidae) has occasionally been reported on "unconventional" host plants from different families, including Solanaceae. In this study, we focus on wild parental (F0 ) adults and semiwild first filial (F1 ) larvae of Z. cucurbitae from multiple sites in La Réunion and explore how the gut microbiome composition changes when this fly is feeding on a noncucurbit host (Solanum melongena). Our analyses show nonobvious gut microbiome responses following the F0 -F1 host shift and the importance of not just diet but also local effects, which heavily affected the diversity and composition of microbiomes. We identified the main bacterial genera responsible for differences between treatments. These data further stress the importance of a careful approach when drawing general conclusions based on laboratory populations or inadequately replicated field samples.

Redescription of the giant Southeast Asian millipede Spirobolusmacrurus Pocock, 1893 and its assignment to the new genus Macrurobolus gen. nov. (Diplopoda, Spirobolida, Pachybolidae).

A new genus of the millipede family Pachybolidae from Southeast Asia is described: Macrurobolus gen. nov., with Spirobolusmacrurus Pocock, 1893 as type species. This latter species is DNA barcoded (COI) and redescribed based on male morphological characters, which hitherto were unknown. The new genus differs from other pachybolid genera by having (1) the preanal ring process long and protruding beyond the anal valves and (2) the anterior gonopod telopodite distally abruptly narrowed, forming an extremely long, slender, elevated process curved caudad. Given that Macrurobolus gen. nov. is a monotypic genus, it is aphyletic and thus requires further taxonomic revision.

Genital anatomy, jaw and radula of Guladentiasubtussulcata (Helicoidea, Cepolidae), endemic to western Cuba.

This study provides the first data on the genital anatomy, jaw and radula of Guladentiasubtussulcata (L. Pfeiffer, 1863). The auxiliary copulatory organ of this species is very peculiar, similar to that of Jeanneretia L. Pfeiffer, 1877, and different from that of other cepolids. It consists of an elongate, pedunculate mucus gland inserted apically on a muscular papilla and an atrial sac, all covered by a sheath. A sheath-like accessory gland is inserted at the base of the atrial sac. Another similarity with Jeanneretia is the presence of a fertilization pouch-spermatheca complex with a single exposed spermatheca. Like Jeanneretia, G.subtussulcata has an oxygnath, highly arched jaw with slight striae over the entire surface and a broad, well-developed median projection. The radula has triangular and monocuspid central and lateral teeth (the central teeth are smaller than the rest). The marginal teeth are multicuspid with the mesocone and ectocones smaller than the endocones. The similar structures of the auxiliary copulatory organ (without dart sac) and spermatheca (simple) strongly suggest that G.subtussulcata and Jeanneretia spp. are closely related. As such, it remains to be decided whether Guladentia Clench & Aguayo, 1951 and Jeanneretia should continue to be treated as separate genera.

MEMO: Monitoring of exotic mosquitoes in Belgium.

'MEMO: Monitoring of Exotic MOsquitoes in Belgium' is a sampling event dataset published by the Institute of Tropical Medicine (ITM) in Antwerp, Belgium. It forms part of the early detection of exotic mosquito species (EMS) along high-risk introduction routes in Belgium, where data are collected at defined points of entry (PoEs) using a standardised protocol. The MEMO dataset contains mosquito sampling counts performed between 2017 and 2020. MEMO+2020, an extension of the MEMO dataset, contains only Aedes albopictus mosquito trap counts performed in 2020. Here, we present these data published as a standardised Darwin Core archive, which includes, for each sampling event, an eventID, date, location and sampling protocol (in the event core); and an occurrenceID for each occurrence (tube), the number of collected individuals per tube, species status (present/absent), information on the identification and scientific name (in the occurrence extension).

Two new millipede species of the genus Coxobolellus Pimvichai, Enghoff, Panha & Backeljau, 2020 (Diplopoda, Spirobolida, Pseudospirobolellidae).

Two new millipede species of the genus Coxobolellus Pimvichai, Enghoff, Panha & Backeljau, 2020 from Thailand are described: Coxobolellussaratani sp. nov. from Loei Province and Coxobolellusserratoligulatus sp. nov. from Uttaradit Province. The descriptions are based on gonopod morphology and two mitochondrial gene fragments (COI and 16S rRNA). The phylogenetic mtDNA analysis assigned the two new species unequivocally to the consistently well-supported Coxobolellus clade, in which they form a fifth subclade that was well supported by maximum likelihood analysis of 16S rRNA, though neither by Bayesian inference nor by COI. The two new Coxobolellus species share four conspicuous gonopodal synapomorphies of the genus: (1) the protruding process on the coxae of the 3rd (and sometimes 4th) pair of male legs, (2) a large, triangular coxae on the 4th-5th pair of legs, (3) a short process of the preanal ring protruding as far as, or slightly beyond, the anal valves, and (4) the posterior gonopod telopodite divided into two parts, with a conspicuous pore opening at the mesal margin at the end of the coxal part of the posterior gonopod. Thus, the two new species provide further evidence of the well-defined monophyly of the genus Coxobolellus. Finally, the paper provides an updated morphological identification key to all currently described Coxobolellus species.

First record of the West Nile virus bridge vector Culex modestus Ficalbi (Diptera: Culicidae) in Belgium, validated by DNA barcoding.

A thorough knowledge of the presence and spatio-temporal distribution patterns of vector species are pivotal to assess the risk of mosquito-borne diseases in Europe. In 2018, a Culex larva was collected during routine monitoring activities to intercept exotic Aedes mosquito species in the port of Antwerp (Kallo, Belgium). The larva, collected from a pond in mid-September, was morphologically identified as Culex modestus, and this identification was subsequently confirmed by COI barcoding. It is the first confirmed record of this West Nile virus bridge vector in Belgium. The present study also demonstrates the value of DNA-based identification techniques to validate the presence of potential vector species.

Population genetic structure of the Asian bush mosquito, Aedes japonicus (Diptera, Culicidae), in Belgium suggests multiple introductions.

BACKGROUND: Aedes japonicus japonicus has expanded beyond its native range and has established in multiple European countries, including Belgium. In addition to the population located at Natoye, Belgium, locally established since 2002, specimens were recently collected along the Belgian border. The first objective of this study was therefore to investigate the origin of these new introductions, which were assumed to be related to the expansion of the nearby population in western Germany. Also, an intensive elimination campaign was undertaken at Natoye between 2012 and 2015, after which the species was declared to be eradicated. This species was re-detected in 2017, and thus the second objective was to investigate if these specimens resulted from a new introduction event and/or from a few undetected specimens that escaped the elimination campaign. METHODS: Population genetic variation at nad4 and seven microsatellite loci was surveyed in 224 and 68 specimens collected in Belgium and Germany, respectively. German samples were included as reference to investigate putative introduction source(s). At Natoye, 52 and 135 specimens were collected before and after the elimination campaign, respectively, to investigate temporal changes in the genetic composition and diversity. RESULTS: At Natoye, the genotypic microsatellite make-up showed a clear difference before and after the elimination campaign. Also, the population after 2017 displayed an increased allelic richness and number of private alleles, indicative of new introduction(s). However, the Natoye population present before the elimination programme is believed to have survived at low density. At the Belgian border, clustering results suggest a relation with the western German population. Whether the introduction(s) occur via passive human-mediated ground transport or, alternatively, by natural spread cannot be determined yet from the dataset. CONCLUSION: Further introductions within Belgium are expected to occur in the near future, especially along the eastern Belgian border, which is at the front of the invasion of Ae. japonicus towards the west. Our results also point to the complexity of controlling invasive species, since 4 years of intense control measures were found to be not completely successful at eliminating this exotic at Natoye.

A century of coping with environmental and ecological changes via compensatory biomineralization in mussels.

Accurate biological models are critical to predict biotic responses to climate change and human-caused disturbances. Current understanding of organismal responses to change stems from studies over relatively short timescales. However, most projections lack long-term observations incorporating the potential for transgenerational phenotypic plasticity and genetic adaption, the keys to resistance. Here, we describe unexpected temporal compensatory responses in biomineralization as a mechanism for resistance to altered environmental conditions and predation impacts in a calcifying foundation species. We evaluated exceptional archival specimens of the blue mussel Mytilus edulis collected regularly between 1904 and 2016 along 15 km of Belgian coastline, along with records of key environmental descriptors and predators. Contrary to global-scale predictions, shell production increased over the last century, highlighting a protective capacity of mussels for qualitative and quantitative trade-offs in biomineralization as compensatory responses to altered environments. We also demonstrated the role of changes in predator communities in stimulating unanticipated biological trends that run contrary to experimental predictive models under future climate scenarios. Analysis of archival records has a key role for anticipating emergent impacts of climate change.

Deciphering mollusc shell production: the roles of genetic mechanisms through to ecology, aquaculture and biomimetics.

Most molluscs possess shells, constructed from a vast array of microstructures and architectures. The fully formed shell is composed of calcite or aragonite. These CaCO3 crystals form complex biocomposites with proteins, which although typically less than 5% of total shell mass, play significant roles in determining shell microstructure. Despite much research effort, large knowledge gaps remain in how molluscs construct and maintain their shells, and how they produce such a great diversity of forms. Here we synthesize results on how shell shape, microstructure, composition and organic content vary among, and within, species in response to numerous biotic and abiotic factors. At the local level, temperature, food supply and predation cues significantly affect shell morphology, whilst salinity has a much stronger influence across latitudes. Moreover, we emphasize how advances in genomic technologies [e.g. restriction site-associated DNA sequencing (RAD-Seq) and epigenetics] allow detailed examinations of whether morphological changes result from phenotypic plasticity or genetic adaptation, or a combination of these. RAD-Seq has already identified single nucleotide polymorphisms associated with temperature and aquaculture practices, whilst epigenetic processes have been shown significantly to modify shell construction to local conditions in, for example, Antarctica and New Zealand. We also synthesize results on the costs of shell construction and explore how these affect energetic trade-offs in animal metabolism. The cellular costs are still debated, with CaCO3 precipitation estimates ranging from 1-2 J/mg to 17-55 J/mg depending on experimental and environmental conditions. However, organic components are more expensive (~29 J/mg) and recent data indicate transmembrane calcium ion transporters can involve considerable costs. This review emphasizes the role that molecular analyses have played in demonstrating multiple evolutionary origins of biomineralization genes. Although these are characterized by lineage-specific proteins and unique combinations of co-opted genes, a small set of protein domains have been identified as a conserved biomineralization tool box. We further highlight the use of sequence data sets in providing candidate genes for in situ localization and protein function studies. The former has elucidated gene expression modularity in mantle tissue, improving understanding of the diversity of shell morphology synthesis. RNA interference (RNAi) and clustered regularly interspersed short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9) experiments have provided proof of concept for use in the functional investigation of mollusc gene sequences, showing for example that Pif (aragonite-binding) protein plays a significant role in structured nacre crystal growth and that the Lsdia1 gene sets shell chirality in Lymnaea stagnalis. Much research has focused on the impacts of ocean acidification on molluscs. Initial studies were predominantly pessimistic for future molluscan biodiversity. However, more sophisticated experiments incorporating selective breeding and multiple generations are identifying subtle effects and that variability within mollusc genomes has potential for adaption to future conditions. Furthermore, we highlight recent historical studies based on museum collections that demonstrate a greater resilience of molluscs to climate change compared with experimental data. The future of mollusc research lies not solely with ecological investigations into biodiversity, and this review synthesizes knowledge across disciplines to understand biomineralization. It spans research ranging from evolution and development, through predictions of biodiversity prospects and future-proofing of aquaculture to identifying new biomimetic opportunities and societal benefits from recycling shell products.

Checklist of the fresh and brackish water snails (Mollusca, Gastropoda) of Bénin and adjacent West African ecoregions.

Currently no comprehensive checklist of fresh and brackish water gastropods from Bénin exists, and those for adjacent West African areas are outdated. Yet, such checklists provide essential biodiversity information and a consistent taxonomic and nomenclatural framework for that biodiversity. Here a first checklist of the fresh and brackish water gastropods from Bénin and adjacent West African ecoregions is presented, based on an extensive literature review and field surveys between September 2014 and June 2019 in six major fresh and brackish water ecosystems in Bénin. This inventory includes information on synonymy, species distribution in West Africa, habitats, and conservation status. The fresh and brackish water gastropod fauna includes 60 species, belonging to 28 genera and 16 families. Pachychilidae, Ampullariidae, Neritidae, and Bulinidae were the most diverse families with 9, 8, 7, and 7 species, respectively. However, literature and field data indicated that 23 species observed in West African basins that extend to Bénin do not occur in the territory of Bénin. These species were not detected in our field surveys, most likely because they are rare at collecting sites. Of the 60 species included, five are classified as "Data Deficient", 43 as "Least Concern", two as "Nearly Threatened", one as "Vulnerable", and six as "Endangered" by the IUCN, whereas the remaining three species were not evaluated. Because the taxonomy of fresh and brackish water gastropods in West Africa is still largely based on morphology, comparative molecular and taxonomic studies may result in substantial revisions of this checklist over the coming years.

Intermediate gastropod hosts of major feline cardiopulmonary nematodes in an area of wildcat and domestic cat sympatry in Greece.

BACKGROUND: The metastrongyloid nematodes Aelurostrongylus abstrusus, Troglostrongylus brevior and Angiostrongylus chabaudi are cardiopulmonary parasites affecting domestic cats (Felis catus) and wildcats (Felis silvestris). Although knowledge on these nematodes has been improved in the past years, gaps in our knowledge of their distribution and role of gastropods as intermediate hosts in Europe still exist. This study reports on the presence of these nematodes and their intermediate hosts in an area in Greece where domestic cats and wildcats occur in sympatry. METHODS: Terrestrial gastropods were collected in the field and identified morphologically and by mitochondrial DNA-sequence analysis. Metastrongyloid larvae were detected by artificial digestion, morphologically identified to the species and stage level and their identity was molecularly confirmed. RESULTS: Aelurostrongylus abstrusus was found in the snails Massylaea vermiculata and Helix lucorum, T. brevior in the slug Tandonia sp., and A. chabaudi in the slug Limax sp. and the snails H. lucorum and M. vermiculata. CONCLUSIONS: To the best of our knowledge this study provides the first reports of (i) terrestrial gastropods being naturally infected with A. chabaudi, (ii) T. brevior naturally infecting terrestrial gastropods in Europe, and (iii) A. abstrusus naturally infecting terrestrial gastropods in Greece. Furthermore, the present study describes for the first time developmental stages of A. chabaudi and T. brevior in naturally infected gastropods. The biological characteristics of various intermediate gastropod hosts that could influence the distribution and expansion of feline cardiopulmonary nematodes are discussed, along with epizootiological implications and perspectives.

Urbanization drives cross-taxon declines in abundance and diversity at multiple spatial scales.

The increasing urbanization process is hypothesized to drastically alter (semi-)natural environments with a concomitant major decline in species abundance and diversity. Yet, studies on this effect of urbanization, and the spatial scale at which it acts, are at present inconclusive due to the large heterogeneity in taxonomic groups and spatial scales at which this relationship has been investigated among studies. Comprehensive studies analysing this relationship across multiple animal groups and at multiple spatial scales are rare, hampering the assessment of how biodiversity generally responds to urbanization. We studied aquatic (cladocerans), limno-terrestrial (bdelloid rotifers) and terrestrial (butterflies, ground beetles, ground- and web spiders, macro-moths, orthopterans and snails) invertebrate groups using a hierarchical spatial design, wherein three local-scale (200 m × 200 m) urbanization levels were repeatedly sampled across three landscape-scale (3 km × 3 km) urbanization levels. We tested for local and landscape urbanization effects on abundance and species richness of each group, whereby total richness was partitioned into the average richness of local communities and the richness due to variation among local communities. Abundances of the terrestrial active dispersers declined in response to local urbanization, with reductions up to 85% for butterflies, while passive dispersers did not show any clear trend. Species richness also declined with increasing levels of urbanization, but responses were highly heterogeneous among the different groups with respect to the richness component and the spatial scale at which urbanization impacts richness. Depending on the group, species richness declined due to biotic homogenization and/or local species loss. This resulted in an overall decrease in total richness across groups in urban areas. These results provide strong support to the general negative impact of urbanization on abundance and species richness within habitat patches and highlight the importance of considering multiple spatial scales and taxa to assess the impacts of urbanization on biodiversity.

Re-description of the type species of the genera Ganesella Blanford, 1863 and Globotrochus Haas, 1935; with description of a new Ganesella species from Thailand (Eupulmonata, Camaenidae).

The taxonomy of the speciose genus Ganesella W.T. Blanford, 1863 and the endemic genus Globotrochus Haas, 1935 is unclear since the anatomical characters of the the type species of these two genera have never been reported before. Therefore, the present paper provides the first anatomical descriptions of the reproductive apparatus, pallial system and radula of Helix capitium Benson, 1848 and Helix onestera Mabille, 1887, the respective type species of Ganesella and Globotrochus. In addition, Ganesella rhombostoma (Pfeiffer, 1861) and Ganesella carinella (Möllendorff, 1902) from Thailand are re-described, and a new species, Ganesella halabalah Sutcharit & Panha, sp. nov., from southern Thailand is described. This new species differs from all others by having a larger shell, an obtuse apex and an aperture lip with a prominent beak-like deflection.

First mitochondrial genomes of five hoverfly species of the genus Eristalinus (Diptera: Syrphidae).

The hoverfly genus Eristalinus (Diptera, Syrphidae) contains many widespread pollinators. The majority of the species of Eristalinus occur in the Afrotropics and their molecular systematics still needs to be investigated. This study presents the first complete and annotated mitochondrial genomes for five species of Eristalinus. They were obtained by high-throughput sequencing of total genomic DNA. The total length of the mitogenomes varied between 15 757 and 16 245 base pairs. Gene composition, positions, and orientation were shared across species, and were identical to those observed for other Diptera. Phylogenetic analyses (maximum likelihood and Bayesian inference) based on the 13 protein coding and both rRNA genes suggested that the subgenus Eristalinus was paraphyletic with respect to the subgenus Eristalodes. An analysis of the phylogenetic informativeness of all protein coding and rRNA genes suggested that NADH dehydrogenase subunit 5 (nad5), cytochrome c oxidase subunit 1, nad4, nad2, cytochrome b, and 16S rRNA genes are the most promising mitochondrial molecular markers to result in supported phylogenetic hypotheses of the genus. In addition to the five complete mitogenomes currently available for hoverflies, the five mitogenomes published here will be useful for broader molecular phylogenetic analyses among hoverflies.

Untangling a mess of worms: Species delimitations reveal morphological crypsis and variability in Southeast Asian semi-aquatic earthworms (Almidae, Glyphidrilus).

Semi-aquatic freshwater earthworms in the genus Glyphidrilus from Southeast Asia are characterized by both an extreme morphological crypsis among divergent phylogenetic lineages and a high morphological variability within the same phylogenetic lineages. The present study provides a new taxonomic framework for this problematic genus in SE Asia by integrating DNA sequence and morphological data. When single-locus and multilocus multispecies coalescent-based (MSC) species delimitation methods were applied to DNA sequence data, they usually yielded highly incongruent results compared to morphology-based species identifications. This suggested the presence of several cryptic species and high levels of intraspecific morphological variation. Applying reciprocal monophyly to the cytochrome c oxidase subunit 1 (COI) gene tree allowed us to propose the existence of 33 monophyletic species. Yet, often substantially more molecular operational taxonomic units (MOTUs) were obtained when species delimitation was based on COI and 16S rRNA sequences. In contrast, the ITS1 and ITS2 sequences suggested fewer MOTUs and did not recover most of the monophyletic species from the Mekong basin. However, several of these latter taxa were better supported when MSC species delimitation methods were applied to the combined mtDNA and ITS datasets. The ITS2 secondary structure retrieved one unnamed Mekong basin species that was not uncovered by the other methods when applied to ITS2 sequences. In conclusion, based on an integrative taxonomic workflow, 26 Glyphidrilus candidate species were retained and two remained to be confirmed. As such, this study provides evidence to suggest nine species new to science and to synonymize 12 nominal morphospecies. It also illustrates that the uncritical use of COI as a universal DNA barcode may overestimate species diversity because COI may be unable to distinguish between divergent conspecific lineages and different candidate species.

Relation between mitochondrial DNA hyperdiversity, mutation rate and mitochondrial genome evolution in Melarhaphe neritoides (Gastropoda: Littorinidae) and other Caenogastropoda.

Mitochondrial DNA hyperdiversity is primarily caused by high mutation rates (µ) and has potential implications for mitogenome architecture and evolution. In the hyperdiverse mtDNA of Melarhaphe neritoides (Gastropoda: Littorinidae), high mutational pressure generates unusually large amounts of synonymous variation, which is expected to (1) promote changes in synonymous codon usage, (2) reflect selection at synonymous sites, (3) increase mtDNA recombination and gene rearrangement, and (4) be correlated with high mtDNA substitution rates. The mitogenome of M. neritoides was sequenced, compared to closely related littorinids and put in the phylogenetic context of Caenogastropoda, to assess the influence of mtDNA hyperdiversity and high µ on gene content and gene order. Most mitogenome features are in line with the trend in Mollusca, except for the atypical secondary structure of the methionine transfer RNA lacking the TΨC-loop. Therefore, mtDNA hyperdiversity and high µ in M. neritoides do not seem to affect its mitogenome architecture. Synonymous sites are under positive selection, which adds to the growing evidence of non-neutral evolution at synonymous sites. Under such non-neutrality, substitution rate involves neutral and non-neutral substitutions, and high µ is not necessarily associated with high substitution rate, thus explaining that, unlike high µ, a high substitution rate is associated with gene order rearrangement.