Integrative Taxonomic Approach Research Articles

Alike but genetically divergent: The resurrection of Urotrygon asterias (Jordan & Gilbert, 1883) from its closest relatives, the Munda and the Blotched stingray.

The genus Urotrygon comprises small- to medium-sized endemic round rays on the American continent and has undergone several synonymization processes. Here, we used an integrative taxonomic approach, including meristic, morphometric, and mtDNA analyses, to resolve the particularly intricate relationship among Urotrygon munda Gill, 1863, Urotrygon chilensis (Günther, 1872), and Urotrygon asterias (Jordan & Gilbert, 1883). The latter species is currently a synonym of U. munda but is also considered the U. chilensis "northern morphotype." These taxonomic entities have historically been confounded, mainly due to their phenotypical resemblance along their geographic distribution in the eastern Pacific. We assessed 78 specimens (43 "northern" and 30 "southern morphotypes" of U. chilensis, as well as 5 U. munda) using 19 external variables for taxonomic and morphometric analysis. Distinct meristic patterns, including pectoral and pelvic ceratotrichia, vertebrae number, and thorn distribution along the dorsal midline, were observed in the series-type specimens of the three taxonomic entities. Our multivariate morphometric analyses consistently differentiated the three groups as distinct taxonomic entities, with an overall classification accuracy of 66.7%. The meristic results also provided reliable information distinguishing the three entities. Based on the nicotinamide adenine dinucleotide (NADH2) and cytochrome oxidase subunit I (COI) genes, our phylogenetic analysis were consistent with the morphometric and meristic data, supporting these three entities as distinct species having their own evolutionary lineages. Our comprehensive approach confidently demonstrated that the northern U. chilensis morphotype matched and corresponded to the description of the Starry round ray, U. asterias, confirming its taxonomic resurrection as a valid species distinct from U. chilensis and U. munda. The geographic distribution of U. asterias spans from the tropical west coast of Mexico (including the Gulf of California) to Costa Rica, revealing that microevolutionary processes have well-defined population clades within this range. Furthermore, U. chilensis is unequivocally established as the sole Urotrygon species occurring south of the Guayaquil marine ecoregion. In addition, the public COI and NADH2 sequences available for Urotrygon hosted in the ad hoc online databases were found to be misidentified, emphasizing the need for rigorous taxonomic scrutiny in this group. Finally, our research underscores the significance of an integrative approach that combines morphometric, meristic, and molecular techniques with historical data to disentangle the complexities of closely related taxa.

Phylogenetics, Molecular Species Delimitation and Geometric Morphometrics of All Reddish-Brown Species in the Genus Neotriplax Lewis, 1887 (Coleoptera: Erotylidae: Tritomini)

To date, five species of reddish-brown Neotriplax have been described, but their highly similar body color and other phenotypic traits make accurate taxonomy challenging. To clarify species-level taxonomy and validate potential new species, the cytochrome oxidase subunit I (COI) was used for phylogenetic analysis and the geometric morphometrics of elytron, pronotum, and hind wing were employed to distinguish all reddish-brown Neotriplax species. Phylogenetic results using maximum likelihood and Bayesian analyses of COI sequences aligned well with the current taxonomy of the Neotriplax species group. Significant K2P divergences, with no overlap between intra- and interspecific genetic distances, were obtained in Neotriplax species. The automatic barcode gap discovery (ABGD), assemble species by automatic partitioning (ASAP), and generalized mixed Yule coalescent (GMYC) approaches concurred, dividing the similar species into eight molecular operational taxonomic units (MOTUs). Geometric morphometric analysis using pronotum, elytron, hind wing shape and wing vein patterns also validated the classification of all eight species. By integrating these analytical approaches with morphological evidence, we successfully delineated the reddish-brown species of Neotriplax into eight species with three new species: N. qinghaiensis sp. nov., N. maoershanensis sp. nov., and N. guangxiensis sp. nov. Furthermore, we documented the first record of N. lewisii in China. This study underscores the utility of an integrative taxonomy approach in species delimitation within Neotriplax and serves as a reference for the taxonomic revision of other morphologically challenging beetles through integrative taxonomy.

Delimiting species, revealing cryptic diversity, and population divergence in Qinghai-Tibet Plateau weevils through DNA barcoding.

The Leptomias group represents one of the most diverse taxonomic group of weevils in the Qinghai-Tibet Plateau and its adjacent areas. Despite the potential of hidden diversity, relatively few comprehensive studies have been conducted on species diversity in this taxonomic group. In this study, we performed DNA barcoding analysis for species of the Leptomias group using a comprehensive DNA barcode dataset that included 476 sequences representing 54 morphospecies. Within the dataset, our laboratory contributed 474 sequences, and 390 sequences were newly generated for this study. The average Kimura 2-parameter distances among morphospecies and genera were 0.76% and 19.15%, respectively. In 94.4% of the species, the minimum interspecific distances exceeded the maximum intraspecific distances, indicating the presence of barcode gaps in most species of Leptomias group. The application of Automatic Barcode Gap Discovery, Assemble Species by Automatic Partitioning, Barcode Index Number, Bayesian Poisson tree processes, jMOTU, and Neighbor-joining tree methods revealed 45, 45, 63, 54, and 55 distinct clusters representing single species, respectively. Additionally, a total of four morphospecies, Leptomias kangmarensis, L. midlineatus, L. siahus, and L. sp.9RL, were found to be assigned to multiple subclade each, indicating the geographical divergences and the presence of cryptic diversity. Our findings of this study demonstrate that Qinghai-Tibet Plateau exhibits a higher species diversity of the Leptomias group, and it is imperative to investigate cryptic species within certain morphospecies using integrative taxonomic approaches in future studies. Moreover, the construction of a DNA barcode reference library presented herein establishes a robust foundational dataset to support forthcoming research on weevil taxonomy, phylogenetics, ecology, and evolution.

Revisions of the clavipes and pruni species groups of the genus Merodon Meigen, 1803 (Diptera, Syrphidae)

This study focuses on the avidus–nigritarsis lineage within the genus Merodon, exploring morphological, genetic, and distributional aspects of two related assemblies within this lineage: the clavipes and pruni species groups. An integrative taxonomic approach was followed to ensure comprehensive species identification and validation, using adult morphology, wing geometric morphometrics, and genetic analysis of the mtDNA COI gene. In the clavipes group, seven species were identified, including three new species: M. aenigmaticus Vujić, Radenković & Likov, sp. nov., M. latens Vujić, Radenković & Likov, sp. nov., and M. rufofemoris Vujić, Radenković & Likov, sp. nov. In the pruni group, our revision revealed a new species, M. aequalis Vujić, Radenković & Likov, sp. nov., and the revalidation of Merodon obscurus Gil Collado, 1929, stat. rev.Merodon pallidus Macquart, 1842 is redescribed. Diagnoses, identification keys to species, and distribution maps are provided, and neotypes for Syrphus clavipes Fabricius, 1781 and Merodon quadrinotatus (Sack, 1931) are designated. Additionally, the following new synonyms are proposed: M. clavipes albussyn. nov., M. clavipes atersyn. nov., M. clavipes nigersyn. nov., and M. splendenssyn. nov. are junior synonyms of M. clavipes; and M. velox armeniacussyn. nov. and M. velox anathemasyn. nov. are junior synonyms of M. velox.

Large‐scale integrative taxonomy of Swedish grass flies (Diptera, Chloropidae) reveals hitherto unknown complexity of a dark taxon

AbstractThe study of biodiversity is fundamental to preserving life on Earth but a challenging endeavour due to the taxonomic neglect associated with hyperdiverse lineages. To overcome this problem, we have implemented for the first time a Large‐scale integrative taxonomic (LIT) approach on the hyperdiverse insect family Chloropidae. This lineage of flies is a great fit for implementing emerging protocols such as LIT due to their ecological relevance, abundance and global distribution, poorly known genetic diversity and challenging taxonomy. In this study, we test cost‐effective barcoding workflows on nonoptimally preserved chloropid flies from Sweden and then apply targeted morphological validation to accelerate the species recognition process. Our dataset yielded DNA barcodes of 63 species that represent almost one‐third of the species diversity from Sweden and 15% of the European diversity. Applying LIT allowed us to uncover two new species and address for the first time three troublesome species complexes through a comprehensive framework. We also provide previously unknown DNA barcodes for 35% of the described species found and address distribution patterns with a focus on grassland environments. We have thus contributed significantly to overcoming the taxonomic neglect of this hyperdiverse lineage by tackling the diversity of chloropids using complementary sources of data, scalable techniques and quickly translating multiple sources of evidence into named species.

A phylogenomic approach to a taxonomic revision: a combination, new synonymies, and a description of two new species within the camel spider genus Chanbria Muma, 1951 (Solifugae: Eremobatidae)

Abstract This study summarizes the taxonomic treatment of the camel spider genus Chanbria Muma, 1951. Taking an integrative taxonomic approach incorporating phylogenomic, morphological, and geographical information, the genus is herein revised. Of the four species currently placed in the genus, two are retained: Chanbria regalis Muma, 1951 and Chanbria serpentinus Muma, 1951. Eremochelis plicatus (Muma, 1962) is transferred to this genus because it is consistently recovered in a clade with Chanbria based on several phylogenetic analyses using hundreds of loci recovered from ultraconserved element data. In this study, we re-analyse previously acquired genomic data to assess former species hypotheses and identify new morphological synapomorphies that support the monophyly of Chanbria. The genetic data support the synonymization of Chanbria rectus Muma, 1962 syn. nov. with C. regalis. Furthermore, we synonymize Chanbria tehachapianus Muma, 1962 syn. nov. with C. regalis because C. tehachapianus was erected based on limited morphological information and lack of geographical separation between other populations of C. regalis. Two new species, Chanbria brookharti sp. nov. and Chanbria mapemes sp. nov., are described. This brings the total number of species of Chanbria described to five recognized species: C. regalis, C. serpentinus, C. plicatus com. nov., C. brookharti sp. nov., and C. mapemes sp. nov.

Unravelling the relationships among Madrepora Linnaeus, 1758, Oculina Lamark, 1816 and Cladocora Ehrenberg, 1834 (Cnidaria: Anthozoa: Scleractinia).

Despite the widespread use of integrative taxonomic approaches, many scleractinian coral genera and species remain grouped in polyphyletic families, classified as incertae sedis or simply understudied. Oculinidae Gray, 1847 represents a family for which many taxonomic questions remain unresolved, particularly those related to some of the current genera, such as Oculina Lamark, 1816 or recently removed genera, including Cladocora Ehrenberg, 1834 and Madrepora Linnaeus, 1758. Cladocora is currently assigned to the family Cladocoridae Milne Edwards & Haime, 1857 and a new family, Bathyporidae Kitahara, Capel, Zilberberg & Cairns, 2024, was recently raised to accommodate Madrepora . However, the name Bathyporidae is not valid because this was not formed on the basis of a type genus name. To resolve taxonomic questions related to these three genera, the evolutionary relationships are explored through phylogenetic analyses of 18 molecular markers. The results of these analyses support a close relationship between the species Oculina patagonica and Cladocora caespitosa , indicating that these may belong to the same family (and possibly genus), and highlighting the need for detailed revisions of Oculina and Cladocora . By contrast, a distant relationship is found between these two species and Madrepora oculata , with the overall evidence supporting the placement of Madrepora in the resurrected family Madreporidae Ehrenberg, 1834. This study advances our knowledge of coral systematics and highlights the need for a comprehensive review of the genera Oculina , Cladocora and Madrepora .

Multiple sources of evidence unravel a complex taxonomic history: the new genus Leonoria of the Spermacoce clade (Spermacoceae-Rubiaceae)

For a long time, the taxonomic status of Denscantia, a genus endemic to the Atlantic Forest along the Brazilian coast, has been a topic of discussion. Phylogenetic analyses using two nuclear and four chloroplast DNA markers from 82 accessions representing 19 genera of the Spermacoce clade (tribe Spermacoceae, Rubiaceae) confirm that the Brazilian genus Denscantia is biphyletic. Together with the recovered phylogenetic relationships, the analyses of reproductive morphological characters, leaf morphology and anatomy, histochemical, geographic distribution ranges, and ecological niche derived from climatic space further demonstrate that Denscantia calcicola is a distinct lineage from the other Denscantia species. Therefore, the above-mentioned species should be taxonomically designated as a new monospecific genus, Leonoria. Significant morphological differences between Leonoria and Denscantia were found in inflorescence organization, style shape, fruit dehiscence and pollen morphology. Morphological and anatomical variation among leaf traits was found in epidermal cells, trichome distribution, mesophyll histochemistry and vascular organization. The analysis of occurrence records of 207 specimens demonstrates a clear ecological distinction between of Denscantia s.s. and Leonoria, which is ecologically confined to limestone outcrops associated with seasonally dry forests. The current study highlights the importance of an integrative taxonomic approach combining multiple sources of evidence to unravel complex taxonomic history within Spermacoceae.

Comprehensive morphological and phylogenetic inferences of star-shaped fungus Astraeus (Diplocystidiaceae) from sal-dominant tropical and subtropical Pinus-Shorea forests in India: an integrative taxonomic analysis

Classifying fungus based on morphological traits is an effective strategy to distinguish between puffballs, earthballs, earthstars, and other gasteroid fungi, as well as for identifying sub-groups of closely related taxon. However, to delimitate taxa it should be addressed with caution, since cryptic species have been described recently in different genera of gasteroid fungi. Astraeus is a star-shaped fungus that has piqued the interest of mycologists worldwide, including India. These endearing fungi have tremendous nutritional and therapeutic benefits, but their molecular and phylogenetic placement in India is uncertain. An integrative taxonomic approach was used to identify and resolve ambiguities within the genus. This study, the first to elucidate the distribution, taxonomy, and phylogeny of the genus Astraeus in the mixed subtropical Pinus-Shorea forest and tropical dry deciduous Shorea dominated forest in India, aims to shed light on these indispensable ectomycorrhizal fungi. The phylogenetic analysis assigned all thirteen ITS DNA barcoding Indian sequences of Astraeus to the Southeast Asian clade, reinforcing the genus Southeast Asian origin. Detailed species descriptions, line diagrams, SEM images of basidiospores, ITS nrDNA based phylogeny, and a dichotomous key are provided. Mycoobservations of the taxon from Pinus-Shorea subtropical and Shorea-dominated forests were additionally reviewed.

Comparison of DNA metabarcoding and microscopy in analysing planktonic protists from the European Arctic

In the era of climate change-related restructuring of planktonic protist communities, it is especially important to identify possible shifts in their taxonomic composition. While traditional microscopy-based morphological classification is time-consuming and requires experienced taxonomists, metabarcoding seems to substantially accelerate the determination of taxonomic composition. In this study, based on samples collected in summer 2019 from the West Spitsbergen Current, we analysed planktonic protists using both methods. Metabarcoding, based on high-throughput sequencing of the V4 region of the 18S rRNA gene, resulted in a much higher number of operational taxonomic units (OTUs) and sample diversity than microscopy, although the resolution of taxonomic identification ranged from species to phyla. Most morphology-based identification was performed at the species or genus level, additionally allowing us to include information about dominants and size fractions. The highest proportion of 45% shared taxa by both methods was recorded at the class level. The composition of dominant protists differed between the approaches, with most similarities being observed in Bacillariophyceae, for which two genera, Thalassiosira and Eucampia, were found to be the most abundant with both methods. For Dinophyceae, the most abundant representatives identified by microscopy were Gymnodinium spp., Prorocentrum minimum and Gonyaulax gracilis, while in the metabarcoding approach, most dinoflagellates were identified to the class level only. Given the different levels of accuracy of taxonomic determinations and possible biases in results connected to the chosen methodology, we advocate using an integrative taxonomic approach for the classification of planktonic protists based on the combination of microscopy and molecular methods.

Two new species of grass flies (Diptera, Chloropidae) from Sweden

This study implements an integrative taxonomy approach to describe two new species of Swedish grass flies Aphanotrigonum palpalis sp. nov. and Tricimba atipica sp. nov. Morphological and molecular diagnoses plus illustration of key morphological features are included, and amended keys to the Fennoscandian species of both genera are given.

Description of a new species of the genus Cultellus Schumacher, 1817 (Bivalvia, Pharidae) from the South China Sea, based on integrative taxonomy

The present study describes a new species within the genus Cultellus Schumacher, 1817 collected from the South China Sea. An integrative taxonomic approach incorporating morphological comparisons, geometric morphometrics and genetic analyses was used to identify and differentiate the new species. Cultellus exilissp. nov. is distinguished from its congeners by its slender, fragile and translucent valves, curved posteroventral margins and relatively large protractor scars. The geometric morphometric analyses, based on outlines data, indicated that samples of Cultellus exilissp. nov. clustered together and were distinctly separated from other species. Multiple species delimitation results, based on the mitochondrial COI gene, support the separation of Cultellus exilissp. nov. from its related congeners. Phylogenetic analyses of a nuclear (28S rRNA) and two mitochondrial (COI, 16S rRNA) genes using Maximum Likelihood and Bayesian Inference methods revealed that the species belongs to the genus Cultellus. The superfamily Solenoidea Lamarck, 1809, which includes the families Solenidae Lamarck, 1809 and Pharidae H. Adams & A. Adams, 1856, exhibits closer affinity to the family Hiatellidae Gray, 1824 than to Solecurtidae d’Orbigny, 1846. Furthermore, we found that the genus Siliqua was clustered alongside the genera Ensiculus and Phaxas as a sister clade, which contradicts the current systematics of the subfamily within the family Pharidae. This work highlights the utility of integrative taxonomy for species identification, recognition and phylogenetic investigation.

An integrative taxonomic revision of the Trimeresurus popeiorum group of pitvipers (Reptilia: Serpentes: Viperidae) with descriptions of two new species from the Indo-Burma Biodiversity Hotspot

Abstract Despite recent progress in our understanding of diversity within the genus Trimeresurus Lacépède, 1804, the subgenus Popeia Malhotra & Thorpe, 2004, distributed across most parts of East and Southeast Asia, remains taxonomically challenging. We applied an integrative taxonomic approach including analyses of morphological data and four mitochondrial genes (12S and 16S rRNA, cytochrome b, and NADH dehydrogenase subunit 4), along with examination of available type material, to address longstanding taxonomic questions in one clade within Popeia, the T. popeiorum group, and reveal a high level of hidden diversity of these snakes in the Indo-Burma Biodiversity Hotspot. We confirm that T. popeiorum Smith, 1937 sensu stricto is restricted to Northeast India, eastern Nepal, southern Bhutan, southeastern Bangladesh, western Yunnan Province (China), and northern and southwestern Myanmar. We further confirm that the recently described species T. yingjiangensis Chen et al., 2019 is a junior synonym of T. popeiorum. In addition, we discovered that the combination Trimesurus [sic] elegans Gray, 1853 is a valid senior synonym of T. popeiorum and threatens the stability of the latter taxon. Therefore, in order to protect the nomen popeiorum and in accordance with Article 23.9 of the International Code of Zoological Nomenclature, we regard the taxon Trimesurus elegans as a nomen oblitum and render Trimeresurus popeiorum a nomen protectum. Examination of a larger series of specimens allows us to describe two new cryptic species of Trimeresurus from the Indo-Burma Region. This study brings the total number of species in the subgenus Popeia to six and also suggests that the subspecific taxonomy of the T. sabahi complex requires further investigation. We urge adequate actions regarding the conservation of the newly discovered species and recommend further studies on their toxicology.

NEW RECORDS OF HELMINTHS OF THE JAGUAR IN MEXICO, WITH AN UPDATED LIST OF SPECIES IN THE AMERICAS.

An inventory of parasites infecting the jaguar (Panthera onca) across its distribution range is relevant for the conservation of this threatened big cat. In this study, we report the occurrence of helminths in a jaguar from Mexico using morphological techniques (cleared and stained mounts and scanning electron microscopy) and partial sequences of the 28S ribosomal RNA (28S rRNA) gene and the cytochrome c oxidase 1 mitochondrial (COI) gene. We also provide an updated list of helminth species reported in jaguars in the Americas. Three helminth taxa are identified in the jaguar examined from Mexico: Toxocara cati, Physaloptera sp., and Taenia sp. The new 28S rRNA sequences of To. cati, Physaloptera sp., and Taenia sp. and the COI sequence of Taenia sp. corroborate the identity of the helminths isolated from this host. One hundred and twenty-nine records of helminths parasitizing jaguars from 49 studies up to May 2023 were identified in the Americas. In most of these studies (73.6%), helminths were identified using coproparasitological techniques. Sixteen helminths (7 nematodes, 5 cestodes, 3 acanthocephalans, and 1 trematode) were identified at the species level in free-ranging and captive jaguars. The study demonstrates the value of an integrative taxonomy approach to increase the accuracy of parasite identification in wildlife, especially when helminth specimens are scarce or poorly fixed.

Vocal repertoire of Microhyla nilphamariensis from Delhi and comparison with closely related M. ornata populations from the western coast of India and Sri Lanka.

Advertisement calls in frogs have evolved to be species-specific signals of recognition and are therefore considered an essential component of integrative taxonomic approaches to identify species and delineate their distribution range. The species rich genus Microhyla is a particularly challenging group for species identification, discovery and conservation management due to the small size, conserved morphology and wide distribution of its members, necessitating the need for a thorough description of their vocalization. In this study, we provide quantitative description of the vocal behaviour of Microhyla nilphamariensis, a widely distributed south Asian species, from Delhi, India, based on call recordings of 18 individuals and assessment of 21 call properties. Based on the properties measured acrossed 360 calls, we find that a typical advertisement call of M. nilphamariensis lasts for 393.5± 57.5 ms, has 17 pulses on average and produce pulses at rate of 39 pulses/s. The overall call dominant frequency was found to be 2.8 KHz and the call spectrum consisted of two dominant frequency peaks centered at 1.6 KHz and 3.6 KHz, ranging between 1.5-4.1 KHz. Apart from its typical advertisement call, our study also reveals the presence of three 'rare' call types, previously unreported in this species. We describe variability in call properties and discuss their relation to body size and temperature. We found that overall dominant frequency 1 (spectral property) was found to be correlated with body size, while first pulse period (temporal property) was found to be correlated with temperature. Further, we compare the vocal repertoire of M. nilphamariensis with that of the congener Microhyla ornata from the western coast of India and Sri Lanka and also compare the call properties of these two populations of M. ornata to investigate intra-specific call variation. We find statistically significant differentiation in their acoustic repertoire in both cases. Based on 18 call properties (out of 20), individuals of each locality clearly segregate on PCA factor plane forming separate groups. Discriminant function analysis (DFA) using PCA factors shows 100% classification success with individuals of each locality getting classified to a discrete group. This confirms significant acoustic differentiation between these species as well as between geographically distant conspecifics. The data generated in this study will be useful for comparative bioacoustic analysis of Microhyla species and can be utilized to monitor populations and devise conservation management plan for threatened species in this group.

Revised description of the blueberry bud mite, Acalitus vaccinii (Acari: Trombidiformes: Eriophyidae), and a key to all Eriophyoidea on Vaccinium

In 2014 the blueberry bud mite, Acalitus vaccinii (Acari: Trombidformes: Eriophyidae), was detected in the Mpumalanga province of South Africa for the first time causing significant damage to cultivated blueberries. This was the first detection of this pest outside of North America, to which it is native. However, its taxonomic description at that time lacked critical detail and omitted characters and life stages important for easy and accurate identification. Using an integrative taxonomic approach, we combined phase contrast light microscopy with low-temperature SEM and DNA barcoding data to revise the description of A. vaccinii using South African specimens. Additional characters not included in previous descriptions but reported here are the h1 (accessory) setae, leg I & II u’ (mesal) setae, and leg II bv (femoral) setae. Detailed descriptions and measurements of all life stages are included, along with a discussion of morphological variation and biology. Two DNA sequences of common barcode regions (nuclear and mitochondrial) are provided to further aid in identification. In addition, a key to all known species of eriophyoid mites present on Vaccinium is provided.

Integrative Taxonomy of Prosogonotrema bilabiatum Vigueras, 1940 (Digenea: Sclerodistomidae): A Parasite in Atlantic Spadefish Chaetodipterus faber (Broussonet, 1782) (Acanthuriformes: Ephippidae) from Brazil.

The present work aims to expand the knowledge of the digenean species Prosogonotrema bilabiatum (Sclerodistomidae), a parasite of Chaetodipterus faber (Acanthuriformes) from Brazil, with an integrative taxonomic approach, using light microscopy, scanning electron microscopy, histology, and molecular biology. Forty-one digenean specimens were stained with hydrochloric carmine for morphological studies. Eleven parasites were dehydrated through a graded ethanol series, critical point dried with carbon dioxide, and coated with gold for scanning electron microscopy analysis. Four specimens were processed following histological routine and stained with hematoxylin and eosin and Gomori trichrome. DNA extracted was amplified using 28S partial primer D1-D3. Maximum likelihood and Bayesian inference were performed for phylogenetic analysis. Morphometric and morphological data of the specimens studied ranged in accordance as observed in previous descriptions of the species. Observations from scanning electron microscopy and histology corroborated with those observed in stained whole mounts. Molecular analysis showed that specimens of P. bilabiatum from Brazil clustered with another two sequences of this species fromdifferent hosts and localities, with a high node support value. The integrative taxonomic approach allowed to record and describe new characteristics of P. bilabiatum related to the tegument, the structure and the arrangement of its tissues. The use of molecular markers confirmed that specimens identified as P. bilabiatum from different hosts and localities are all conspecific. Further studies, mainly molecular with less conserved genetic markers, should be carried out to better understand the phylogenetic relationships of Prosogonotrema with Hemiuroidea.

Novel molecular resources for single-specimen barcoding of enigmatic crustacean y-larvae.

Despite discovery more than 100years ago and documented global occurrence from shallow waters to the deep sea, the life cycle of the enigmatic crustacean y-larvae isincompletely understood and adult forms remain unknown. To date, only 2 of the 17 formally described species, all based on larval stages, have been investigated using an integrative taxonomic approach. This approach provided descriptions of the morphology of the naupliar and cyprid stages, and made use of exuvial voucher material and DNA barcodes. To improve our knowledge about the evolutionary history and ecological importance of y-larvae, we developed a novel protocol that maximises the amount of morpho-ecological and molecular data that can be harvested from single larval specimens. This includes single-specimen DNA barcoding and daily imaging of y-nauplii reared in culture dishes, mounting of the last naupliar exuviae on a slide as a reference voucher, live imaging of the y-cyprid instar that follows, and fixation, DNA extraction, amplification and sequencing of the y-cyprid specimen. Through development and testing of a suite of new primers for both nuclear and mitochondrial protein-coding and ribosomal genes, we showcase how new sequence data can be used to estimate the phylogeny of Facetotecta. We expect that our novel procedure will help to unravel the complex systematics of y-larvae and show how these fascinating larval forms have evolved. Moreover, we posit that our protocols should work on larval specimens from a diverse array of moulting marine invertebrate taxa.

Integrative taxonomy of metazoan parasites of the bluntnose sixgill shark Hexanchus griseus (Bonnaterre, 1788) in the Mediterranean Sea, with the resurrection of Grillotia acanthoscolex Rees, 1944 (Cestoda: Trypanorhyncha).

Appropriate diagnoses of parasites of apex marine predators are crucial to understand their biodiversity, host specificity, biogeography, and life cycles. Such diagnoses are also informative of ecological and biological characteristics of both host and environment in which the hosts and their parasites live. We here (i) investigate the parasite fauna of a bluntnose sixgill shark Hexanchus griseus (Bonnaterre, 1788) obtained from the Gulf of Naples (Tyrrhenian Sea), (ii) characterize molecularly all its metazoan parasites, and (iii) resurrect and report the main morphological features and phylogenetic position of Grillotia acanthoscolex, a cestode species previously synonymized with Grillotia adenoplusia. A rich parasite fauna represented by eight different taxa was found, including two monogeneans (Protocotyle grisea and Protocotyle taschenbergi), one digenean (Otodistomum veliporum), four cestodes (Crossobothrium dohrnii, Clistobothrium sp., G. acanthoscolex, and G. adenoplusia), and one copepod (Protodactylina pamelae). Sequencing of these samples accounts for an important molecular baseline to widen the knowledge on the parasitic fauna of bluntnose sixgill sharks worldwide and to reconstruct their correct food chains. The bluntnose sixgill shark was found to be a definitive host for all endoparasites found here, confirming that it occupies an apex trophic level in the Mediterranean Sea. The taxa composition of the trophic parasite fauna confirms that the bluntnose sixgill shark mostly feeds on teleost fish species. However, the occurrence of two phillobothrid cestodes (C. dohrnii and Clistobothrium sp.) suggests that it also feeds on squids. Finally, we emphasize the importance of using integrative taxonomic approaches in the study of parasites from definitive and intermediate hosts to elucidate biology and ecology of taxa generally understudied in the Mediterranean Sea.

Microallopatric speciation in the relict dung beetle genus Grebennikovius (Coleoptera: Scarabaeidae) in the Eastern Arc Mountains

Abstract The Eastern Arc Mountains (EAMs) in Tanzania and Kenya are renowned for their exceptional species endemism often restricted to isolated mountain blocks. Forest fragmentation during the Pliocene–Pleistocene played a significant role in shaping the EAMs’ biodiversity by facilitating allopatric speciation between different mountains. However, only a few studies focused on species diversification within the same mountain block. In this article, we investigated the taxonomy and evolution of the dung beetle genus Grebennikovius, endemic to the Uluguru Mountains. Our goal was to unravel factors promoting allopatric speciation within a confined geographic area like the Ulugurus. We used an integrative taxonomic approach, combining phenotypic data and COI barcodes to delimit species and reconstructed a timetree of the genus. Molecular and morphological evidence consistently recovered 4 distinct Grebennikovius species, of which 3 are new to science: G. armiger n. sp., G. basilewskyi (Balthasar, 1960), G. lupanganus n. sp., and G. pafelo n. sp. Grebennikovius represents a rare case of microallopatry, with all species occurring in close proximity within different regions of the limited Uluguru forest (∼230 km2). We infer that speciation was driven by the interplay between climate fluctuations during the Miocene and mid-Pleistocene, Uluguru topography, and the stability of rainforest ecological conditions. The latter factor probably favored the loss of wings and a specialization for Uluguru microhabitats, reinforcing population isolation and divergence. This study highlights a unique instance of diversification resulting from small-scale speciation events, providing valuable insights into the evolution of Eastern African rainforest arthropods and underscoring the importance of microhabitats conservation.