Mitochondrial 16S rRNA Research Articles

Communicator whistles: A Trek through the taxonomy of the Boophis marojezensis complex reveals seven new, morphologically cryptic treefrogs from Madagascar (Amphibia: Anura: Mantellidae)

Abstract The Malagasy stream-breeding treefrog species Boophis marojezensis contains bioacoustically and genetically highly divergent populations. Some of these populations have been defined as candidate species and emit somewhat bizarre advertisement calls consisting of multiple whistle-notes. We here enable a long-overdue taxonomic revision of this species complex by applying a museomics approach to sequence DNA from the holotype of B. marojezensis. Based on an integrative approach that combines divergence levels in mitochondrial DNA and in three nuclear-encoded genes, morphological data, and bioacoustic comparisons, we conclude that eight different species exist in this complex, seven of which are formally described herein as new. Although morphological differences between species are small and mainly separate small-sized from larger-sized species, conclusive evidence for the new species comes from their sympatric and sometimes syntopic occurrence without haplotype sharing in three nuclear genes and under maintenance of bioacoustic differences. Uncorrected genetic divergences in the mitochondrial 16S rRNA gene are >3% in almost all cases, and in some cases up to 8%. In reference to the otherworldly sounds by which these frogs fill Malagasy rainforests, some of them reminiscent of sounds of technical equipment in the fictional “Star Trek” universe, we here name and describe the seven new species in honor of fictional captains of starships, namely B. kirkisp. nov., B. picardisp. nov., B. siskoisp. nov., B. janewayaesp. nov., B. archerisp. nov., B. pikeisp. nov., and B. burnhamaesp. nov. The majority of these species occur in northern Madagascar, where up to three species can occur in immediate geographical proximity, e.g., B. marojezensis, B. burnhamaesp. nov. and B. pikeisp. nov. at different elevations in the Marojejy Massif. South of 16°S latitude, only B. janewayaesp. nov., B. picardisp. nov., and B. kirkisp. nov. are found, with the latter extending southwards to Ranomafana National Park. Our study confirms the existence of numerous morphologically cryptic and microendemic species among Madagascar’s amphibians, some of which are known only from unprotected sites and require adequate conservation management.

Revision of the Chinese termite genus Peribulbitermes (Blattodea, Isoptera, Termitidae): A new synonym of Ahmaditermes

The genus Peribulbitermes is a small genus in Nasutitermitinae comprising three species distributed only in China. Type specimens of Peribulbitermes dinghuensis Li, 1985 and Peribulbitermes jinghongensis Li, 1985 are obtained in our study. After examining the morphological characters of the specimens, it is found that P. jinghongensis cannot be differentiated from P. dinghuensis by the diagnostic characters reported in original description. We propose that P. jinghongensis is the junior synonym of P. dinghuensis. Additionally, molecular phylogenetic relationships of Peribulbitermes, with its morphologically similar genera, Bulbitermes, Ahmaditermes and Nasutitermes, as well as other nine genera in Nasutitermes Group of Nasutitermitinae were analysed based on mitochondrial COI, COII and 16S rRNA genes. The results show that the relationship between Peribulbitermes and Ahmaditermes is the closest. Peribulbitermes should be downgraded to synonymy of Ahmaditermes. P. dinghuensis and Peribulbitermes parafulvus (Tsai and Chen, 1963) should be transferred to Ahmaditermes as Ahmaditermes dinghuensis and Ahmaditermes parafulvus.

Improvement in Microbiota Recovery Using Cas-9 Digestion of Mānuka Plastid and Mitochondrial DNA

Understanding host-microbe interactions in planta is an expanding area of research. Amplicon sequencing of the 16S rRNA gene is a powerful and common method to study bacterial communities associated with plants. However, the co-amplification of mitochondrial and plastid 16S rRNA genes by universal primers impairs the sensitivity and performance of 16S rRNA sequencing. In 2020, a new method, Cas-16S-seq, was reported in the literature to remove host contamination for profiling the microbiota in rice, a well-studied domestic plant, by engineering RNA-programmable Cas9 nuclease in 16S rRNA sequencing. For the first time, we tested the efficiency and applicability of the Cas-16S-seq method on foliage, flowers, and seed of a non-domesticated wild plant for which there is limited genomic information, Leptospermum scoparium (mānuka). Our study demonstrated the efficiency of the Cas-16S-seq method for L. scoparium in removing host contamination in V4-16S amplicons. An increase of 46% in bacterial sequences was found using six guide RNAs (gRNAs), three gRNAs targeting the mitochondrial sequence, and three gRNAs targeting the chloroplast sequence of L. scoparium in the same reaction. An increase of 72% in bacterial sequences was obtained by targeting the mitochondrial and chloroplast sequences of L. scoparium in the same sample at two different steps of the library preparation (DNA and 1st step PCR). The number of OTUs (operational taxonomic units) retrieved from soil samples was consistent when using the different methods (Cas-16S-seq and 16S-seq) indicating that the Cas-16S-seq implemented for L. scoparium did not introduce bias to microbiota profiling. Our findings provide a valuable tool for future studies investigating the bacterial microbiota of L. scoparium in addition to evaluating an important tool in the plant microbiota research on other non-domesticated wild species.

A molecular approach to identify parrotfish (Sparisoma) species during early ontogeny.

Sparisoma species (parrotfish) comprise an important functional group contributing to coral-reef resilience. The morphological diagnostic characteristics for species identification are clearly described for adult forms but not for the early stages. Consequently, many taxonomical listings of Sparisoma larvae are restricted to the genus level. The aims of this study are to determine whether the morphological and molecular identification techniques are useful to assign the species taxonomic level to Sparisoma larvae occurring in the Gulf of Mexico and whether there is a set of diagnostic features that could be used to discriminate between species in larvae of different developmental stages. Morphological assignment of Sparisoma was performed based on morphological and meristic features for 30 larvae collected in the Gulf of Mexico from late August to mid-September 2015. To corroborate and complement the morphological assignments, molecular identification was carried out using DNA sequences from regions of two mitochondrial genes, mitochondrial cytochrome oxidase I (mtDNA COI) and mitochondrial 16S rRNA (mtDNA 16S rRNA). COI and 16S gene trees for Sparisoma and related fish taxa were constructed using sequences available in the NCBI (National Center for Biotechnology Information) GenBank and BOLD (Barcode of Life Data) databases. Two morphotypes were identified based on morphology, but no diagnostic characteristics for species discrimination were found. Molecular identification, in contrast, successfully discriminated four early development stages of Sparisoma atomarium, three stages of Sparisoma radians, and two stages of Sparisoma chrysopterum and Sparisoma aurofrenatum, therefore demonstrating the successful and necessary application of molecular taxonomic approaches for species-level identifications of Sparisoma larvae.

Carcinoplaxmistio Ng & Mitra, 2019 (Crustacea, Decapoda, Goneplacidae): additional records and genetic differentiation of allied taxa.

The goneplacid crab, Carcinoplaxmistio Ng & Mitra, 2019, was originally described from West Bengal, India, in the northern Indian Ocean. Additional material of C.mistio from off Tamil Nadu in the southeast of India revealed a high degree of size-associated variation in the structures of the anterolateral tooth of the carapace, chelipeds, and male and female pleons. In addition to an in-depth morphological examination of C.mistio, this study also records the natural coloration of the species and conducts a genetic comparison (with mitochondrial COI and 16S rRNA genes) with its close relatives, C.haswelli (Miers, 1884) and C.purpurea Rathbun, 1914. Molecular comparison of C.mistio with its morphologically closest congener, C.haswelli from northern Australia and the western Pacific, corroborates their status as separate species. The genetic sequence of C.mistio, however, is similar to that of C.purpurea from the West Pacific, although these two species can easily be distinguished by distinct carapace and ambulatory leg characters. The present study provides some possible explanations for the genetic and morphological incongruence observed between C.mistio and C.purpurea and highlights the need for a detailed molecular study for Carcinoplax H. Milne Edwards, 1852, to appreciate the evolution of various morphological characters in the genus.

Fine-scale phylogeography of Tripneustes gratilla revealed a core-periphery pattern in the South China sea

The Core-Periphery Hypothesis (CPH) is a theoretical framework in phylogeography used to examine the spatial distribution of genetic diversity. The tropical Indian-Western Pacific (IWP), serving as a prominent marine biodiversity hotspot, constantly exports species and evolutionary novelties to peripheral habitats. Many species display genetic patterns consistent with CPH predictions from the IWP to periphery regions. However, the presence of CPH’s genetic signals in intermediate distribution zones remains underexplored. Here, we collected four sea urchin populations of Tripneustes gratilla in the South China Sea and analysed their population structure and evolutionary patterns using 13 morphological parameters, one mitochondrial 16S rRNA gene and nine polymorphic microsatellites. The results revealed substantial genetic homogeneity among the populations, implying a single origin from a glacial refugium. Nonetheless, morphological and microsatellite data distinctly differentiate the northernmost Fengjiawan population from the others, which could be attributed to increased environmental selection pressures such as temperature. Analysis of genetic diversity variation along latitudinal gradients and the relationship between geographic and genetic distances align with CPH expectations for both 16S and microsatellites, albeit to varying degrees of significance. This study enhances our understanding of tropical marine invertebrate evolution and supports the applicability of the CPH model at a fine-scale level.

A systematic survey of environmental DNA in Palau's lakes and waterfalls reveals an increase in Leptospira levels after flooding

ObjectiveLeptospirosis is an important bacterial zoonosis which is widespread in tropical and subtropical islands and influences human and animal health which has secondary economic effects. Although leptospirosis is endemic in Palau, an Oceanian Pacific Island country, few systematic surveys of potential risk factors for Leptospira infection, such as weather and host animals, have been conducted in the natural environment. We used environmental DNA metabarcoding to assess the distribution, species diversity, and abundance of pathogenic Leptospira in this endemic region to investigate the potential environmental risks. MethodsForty-two paired water samples, representing fine and rainy weather conditions, were collected from four representative waterfalls and lakes on Babeldaob Island, the largest island in Palau. High-throughput sequencing analysis was conducted for polymerase chain reaction products of leptospiral 16S rRNA and vertebrate animal mitochondrial 12S rRNA genes. ResultsWe revealed greater Leptospira diversity and abundance in samples collected after continuous rain, particularly in the presence of flooding, compared with samples collected under typhoon, monsoon, or fine weather conditions. From same samples, six mammalian species including cats (Felis catus), mice (Mus musculus), Yap flying fox (Pteropus yapensis), rats (Rattus spp.), and pigs (Sus scrofa) were repeatedly detected. These may be candidates of host animals of Leptospira in Palau; however, their detection was not clearly correlated with that of Leptospira. ConclusionWe repeatedly detected several species of pathogenic Leptospira from water samples of a wide region of Babeldaob Island. We confirmed that Leptospira contamination in freshwater environments increased under rainy conditions, particularly in the presence of flooding. This information could be used to improve public health control measures in this region.

Expanded range of Nasutitermes callimorphus Mathews, 1977 (Isoptera: Termitidae: Nasutitermitinae), comparison with N. corniger (Motschulsky, 1855) and N. ephratae (Holmgren, 1910), and synonymy of N. dasyopsis Thorne, 1989 into N. nigriceps (Haldeman, 1854)

The imago of N. callimorphus is described for the first time. Nasutitermes callimorphus occurs from Mexico to Paraguay. Nasutitermes callimorphus is smaller in all measurements but generally resembles its widely distributed sympatric congeners, N. corniger (Motschulsky, 1855) and N. ephratae (Holmgren, 1910). Molecular phylogenetic analysis of a portion of the mitochondrial 16S rRNA gene and the COII mtDNA marker revealed that N. callimorphus forms a distinct clade using both maximum parsimony and maximum likelihood analysis. Unlike N. corniger and N. ephratae, N. callimorphus does not build epigeal or arboreal carton nests. We further regard N. dasyopsis Thorne, 1989 as a junior synonym of N. nigriceps (Haldeman, 1854).

From ctenophores to scyphozoans: parasitic spillover of a burrowing sea anemone

Most host-parasite associations are explained by phylogenetically conservative capabilities for host utilization, and therefore parasite switches between distantly related hosts are rare. Here we report the first evidence of a parasitic spillover of the burrowing sea anemone Edwardsiella carnea from the invasive ctenophore Mnemiopsis leidyi to two scyphozoan hosts: the native Mediterranean barrel jellyfish Rhizostoma pulmo and the invasive Indo-Pacific nomad jellyfish Rhopilema nomadica, collected from the Eastern Mediterranean Sea. Edwardsiella carnea planulae found in these jellyfish were identified using molecular analyses of the mitochondrial 16S and nuclear 18S rRNA genes. Overall, 93 planulae were found on tentacles, oral arms, and inside of the gastrovascular canals of the scyphomedusae, whereas no infection was observed in co-occurring ctenophores. DNA metabarcoding approach indicated seasonal presence of Edwardsiella sp. in the Eastern Mediterranean mesozooplankton, coinciding with jellyfish blooms in the region. Our findings suggest a non-specific parasitic relationship between Edwardsiella carnea and various gelatinous hosts based on shared functionality rather than evolutionary history, potentially driven by shifts in host availability due to jellyfish blooms. This spillover raises questions about the ecological impacts of parasitism on native and invasive scyphozoan hosts and the potential role of Edwardsiella in controlling their populations.

Crossroad between the sylvatic and domestic cycles of Mesocestoides litteratus: mesocestodiasis in dogs adopted from shelters in Türkiye.

This study aimed to report the presence of Mesocestoides litteratus in dogs adopted from shelters in Türkiye. Gravid segments were examined microscopically in the faeces of dogs from different shelters located in Ankara and Kirikkale provinces in the central region of Türkiye. Then, genomic DNA obtained from these segments, a 446-bp fragment of the mitochondrial cytochrome C oxidase subunit 1 gene, and a 350-bp fragment of mitochondrial 12S rRNA were amplified and sequenced. BLASTn search was performed. During light microscopic examination, an egg-filled paruterine organ was observed in the middle part of the segment. Thin-shelled, oval, 35-µm-diameter parasite eggs containing an oncosphere with three pairs of hooklets were observed. The gravid segments were determined as Mesocestoides spp. based on the appearance of the typical paruterine organ. PCR results supported our diagnosis; moreover, according to the BLAST results, it was detected that the species infecting two dogs was 98.01-100% similar to M. litteratus. Praziquantel-containing medication was administered to the infected dogs at a dosage of 5mg/kg. Foxes act as the final host of M. litteratus and the parasite is prevalent in wildlife; however, these animals may disperse the parasite in urban life. Veterinarians need to be made more aware of this parasite, especially if the dogs are owned from shelters.

Generalist species exhibit more genetic structure in comparison to a habitat specialist: Evidence from a phylogeographic study of two freshwater crabs (Decapoda: Potamonautidae: Potamonautes), implications for habitat conservation.

During the present study, the phylogeography of the wetland specialist, freshwater crab, Potamonautes flavusjo, was investigated and compared to that of the ubiquitous, generalist, P. sidneyi, using DNA sequence data from the mitochondrial cytochrome oxidase subunit one (COI). We inferred the evolutionary history of each species and compared their population-level genetic structure by constructing haplotype networks and using an analysis of molecular variation. Additionally, we explored the evolutionary relationship between southern African lentic and lotic freshwater crab species by examining the usefulness of carapace attributes in relation to genetic indices and a species' assumed dispersal capacity. In the lentic species, P. flavusjo, a single interconnected haplocluster characterized by shared haplotypes was observed, suggesting marked maternal dispersal, a result corroborated by the low F ST values. In contrast, for the lotic species, P. sidneyi two distinct haploclusters and marked genetic differentiation was observed indicating the absence of maternal dispersal, a result corroborated by the high F ST values. The phylogenetic relationship in P. sidneyi was further investigated using a maximum likelihood and Bayesian inference analyses with the addition of sequence data from the mitochondrial 16S rRNA locus to estimate divergence times. Three species delimitation methods (ASAP, PTP, and bGMYC) were used to explore the presence of distinct lineages in P. sidneyi. The phylogenetic results indicated that within P. sidneyi two clades were present, while divergence time estimates suggest cladogenesis during the middle to late Pleistocene. The species delimitation methods used showed moderate congruence, however all oversplit the number of putative lineages. Our results indicated that P. sidneyi is a species complex comprised of two cryptic lineages, occurring in sympatry contemporarily possibly alluding to secondary contact. Carapace height was found to have no discernible influence on the genetic indices and presumed dispersal capabilities of mainland southern African freshwater crab species. The importance of our results are discussed in terms of conservation of freshwater habitats.

First Evidence of Rickettsia conorii Infection in Dogs in Northern Tunisia.

A cross-sectional study was carried out, between April 2021 and June 2022, to understand the role of dogs in the circulation of rickettsiosis in Tunisia. The presence of specific IgG antibodies against Rickettsia conorii was analyzed by indirect immunofluorescence test. By qPCR, blood and ticks were collected from 136 dogs examined at the Canine Department of National School for Veterinary Medicine of Tunisia. These dogs were also analyzed to detect Rickettsia DNA. The rate of Rickettsia seropositivity in 136 dogs was 55.14%. A total of 51 (53%) seropositive dogs showed clinical and biological signs such as fever and anorexia as well as thrombocytopenia and anemia. By qPCR, targeting the mitochondrial 16S rRNA gene, no Rickettsia DNA was detected in the blood. On the other hand, qPCR followed by sequencing revealed the presence of R. conorii subsp. raoultii in 7 tick pools of the 51 pools composed of the 227 ticks collected. A One Health approach to raise the awareness of dog owners to control tick infestations is imperative, given the dangers of canine zoonoses.

A new species of colorful Platypelis (Amphibia: Microhylidae) from the Tsaratanàna and Bemanevika massifs in northern Madagascar

Frogs of the genus Platypelis are known to have their center of species richness in the mountain massifs of northern Madagascar. We here formally describe a new species of Platypelis from this region. Platypelis saikamavo sp. nov. has been known for over 20 years but its taxonomic identity has only recently been robustly clarified as a result of ongoing taxonomic revisions of other Platypelis species. The new species is known from rainforest on the Tsaratanàna Massif and Bemanevika, at elevations of 1000–1530 m above sea level. It is characterized by an isolated phylogenetic position and is genetically highly divergent, with an uncorrected pairwise distance ≥4.5% in the mitochondrial 16S rRNA gene to all other nominal species of Platypelis. It has a unique combination of morphological and chromatic character states such as a relatively large body size of 28–31 mm in snout–vent length, distinct superciliary tubercles, and bright yellow color around the forelimb insertion and on the posterior abdomen where this color extends in the form of flash marks onto the inguinal region.

Identification of Fish Species Involved with Ciguatera Food Poisoning in Okinawan Waters by Using PCR-RFLP analysis

Ciguatera fish poisoning (CFP), known as a seafood-borne disease, is caused by consumption of fish contaminated with ciguatoxins in tropical and subtropical sea. The ciguatera fishes, Variola louti, Lutjanus monostigma and L. bohar have an absolute majority in the Ryukyu Archipelago, southwestern Japan. We developed the cluster analysis of phylogenetic tree by using mitochondrial (mt) DNA 16S rRNA sequences of V. louti, L. monostigma and L. bohar and differentiate them from morphologically similar species (L. fulviflamma, L. russellii, L. argentimaculatus, Plectropomus leopardus and V. albimarginata) in our previous study. The fish were acquired from the coastal waters of the Ryukyu Archipelago, and a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) marker of the mtDNA 16S rRNA region was used, employing the restriction enzymes BmgT120 I, Dde I, and SnaB I, to identify the fish species responsible for CFP. These results showed that a PCR-RFLP marker can be obtained more easily than a nucleotide sequence.

Further evidence for diversification within the Diogenes pugilator complex (Anomura, Diogenidae) in the Mediterranean and Black Seas

Abstract We document an unrecognised genetic lineage within the European hermit crab Diogenes pugilator species complex. Specifically, we propose the revalidation of the previously synonymised species D. ponticus, for specimens previously identified as D. pugilator from the Adriatic, Ionian, Aegean, and Black Seas. Analyses of the mitochondrial genes COI and 16S rRNA resulted in a clear genetic separation to East Atlantic and Western Mediterranean populations of Diogenes. Morphological analyses confirmed differences in the individuals assigned to D. ponticus compared to the congeners D. curvimanus and D. pugilator and thereby a concordance between molecular and morphological characters. Despite the relatively high morphological resemblance to D. armatus, the molecular differentiation and the remarkable geographic segregation within the species complex suggest at least one speciation or radiation event in the past, underlining the growing importance of molecular methods in the identification of species and evolutionarily significant units. Major oceanographic processes that are known to shape genetic variability and population genetic structure in the marine environment are pointed out as possible structuring factors for the observed intrageneric divergence. The evidence of long evolutionary independence is therefore considered sufficient to re-establish the validity of D. ponticus as a valid species in the northern-central and north-eastern Mediterranean Sea and the Black Sea and is redescribed accordingly.

Reassessment and phylogenetic position of the overlooked limacoid land snail Trochomorpha sculpticarina Martens, 1883 (Eupulmonata, Ariophantidae), with the description of a new genus

The Malay Peninsula has traditionally been considered to harbour a diverse land-snail fauna, both in terms of a high species richness and a wide variety of conchological traits, especially within the limacoid land snails. A recent survey along the Malay Peninsula of southern Thailand discovered an overlooked limacoid taxon “Trochomorpha” sculpticarina Martens, 1883, previously assigned to genus Trochomorpha of the Trochomorphidae. This genus is herein described as Janbinmorphagen. nov. based on comparative studies of shell morphology, external features, genital anatomy and radular morphology, as well as analyses of partial sequences of two mitochondrial markers, COI and 16S rRNA, and of one nuclear marker, 28S rRNA. This new genus is characterised by a combination of distinct morphological and anatomical features. The most distinguishing features are a depressed trochiform shell with a keeled last whorl, gametolytic organ without a duct, gametolytic sac with two lobes, and proximal epiphallus encircled with a thick sheath and attached by the penial retractor muscle. In addition, an analysis of the differentiation in mitochondrial and nuclear markers confirmed that this new genus, first recognised by morphology, is also genetically distinct. The molecular data also confirm that J. sculpticarinacomb. nov. is a member of the Ariophantidae and has a close evolutionary relationship to Hemiplecta and Maelamaodiscus.

Development and characterization of primary cell culture from the spinal cord of Asian seabass, Lates calcarifer.

Asian seabass, Lates calcarifer, is one of the most important fish species in aquaculture. An attempt was made to develop a primary cell culture from the spinal cord of Lates calcarifer by the enzymatic and mechanical dissociation method. The primary cell culture was sub-cultured for 20 times in Leibovitz's L-15 medium with 20% fetal bovine serum (FBS) and 0.5nM of human neurotrophin-3 at 28°C. The primary cell culture was cryopreserved at different passage levels and recovery of cells after long-term storage was estimated about 75-85%. The authenticity of origin of primary cell culture from L. calcarifer was confirmed by polymerase chain reaction assay using species-specific mitochondrial 12S rRNA primer. The primary cell culture was designated as seabass spinal cord cells (SBSC). The cells morphologically resembled the neurons due to their neural-like prolongations and star-like structure. Immunophenotypic analysis of the SBSC revealed that they are of neuronal origin. The SBSC were found to be highly susceptible to striped jack nervous necrosis virus (SJNNV) and infection in the cells was confirmed by RT-PCR. In conclusion, this is the first innovative euryhaline fish neuronal primary cell culture of L. calcarifer now available for neurophysiological and neurotoxicological studies.

Population structure and haplotype network analyses of Hyalomma anatolicum based on the large subunit ribosomal RNA (16S rRNA) gene.

Hyalomma anatolicum, an Anatolian hard tick is a well-recognized vector involved in the transmission of various pathogens to animals and humans. The present study elucidated the population structure and haplotype network of H. anatolicum based on the mitochondrial large subunit ribosomal RNA (16S rRNA) gene sequence. The population structure and haplotype network analysis of 75 sequences archived in the GenBank, including the 15 sequences generated herein, yielded 24 haplotypes. Haplotype 1 (Hap_1) was the predominant haplotype consisting of 45 sequences from India, China, Pakistan, Turkey, Egypt, Iraq, and Tajikistan. The complete haplotype network exhibited a stellate conformation, highlighting a recent population expansion. The overall dataset, together with the sequences corresponding to India, China, and Pakistan, showed a high haplotype (0.638 ± 0.065, 0.671 ± 0.103, 0.753 ± 0.099, and 0.854 ± 0.061, respectively) and low nucleotide (0.00407 ± 0.00090, 0.00525 ± 0.00196, 0.00680 ± 0.00233, and 0.00453 ± 0.00056, respectively) diversity, further emphasized a recent population expansion. The neutrality indices including Tajima's D, Fu and Li's D, and Fu and Li's F for the complete dataset (- 2.661, - 6.008, and - 5.649, respectively) as well as for the sequences from India (- 2.223, - 3.414, and - 3.567, respectively) were negative, suggesting deviation from neutrality and a recent population expansion. The present study provided novel insights into the population structure and haplotype networks of H. anatolicum based on the mitochondrial 16S rRNA gene, and the different tests inferred a low genetic differentiation and suggested a recent population expansion of this economically important tick species.

Molecular delimitation of cryptic Australian squid species of the genus Uroteuthis Rehder, 1945 (Cephalopoda: Loliginidae), provides a baseline of diversity to resolve classification challenges throughout the Indo-Pacific

ABSTRACT This study provides a comprehensive molecular phylogenetic analysis of Uroteuthis squid from the Indo-Pacific region. The main aim was to increase sample coverage from northern and eastern Australian waters to resolve the identity and distribution of Uroteuthis species taken by local fisheries. Two mitochondrial regions, cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA), were sequenced from 220 new specimens and analysed with a further 51 sequences from GenBank to create a combined phylogeny for the genus. Three nuclear regions, 18S ribosomal DNA (18S rDNA), 28S ribosomal DNA (28S rDNA) and rhodopsin, were also sequenced from representatives of each species. Based on the mitochondrial phylogeny plus distance and tree-based delimitation models, a COI species barcode gap of 4–5% is proposed for discriminating Uroteuthis species. Applying this gap partitioned many described species into species complexes; for example, U. duvaucelii, U. noctiluca and U. edulis resolved into 10 species. Although more conserved, mitochondrial 16S rRNA sequences differentiated all new species clades, whereas none of the nuclear markers resolved the closest species. Results confirm that neither U. chinensis nor U. edulis occurs in Australian waters. Five undescribed species are identified from northern and eastern Australia, of which four are consistent with earlier allozyme studies (and two align with existing DNA sequences). One is a new southeastern, deeper shelf species differentiated in this study, along with a sixth undescribed species from Indonesian waters. Results of the molecular analysis are now being used to inform complementary morphometric analyses for new species descriptions, and genetic stock structure assessments of these important fisheries resources.

Lebbeus dentatus n. sp. (Decapoda, Thoridae) described from a seamount of the Caroline Plate, NW Pacific

Based on the female specimen collected from an unnamed seamount on the Caroline Plate in the NW Pacific, a new species of the thorid genus Lebbeus White, 1847, L. dentatus n. sp., is described and illustrated. The new species belongs to a species group characterized by the possession of epipods on the pereopods 1 and 2, but is characteristic in the carapace having a strong supraorbital tooth extending to 0.3 length of the rostrum and largely unarmed dorsal margin of the rostrum. Integrative approach using morphological comparison and genetic analyses with the mitochondrial COI and 16S rRNA genes is taken to clarify the specific identities of the new taxon. An identification key to species belonging to the species group of Lebbeus characterized by the possession of epipods on the pereopods 1 and 2 is presented.