Isolation, molecular identification, and genomic analysis of Mangrovibacter phragmitis strain ASIOC01 from activated sludge harboring the bioremediation prowess of glycerol and organic pollutants in high-salinity.

The physiological and genotypic characteristics of Mangrovibacter (MGB) remain largely unexplored, including their distribution and abundance within ecosystems. M. phragmitis (MPH) ASIOC01 was successfully isolated from activated sludge (AS), which was pre-enriched by adding 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol as carbon sources. The new isolate, MPH ASIOC01, exhibited resilience in a medium containing sodium chloride concentration up to 11% (with optimal growth observed at 3%) and effectively utilizing glycerol as their sole carbon source. However, species delimitation of MGBs remains challenging due to high 16S rRNA sequence similarity (greater than 99% ANI) among different MGBs. In contrast, among the housekeeping gene discrepancies, the tryptophan synthase beta chain gene can serve as a robust marker for fast species delimitation among MGBs. Furthermore, the complete genome of MPH ASIOC01 was fully sequenced and circlized as a single contig using the PacBio HiFi sequencing method. Comparative genomics revealed genes potentially associated with various phenotypic features of MGBs, such as nitrogen-fixing, phosphate-solubilizing, cellulose-digesting, Cr-reducing, and salt tolerance. Computational analysis suggested that MPH ASIOC01 may have undergone horizontal gene transfer events, possibly contributing unique traits such as antibiotic resistance. Finally, our findings also disclosed that the introduction of MPH ASIOC01 into AS can assist in the remediation of wastewater chemical oxygen demand, which was evaluated using gas chromatograph-mass spectrometry. To the best of our knowledge, this study offers the most comprehensive understanding of the phenotypic and genotypic features of MGBs to date.

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.

Diversity of Orbiniella (Orbiniidae, Annelida) in the North Atlantic and the Arctic.

In this work, the diversity of the genus Orbiniella in the Nordic Seas and the North Atlantic waters south of Iceland is studied based on the analyses of molecular markers (mitochondrial COI, 16S rDNA and nuclear ITS2) and morphological characters. Our results showed the presence of at least five genetic lineages in the studied material which could also be morphologically identified by their segmental annulation patterns, the number and the shape of acicular spines, and the length and the shape of pygidial lobes. The species name Orbiniellapetersenae is assigned to one of the lineages restricting its geographical and vertical distribution to the deep-sea areas north of Iceland and Jan Mayen, and three lineages are described as new species (i.e., Orbiniellagriegi Meca & Budaeva, sp. nov., Orbiniellamayhemi Meca & Budaeva, sp. nov., and Orbiniellaparapari Meca & Budaeva, sp. nov.) elevating the number of known species in the genus to 25. Three deep-sea species of Orbiniella in our study are reported only north of the Greenland-Iceland-Scotland Ridge, one deep-sea species found south of the ridge. A single shallow-water species is distributed along the ridge and on the Norwegian shelf.

Molecular diversity of Diplura in southern High Appalachian leaf litter.

The fauna of Diplura, the two-pronged bristletails (Hexapoda), of the southern Appalachians has received little focused systematic attention. Existing literature suggests the fauna to comprise around a dozen species. Based on a broader DNA barcode-based survey of high elevation litter arthropods in the region, we suggest the fauna to be much richer, with automated species delimitation methods hypothesising as many as 35 species, most highly restricted to single or closely proximate localities. Such a result should not be very surprising for such small, flightless arthropods, although it remains to be seen if other markers or morphology support such high diversity. The region still remains sparsely sampled for these more cryptic elements of the arthropod fauna and much larger numbers of species undoubtedly remain to be discovered.

New evidence on the identity of the European Helorus species (Hymenoptera, Proctotrupoidea, Heloridae).

Background: Species of Helorus Latreille 1802 are rarely collected endoparasitoids of Chrysopidae larvae (Neuroptera). Previous work on the limits between the European species of this species-poor genus, based on morphology only, has left some uncertainties. Here, we approach these cases and revisit previous taxonomic decisions using freshly collected and museum material. New information: We generated the first large-scale Heloridae DNA barcode dataset, combined these with morphological data in an integrative taxonomic approach, and added information from studying all relevant type material. We found five species, Helorusanomalipes (Panzer, 1798), H.coruscus Haliday, 1857 stat. rev., H.nigripes Förster, 1856, H.ruficornis Förster, 1856, and H.striolatus Cameron, 1906, for which we provide an updated identification key. DNA barcode data are added to publicly available DNA barcode reference databases, for all species, except H.nigripes.

Integrative taxonomy, phylogenetics and historical biogeography of subgenus Aeschyntelus Stål, 1872 (Hemiptera: Heteroptera: Rhopalidae).

The subgenus Aeschyntelus includes six species that show variations in body color and shape, thus making it difficult to identify them based on morphological identification alone. To date, no genetic study has evaluated species within this genus. Herein, we collected 171 individuals from 90 localities of Rhopalus and employed an integrative taxonomic approach that incorporated morphological data, mitochondrial genomic data (COI, whole mitochondrial data) and nuclear genomic data (18S + 28S rRNAs, nuclear genome-wide SNPs) to delineate species boundaries. Our analyses confirmed the status of nine described species of Rhopalus and proposed the recognition of one new species known as Rhopalus qinlinganus sp. nov., which is classified within the subgenus Aeschyntelus. Discrepancies arising from nuclear and mitochondrial data suggest the presence of mito-nuclear discordance. Specifically, mitochondrial data indicated admixture within Clade A, comprising R. kerzhneri and R. latus, whereas genome-wide SNPs unambiguously identified two separate species, aligning with morphological classification. Conversely, mitochondrial data clearly distinguished Clade B- consisting of R. sapporensis into two lineages, whereas genome-wide SNPs unequivocally identified a single species. Our study also provides insights into the evolutionary history of Aeschyntelus, thus indicating that it likely originated in East Asia during the middle Miocene. The development of Aeschyntelus biodiversity in the southwestern mountains of China occurred via an uplift-driven diversification process. Our findings highlight the necessity of integrating both morphological and multiple molecular datasets for precise species identification, particularly when delineating closely related species. Additionally, it reveals the important role of mountain orogenesis on speciation within the southwestern mountains of China.

Diversity of Rhizophydiales (Chytridiomycota) in Thailand: unveiling the hidden gems of the Kingdom.

Chytrids, often overshadowed by their other fungal counterparts, take center stage as we unravel the mysteries surrounding new species within Rhizophydiales and explore their unique characteristics. In the broader spectrum of chytrids, their significance lies not only in their roles as decomposers but also as key players in nutrient cycling within aquatic ecosystems as parasites and saprobes. Baited soil and aquatic samples collected from various provinces of Thailand, yielded new species of the Rhizophydiales (Chytridiomycota), some of which expanded previously single species genera. Our investigation incorporated a combination of morphological and phylogenetic approaches, enabling us to identify these isolates as distinct taxa. The novel isolates possess distinguishing features, such as variations in size and shape of the sporangium and zoospores, that somewhat differentiate them from described taxa. To confirm the novelty of the species, we employed robust phylogenetic analyses using maximum likelihood and bayesian methods. The results provided strong support for the presence of eight distinct lineages within the Rhizophydiales, representing our newly discovered species. Furthermore, we employed Poisson Tree Processes to infer putative species boundaries and supplement evidence for the establishment of our new Rhizophydiales species. By meticulously exploring their morphological characteristics and genetic makeup, we expand the known catalogue of fungal diversity by describing Alphamyces thailandicus, Angulomyces ubonensis, Gorgonomyces aquaticus, G. chiangraiensis, G. limnicus, Pateramyces pingflumenensis, Terramyces aquatica, and T. flumenensis and also provide valuable insights into the intricacies of this order. This newfound knowledge not only enriches our understanding of Rhizophydiales but also contributes significantly to the broader field of mycology, addressing a critical gap in the documentation of fungal species. The identification and characterization of these eight novel species mark a noteworthy stride towards a more comprehensive comprehension of fungal ecosystems and their vital role.

One Step beyond Species Description: Unveiling a Fine-Scale Diversity within the Genus Dzhanokmenia Kostjukov (Hymenoptera: Eulophidae).

Although Chalcidoidea is one of the megadiverse superfamilies in Hymenoptera, numerous species are still being discovered and described. However, the difficulties in delimiting intra- and interspecific variation hinder this process. In this study, DNA barcoding methods using the COI gene were employed to investigate the morphological variation within Dzhanokmenia Kostjukov, 1977. The nuclear locus, 28S D2, was used to infer a phylogeny to gain an understanding of the relationship of Dzhanokmenia with other potentially close genera. Through a preliminary DNA barcode library established here, including eight species, we calibrated the intraspecific variation in certain diagnostic characters for the new species described here, D. brevifunis Ganbaatar & Cao sp. nov. Maximum likelihood results show that Dzhanokmenia is clustered with the genera associated with Tetrastichus, such as Chaenotetrastichus Graham, 1987, Baryscapus Förster, 1856, Tetrastichus Haliday, 1844, and Oomyzus Rondani, 1870 involved in this study. Our results indicate that the species diversity of Dzhanokmenia is understudied and tentatively confirm that Dzhanokmenia has a potential close relationship with Baryscapus. Along with the DNA barcode library, the referenced phylogeny datasets improve the understanding of the systematic position of Dzhanokmenia within the subfamily Tetrastichinae and the definition of this genus in terms of morphology, thereby facilitating species delimitation, discovery, and description within Dzhanokmenia.

The expansion of invasive species to the East: new sites of the bullheads (genus Ameiurus Rafinesque 1820) in Ukraine with morphological and genetic identification.

This study confirms the extended distribution of two invasive species of the genus Ameiurus in Ukraine. Specifically, A. melas is recorded for the first time in the Southern Buh basin and A. nebulosus has expanded further eastward within the Dnipro basin. Material collected in 2019 and 2022 was identified by morphological features and confirmed by molecular genetic analysis. The most reliable morphological characters for distinguishing these two species include anal-fin membrane pigmentation (light or black), gill raker count (fewer or more than 16), and serrations on the pectoral-fin spine (well-developed along the full length or small, absent near the tip). The analysis of the cytochrome oxidase subunit I barcoding marker identified all samples from the Dnipro Basin (Tnia and Velykyi Luh localities) as A. nebulosus, while all specimens from the Vinnytsia region within the Southern Buh basin (Sotskoho and Vyshenske lakes) were attributed to A. melas. The maximum-likelihood analysis revealed clearly separated clades with high bootstrap support (>75%), strongly supporting the presence of the two separate species. This study suggests the potential for further eastward expansion of both species within Ukraine: A. nebulosus in the northern direction and A. melas in the southern direction.

Exploring Barbronia species diversity and phylogenetic relationship within Suborder Erpobdelliformes (Clitellata: Annelida).

Background: Barbronia, a genus of freshwater macrophagous leeches, belongs to Erpobdelliformes (Salifidae: Clitellata: Annelida), and B. weberi, a well-known leech within this genus, has a worldwide distribution. However, the systematics of Barbronia have not yet been adequately investigated, primarily due to a few molecular markers, and only 20 Barbronia sequences available in the GenBank database. This gap significantly limits our understanding of the Barbronia species identification, as well as the phylogenetic placement of the genus Barbronia within Salifidae. Methods: Next-generation sequencing (NGS) was used to simultaneously capture the entire mitochondrial genome and the full-length 18S/28S rDNA sequences. The species boundary of Barbronia species was estimated using bGMYC and bPTP methods, based on all available Barbronia COI sequences. Uncorrected COI p-distance was calculated in MEGA. A molecular data matrix consisting of four loci (COI, 12S, 18S, and 28S rDNA) for outgroups (three Haemopis leeches) and 49 erpobdellid leeches, representing eight genera within the Suborder Erpobdelliformes was aligned using MAFFT and LocARNA. This matrix was used to reconstruct the phylogenetic relationship of Barbronia via Bayesian inference (BI) and the maximum likelihood (ML) method. Results: The full lengths of the mitochondrial genome, 18S and 28S rDNAs of B. cf. gwalagwalensis, are 14847 bp, 1876 bp 1876 bp, and 2863 bp, respectively. Both bGMYC and bPTP results based on COI data are generally congruent, suggesting that the previously proposed taxa (B. arcana, B. weberi formosana, and B. wuttkei or Erpobdella wuttkei) are synonyms of B. weberi. The specimens listed in the B. gwalagwalensis group, however, are split into at least two Primary Species Hypotheses (PSHs). The p-distance of the first PSH is less than 1.3% but increased to 4.5% when including the secondary PSH (i.e., B. cf. gwalagwalensis). In comparison, the interspecific p-distance between the B. weberi group and the B. gwalagwalensis group ranged from 6.4% to 8.7%, and the intraspecific p-distance within the B. weberi group is less than 0.8%. Considering the species delimitation results and the sufficient large p-distance, the specimen sampled in China is treated as B. cf. gwalagwalensis. The monophyly of the four Erpobdelliformes families Salifidae, Orobdellidae, Gastrostomobdellidae sensu stricto and Erpobdellidae is well supported in ML and BI analysis based on a data of four markers. Within the Salifidae, a well-supported Barbronia is closely related to a clade containing Odontobdella and Mimobdella, and these three genera are sister to a clade consisted of Salifa and Linta. According to the results of this study, the strategy of simultaneous obtaining both whole mitochondria and nuclear markers from extensively sampled Salifids species using NGS is expected to fathom both the species diversity of B. gwalagwalensis and the evolutionary relationship of Salifidae.

Species delimitation 4.0: integrative taxonomy meets artificial intelligence.

Although species are central units for biological research, recent findings in genomics are raising awareness that what we call species can be ill-founded entities due to solely morphology-based, regional species descriptions. This particularly applies to groups characterized by intricate evolutionary processes such as hybridization, polyploidy, or asexuality. Here, challenges of current integrative taxonomy (genetics/genomics + morphology + ecology, etc.) become apparent: different favored species concepts, lack of universal characters/markers, missing appropriate analytical tools for intricate evolutionary processes, and highly subjective ranking and fusion of datasets. Now, integrative taxonomy combined with artificial intelligence under a unified species concept can enable automated feature learning and data integration, and thus reduce subjectivity in species delimitation. This approach will likely accelerate revising and unraveling eukaryotic biodiversity.

Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield.

The nine-banded armadillo (Dasypus novemcinctus) is the most widespread xenarthran species across the Americas. Recent studies have suggested it is composed of four morphologically and genetically distinct lineages of uncertain taxonomic status. To address this issue, we used a museomic approach to sequence 80 complete mitogenomes and capture 997 nuclear loci for 71 Dasypus individuals sampled across the entire distribution. We carefully cleaned up potential genotyping errors and cross contaminations that could blur species boundaries by mimicking gene flow. Our results unambiguously support four distinct lineages within the D. novemcinctus complex. We found cases of mito-nuclear phylogenetic discordance but only limited contemporary gene flow confined to the margins of the lineage distributions. All available evidence including the restricted gene flow, phylogenetic reconstructions based on both mitogenomes and nuclear loci, and phylogenetic delimitation methods consistently supported the four lineages within D. novemcinctus as four distinct species. Comparable genetic differentiation values to other recognized Dasypus species further reinforced their status as valid species. Considering congruent morphological results from previous studies, we provide an integrative taxonomic view to recognise four species within the D. novemcinctus complex: D. novemcinctus, D. fenestratus, D. mexicanus, and D. guianensis sp. nov., a new species endemic of the Guiana Shield that we describe here. The two available individuals of D. mazzai and D. sabanicola were consistently nested within D. novemcinctus lineage and their status remains to be assessed. The present work offers a case study illustrating the power of museomics to reveal cryptic species diversity within a widely distributed and emblematic species of mammals.

Integrative taxonomy reveals a new species of the glass knifefish genus Eigenmannia Jordan & Evermann, 1896 (Teleostei: Gymnotiformes: Sternopygidae) from the Rio Branco basin, Brazil.

A new species of Eigenmannia is described from the Rio Branco basin, Roraima, Brazil, based on morphological and molecular datasets. It is distinguished from all congeners by the following combination of characters: lateral line stripe extending from first perforated lateral line scale to distal portion of caudal filament, presence of superior midlateral stripe with origin posterior to end of body cavity anal-fin hyaline, caudal filament corresponding to 15.2%-43.1% LEA, subterminal mouth, ii,14-16 pectoral-fin rays, 166-219 anal-fin rays, 10-13 scale rows above lateral line at vertical through posterior tip of pectoral fin, 100-128 scales on lateral line, 22-28 premaxillary teeth, 19-23 dentary teeth, 7-10 endopterygoid teeth, depth of posterodorsal expansion on infraorbitals 1 + 2 half as long as infraorbitals 1 + 2 length, basibranchial 1 unossified, 13 precaudal vertebrae, and length of coronomeckelian bone corresponding to 20% of Meckel's cartilage length. The new species has significant genetic divergence from species with accessible DNA sequences in public repositories, ranging from 10.8% to 17.7%. An osteological description of the new species, a review of Eigenmannia cytochrome c oxidase subunit I (COI) sequences available in public repositories based on voucher examination, and a hypothesis of phylogenetic relationships for the new species based on COI are provided. The critical importance of including voucher examination as one of the steps in the pipeline for using DNA sequences present in public repositories in taxonomic and phylogenetic studies is discussed.

Morphological and Molecular Analysis Identified a Subspecies of Crassostrea ariakensis (Fujita, 1913) along the Coast of Asia.

Crassostrea ariakensis (Fujita, 1913) is one of the most important economic and ecological oysters that is naturally distributed along the coast of Asia, separated by the Yangtze River estuary. They are usually compared as different populations, while there is no consensus on whether C. ariakensis in northern and southern areas should be considered as two species or subspecies. Here, we analyzed morphological characteristics, COI, 16s rRNA, mitogenome sequences, and species delimitation analysis (ASAP and PTP) to resolve the intraspecific taxonomic status of the C. ariakensis. Phylogenetic and ASAP analysis highlight that C. ariakensis was divided into N-type and S-type. PTP was unable to differentiate between the two types of C. ariakensis. The divergence time of N-type and S-type C. ariakinsis is estimated to be 1.6 Mya, using the relaxed uncorrelated lognormal clock method. Additionally, significant morphological differences exist between the two groups in terms of the adductor muscle scar color. Despite these differences, the COI (0.6%) and 16S rRNA (0.6%) genetic distance differences between N-type and S-type C. ariakensis has not yet reached the interspecific level. These results suggest that N-type and S-type C. ariakensis should be treated as different subspecies and renamed as C. ariakensis ariakensis subsp. nov and C. ariakensis meridioyangtzensis subsp. nov.

Dragons in the tropics - Phylogeography and speciation in Diporiphora lizards and common geographic breaks in co-distributed taxa.

Co-distributed taxa can respond both similarly or differently to the same climatic and geological events, resulting in a range of phylogeographic patterns across the region. Using a nested approach on a taxonomically diverse yet morphologically conservative group of agamid lizards, we first aimed to evaluate more precisely the extent of phylogeographic structuring within the genus. Then, focusing on four lineages within the more widespread species, we assessed the impact of biogeographic barriers on phylogeographic structuring and demographic history of species, comparing to patterns previously observed in co-distributed taxa. These species occur in the Australian Monsoonal Tropics, a vast tropical savanna system with high richness and endemism associated with environmental heterogeneity and past climate fluctuations. The employment of genomic data helped to determine the relationships between specific taxa that were previously difficult to place. We found a local influence of biogeographic and climatic breaks on population dynamics, analogous to other species. We detected high levels of population structure in the West Kimberley and Arnhem Plateau, which are already known for high endemism. However, we also highlighted unique lineages in areas that have been overlooked until recently, in the South Kimberley and West Top End. Climatic and geographical features in the Arnhem Plateau act as a soft barrier between populations in the east and west regions of the Top End. These observations reflect patterns observed for other vertebrates across this rich biome, indicating how climatic variation, species' ecology, and landscape features interact to shape regional diversity and endemism.

Oxynoemacheilus fatmae, a new species from the Güzelhisar Stream in the Aegean Sea basin, Türkiye (Teleostei: Nemacheilidae).

Oxynoemacheilus fatmae, a new species, is found in the Güzelhisar Stream in the northern Aegean Sea basin. It is differentiated from all other species of Oxynoemacheilus in the northern Aegean Sea and adjacent basins by having four to eight irregularly shaped narrow black bars on the posterior part of flank, and anterior parts of the flank with a marbled pattern. O. fatmae is differentiated from the closest species Oxynoemacheilus theophilii by having 14 fixed diagnostic nucleotide substitution sites, and the pair-wise genetic distance is 2.22%. It further differs from O. theophilii by having a slenderer body (body at dorsal-fin origin: 15%-17% standard length [SL] vs. 17%-18%), a slenderer caudal peduncle (10%-12% SL vs. 12%-13%), a more forked caudal fin (length of middle caudal-fin lope: 16%-19% SL vs. 19%-23%), and the absence the dorsal and ventral adipose crests on the caudal peduncle behind the vertical of the posterior anal-fin base (vs. present). Three species delimitation tests (assemble species by automatic partitioning (ASAP), automatic barcode gap discovery (ABGD) and generalized mixed yule-coalescent (GMYC)) and phylogenetic analyses reinforce the validity of O. fatmae as a distinct species.

A review of Nearctic Lathrobium (Coleoptera, Staphylinidae), with revision and descriptions of new flightless species from the mountains of the southeastern U.S.

Species of the genus Lathrobium Gravenhorst (Coleoptera: Staphylinidae: Paederinae) from North America north of Mexico are reviewed and 41 species are recognized. Morphology and mitochondrial COI sequence data were used to guide species designations in three flightless lineages endemic to the southern Appalachian Mountains, a biologically diverse region known for cryptic diversity. Using a combination of phylogeny, algorithm-based species delimitation analyses, and genitalic morphology, five new cryptic species are described and possible biogeographic scenarios for their speciation hypothesized: L.balsamense Haberski & Caterino, sp. nov., L.camplyacra Haberski & Caterino, sp. nov., L.islae Haberski & Caterino, sp. nov., L.lividum Haberski & Caterino, sp. nov., L.smokiense Haberski & Caterino, sp. nov. Five additional species are described: L.absconditum Haberski & Caterino, sp. nov., L.hardeni Haberski & Caterino, sp. nov., L.lapidum Haberski & Caterino, sp. nov., L.solum Haberski & Caterino, sp. nov., and L.thompsonorum Haberski & Caterino, sp. nov. Two species are transferred from Lathrobium to Pseudolathra Casey: Pseudolathraparcum (LeConte, 1880), comb. nov. and Pseudolathratexana (Casey, 1905), comb. nov. Twenty-six names are reduced to synonymy. Lectotypes are designated for 47 species. Larvae are described where known, and characters of possible diagnostic value are summarized. Species diagnoses, distributions, illustrations of male and female genitalia, and a key to Lathrobium species known from the Nearctic region (including several introduced species) are provided.

A DNA barcode reference library of Croatian mosquitoes (Diptera: Culicidae): implications for identification and delimitation of species, with notes on the distribution of potential vector species.

BACKGROUND: Mosquitoes pose a risk to human health worldwide, and correct species identification and detection of cryptic species are the most important keys for surveillance and control of mosquito vectors. In addition to traditional identification based on morphology, DNA barcoding has recently been widely used as a complementary tool for reliable identification of mosquito species. The main objective of this study was to create a reference DNA barcode library for the Croatian mosquito fauna, which should contribute to more accurate and faster identification of species, including cryptic species, and recognition of relevant vector species. METHODS: Sampling was carried out in three biogeographical regions of Croatia over six years (2017-2022). The mosquitoes were morphologically identified; molecular identification was based on the standard barcoding region of the mitochondrial COI gene and the nuclear ITS2 region, the latter to identify species within the Anopheles maculipennis complex. The BIN-RESL algorithm assigned the COI sequences to the corresponding BINs (Barcode Index Number clusters) in BOLD, i.e. to putative MOTUs (Molecular Operational Taxonomic Units). The bPTP and ASAP species delimitation methods were applied to the genus datasets in order to verify/confirm the assignment of specimens to specific MOTUs. RESULTS: A total of 405 mosquito specimens belonging to six genera and 30 morphospecies were collected and processed. Species delimitation methods assigned the samples to 31 (BIN-RESL), 30 (bPTP) and 28 (ASAP) MOTUs, with most delimited MOTUs matching the morphological identification. Some species of the genera Culex, Aedes and Anopheles were assigned to the same MOTUs, especially species that are difficult to distinguish morphologically and/or represent species complexes. In total, COI barcode sequences for 34 mosquito species and ITS2 sequences for three species of the genus Anopheles were added to the mosquito sequence database for Croatia, including one individual from the Intrudens Group, which represents a new record for the Croatian mosquito fauna. CONCLUSION: We present the results of the first comprehensive study combining morphological and molecular identification of most mosquito species present in Croatia, including several invasive and vector species. With the exception of some closely related species, this study confirmed that DNA barcoding based on COI provides a reliable basis for the identification of mosquito species in Croatia.

Species boundaries of the whiptail catfish Rineloricaria (Siluriformes: Loricariidae) from the Paraíba do Sul River drainage, southeastern Brazil, with species redescriptions and description of a new species.

Species of the catfish genus Rineloricaria are common in the Paraíba do Sul River basin, in southeastern Brazil; here we present a revision of the taxonomic diversity and geographic distribution of the species of the genus inhabiting the basin, based on novel morphologic and molecular data. Five species delimitation methods based on cytochrome C oxidase subunit 1 nucleotide sequences yielded comparable molecular operational taxonomic units. The automatic barcode gap discovery, assemble species by automatic partitioning, barcode index number, and Bayesian implementation of the Poisson tree process methods supported the recognition of five evolutionary lineages. These taxonomic units were assigned to the previously described Rineloricaria nigricauda, Rineloricaria steindachneri, Rineloricaria zawadzkii, and Rineloricaria nudipectoris, and an additional undescribed species. R. zawadzkii was further divided into two intraspecific geographically structured lineages using the generalized mixed Yule coalescent delimitation method. A maximum likelihood phylogenetic analysis revealed that the five lineages from the Paraíba do Sul have closer relationships to different species from southern and southeastern Brazil (Ribeira de Iguape, Lagoa dos Patos, Uruguay, Paraguay, and Parana river basins) than to each other. Based on the analysis of lectotypes, recently collected material, and specimens from ichthyological collections, the poorly described R. nigricauda and R. steindachneri are redescribed following current descriptive standards. The undescribed species from the middle and upper Paraíba do Sul River basin is formally described. The description of a new species, along with the description of species boundaries in R. nigricauda and R. steindachneri, contributes to the knowledge of the ichthyofauna of the Paraíba do Sul River basin and adjacent coastal drainages of southeastern Brazil. An identification key for the species of Rineloricaria occurring in the Paraíba do Sul River basin is provided.

Refining taxonomic identification of microalgae through molecular and genetic evolution: a case study of Prorocentrum lima and Prorocentrum arenarium.

Species delimitation based on lineage definition has become increasingly popular. However, these methods have been limited, especially for species that lack genomic data and are morphologically similar. The trickiest part for the species identification is that the interspecific and intraspecific boundaries are vague. Taking Prorocentrum (Dinophyta) as an example, analysis of cell morphology, growth, and toxin synthesis in both species of P. lima and P. arenarium does not provide a reliable basis for species delineation. However, through phylogenetic and genetic distance analyses of their ITS and LSU sequences, establishment of evolutionary tree based on orthologous gene sequences, and combining the results of automatic barcode gap discovery and Poisson tree processes models, it was sustained that P. arenarium does not belong to the P. lima complex and should be considered as an independent species. Interspecies genetic evolution analysis revealed that P. lima and P. arenarium may contribute to evolutionary direction that favors combating reverse environmental factors. In P. lima, viral invasion may be one of the reasons for its large genome size. In the study, P. lima complex has been selected as an example to enhance the taxonomic identification of microalgae through molecular and genetic evolution, offering valuable insights into refining taxonomic identification and promoting microbial biodiversity research in other species.IMPORTANCEMicroalgae, especially the species known as Prorocentrum, have received significant attention due to their ability to trigger harmful algal blooms and produce toxins. However, the boundaries between species and within species are ambiguous. Clear and comprehensive species delineation indicates that Prorocentrum arenarium should be considered as an independent species, separate from the Prorocentrum lima complex. Improving the classification and identification of microalgae through molecular and genetic evolution will provide reference points for other cryptic species. Prorocentrum occupy multiple ecological niches in marine environments, and studying their evolutionary direction contributes to understanding their ecological adaptations and community succession.