Molecular Operational Taxonomic Units Research Articles

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

BackgroundMosquitoes 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.MethodsSampling 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.ResultsA 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.ConclusionWe 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.Graphical

Towards holistic insect monitoring: species discovery, description, identification and traits for all insects.

Holistic insect monitoring needs scalable techniques to overcome taxon biases, determine species abundances, and gather functional traits for all species. This requires that we address taxonomic impediments and the paucity of data on abundance, biomass and functional traits. We here outline how these data deficiencies could be addressed at scale. The workflow starts with large-scale barcoding (megabarcoding) of all specimens from mass samples obtained at biomonitoring sites. The barcodes are then used to group the specimens into molecular operational taxonomic units that are subsequently tested/validated as species with a second data source (e.g. morphology). New species are described using barcodes, images and short diagnoses, and abundance data are collected for both new and described species. The specimen images used for species discovery then become the raw material for training artificial intelligence identification algorithms and collecting trait data such as body size, biomass and feeding modes. Additional trait data can be obtained from vouchers by using genomic tools developed by molecular ecologists. Applying this pipeline to a few samples per site will lead to greatly improved insect monitoring regardless of whether the species composition of a sample is determined with images, metabarcoding or megabarcoding. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

The Tree of Life eDNA metabarcoding reveals a similar taxonomic richness but dissimilar evolutionary lineages between seaports and marine reserves.

Coastal areas host a major part of marine biodiversity but are seriously threatened by ever-increasing human pressures. Transforming natural coastlines into urban seascapes through habitat artificialization may result in loss of biodiversity and key ecosystem functions. Yet, the extent to which seaports differ from nearby natural habitats and marine reserves across the whole Tree of Life is still unknown. This study aimed to assess the level of α and β-diversity between seaports and reserves, and whether these biodiversity patterns are conserved across taxa and evolutionary lineages. For that, we used environmental DNA (eDNA) metabarcoding to survey six seaports on the French Mediterranean coast and four strictly no-take marine reserves nearby. By targeting four different groups-prokaryotes, eukaryotes, metazoans and fish-with appropriate markers, we provide a holistic view of biodiversity on contrasted habitats. In the absence of comprehensive reference databases, we used bioinformatic pipelines to gather similar sequences into molecular operational taxonomic units (MOTUs). In contrast to our expectations, we obtained no difference in MOTU richness (α-diversity) between habitats except for prokaryotes and threatened fishes with higher diversity in reserves than in seaports. However, we observed a marked dissimilarity (β-diversity) between seaports and reserves for all taxa. Surprisingly, this biodiversity signature of seaports was preserved across the Tree of Life, up to the order. This result reveals that seaports and nearby marine reserves share few taxa and evolutionary lineages along urbanized coasts and suggests major differences in terms of ecosystem functioning between both habitats.

A description of Aliichlorella ignota gen. et sp. nov. and a comparison of the efficiency of species delimitation methods in the Chlorella‐clade (Trebouxiophyceae, Chlorophyta)

AbstractThe present study reports a new representative of Chlorella‐clade that was newly isolated from the Lake Vos'merka (Samara region, Russian Federation). 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strain ACSSI 368 and related Chlorella chlorelloides, Chlorella pulchelloides, formed an separate cluster in the Chlorella‐clade, unrelated to the holotype of the genus Chlorella – Chlorella vulgaris. The independent place of the genus was also confirmed by genetic distances. The individual species status of the studied strain ACSSI 368, compared with the sister C. chlorelloides, was confirmed by morphological differences (cell number per colony, type of chloroplast, number of autospores), genetic distances of the interspecific level by 18S–ITS1–5.8S–ITS2 fragment, alone by internal transcribed spacer 1 (ITS1) and internal transcribed spacer 2 (ITS2) sequences, one complementary base change in ITS1, and the results of species delimitation using five methods. A description of studied strain ACSSI 368 was provided as an authentic strain of the type species Aliichlorella ignota. Currently, the new genus Aliichlorella also includes Aliichlorella chlorelloides comb. nov. and Aliichlorella pulchelloides comb. nov. In general, six different delimitation algorithms were used in the present study. The ASAP (i.e. assemble species by automatic partitioning) and LocMin (i.e. ‘local minimum’ function) algorithms showed synchronous results, but their accuracy with respect to closely related species was somewhat lower. The mlPTP algorithm simultaneously aimed to combine closely related species in one and at the same time divide slightly distinguishing strains within a species into several molecular operational taxonomic units. The bPTP algorithm separated excessively Chlorella‐clade representatives into individual species. The generalized mixed Yule coalescent (GMYC) algorithm results were consistent the most with the modern understanding of the Chlorella‐clade taxonomy. However, the same method is one of the most time‐consuming because, for its implementation, it is necessary to carry out long preparatory work. The KoT results were less accurate than the GMYC results, although this is less time‐consuming because it does not require the preliminary construction of ultrametric trees.

Multiple and frequent trypanosomatid co-infections of insects: the Cuban case study.

Trypanosomatids are obligate parasites of animals, predominantly insects and vertebrates, and flowering plants. Monoxenous species, representing the vast majority of trypanosomatid diversity, develop in a single host, whereas dixenous species cycle between two hosts, of which primarily insect serves as a vector. To explore in-depth the diversity of insect trypanosomatids including their co-infections, sequence profiling of their 18S rRNA gene was used for true bugs (Hemiptera; 18% infection rate) and flies (Diptera; 10%) in Cuba. Out of 48 species (molecular operational taxonomic units) belonging to the genera Vickermania (16 spp.), Blastocrithidia (7), Obscuromonas (4), Phytomonas (5), Leptomonas/Crithidia (5), Herpetomonas (5), Wallacemonas (2), Kentomonas (1), Angomonas (1) and two unnamed genera (1 + 1), 38 species have been encountered for the first time. The detected Wallacemonas and Angomonas species constitute the most basal lineages of their respective genera, while Vickermania emerged as the most diverse group. The finding of Leptomonas seymouri, which is known to rarely infect humans, confirms that Dysdercus bugs are its natural hosts. A clear association of Phytomonas with the heteropteran family Pentatomidae hints at its narrow host association with the insect rather than plant hosts. With a focus on multiple infections of a single fly host, using deep Nanopore sequencing of 18S rRNA, we have identified co-infections with up to 8 trypanosomatid species. The fly midgut was usually occupied by several Vickermania species, while Herpetomonas and/or Kentomonas species prevailed in the hindgut. Metabarcoding was instrumental for analysing extensive co-infections and also allowed the identification of trypanosomatid lineages and genera.

Two new records and description of a new Perinereis (Annelida, Nereididae) species for the Saudi Arabian Red Sea region.

Annelid biodiversity studies in the Red Sea are limited and integrative taxonomy is needed to accurately improve reference libraries in the region. As part of the bioblitz effort in Saudi Arabia to assess the invertebrate biodiversity in the northern Red Sea and Gulf of Aqaba, Perinereis specimens from intertidal marine and lagoon-like rocky environments were selected for an independent assessment, given the known taxonomic ambiguities in this genus. This study used an integrative approach, combining molecular with morphological and geographic data. Our results demonstrate that specimens found mainly in the Gulf of Aqaba are not only morphologically different from other five similar Perinereis Group I species reported in the region, but phylogenetic analysis using available COI sequences from GenBank revealed different molecular operational taxonomic units, suggesting an undescribed species, P.kaustianasp. nov. The new species is genetically close and shares a similar paragnath pattern to the Indo-Pacific distributed P.helleri, in particular in Area III and Areas VII-VIII. Therefore, we suggest it may belong to the same species complex. However, P.kaustianasp. nov. differs from the latter mainly in the shorter length of the postero-dorsal tentacular cirri, median parapodia with much longer dorsal Tentacular cirri, posteriormost parapodia with much wider and greatly expanded dorsal ligules. Additionally, two new records are reported for the Saudi Neom area belonging to P.damietta and P.suezensis, previously described only for the Egyptian coast (Suez Canal) and are distributed sympatrically with the new species, but apparently not sympatric with each other.

Morphological and molecular data combined reveal inter- and intraspecific cranial shape variations in bats of Artibeus Leach, 1821 (Chiroptera: Phyllostomidae)

Abstract Since morphological traits may overlap among species within a complex, the integration of morphological and mitochondrial data could provide crucial insights for distinguishing species, as observed in fruit-eating bats of the genus Artibeus. Therefore, the application of geometric morphometric (GM) techniques could yield more refined and robust analyses of inter- and intraspecific variations. In this study, we generated two datasets: one with molecular delimitation data based on the barcode region (COI) and the other with cranial size and shape data using GM methods. Our aims were to investigate variations between large and small species within the genus Artibeus, as well as to explore potential factors influencing such variations. The results from species delimitation revealed molecular operational taxonomic units (MOTUs) supporting the morphological identification. Analyses using GM techniques demonstrated significant inter- and intraspecific variations in cranial size and shape among Artibeus species. The combined outcomes suggest the absence of a significant phylogenetic signal influencing skull variation. This scenario indicates that potential historical ecological factors may have directly influenced the cranial morphology of these species, acting as significant selection forces in geographical space and generating intraspecific variations in Artibeus planirostris.

Spatial patterns in the diet of Gyps vultures in India and their implications for conservation

Dietary analyses are particularly useful for developing conservation programmes for species threatened by resource depletion, poisoning and environmental pollution. Gyps vultures in South Asia represent one such case, having undergone a population collapse caused by feeding on carcasses of cattle treated with the non-steroidal anti-inflammatory drug diclofenac. Following a ban on the veterinary use of diclofenac, populations of vultures remain low and mostly concentrated near protected areas. Understanding the role of protected areas in the recovery of these critically endangered species requires analyses of spatial variation in their diet. We used faecal metabarcoding to investigate the spatial variation in the diet of Gyps vultures across four landscapes from sites located inside and outside protected areas. We collected faecal 642 samples, of which 419 yielded adequate molecular data to identify the vulture species and 30 molecular operational taxonomic units corresponding to at least 28 diet species. The diet was dominated by large ungulates and varied across landscapes, protection status and vulture species, with the observed variation largely explained by differential intake of wild and domestic species. Domestic livestock was present in >95 % of the samples from Central, West and North India, but 77 % of the samples had only wild species in South India. This variation was explained by livestock density within 100 km radius of the sampling site. Our results imply that protected areas may not offer a respite from possibility of diclofenac-poisoning across most parts of the country and efforts should continue unabated to remove nephrotoxic drugs from veterinary use.

Species delimitation of tea plants (Camellia sect. Thea) based on super-barcodes

BackgroundThe era of high throughput sequencing offers new paths to identifying species boundaries that are complementary to traditional morphology-based delimitations. De novo species delimitation using traditional or DNA super-barcodes serve as efficient approaches to recognizing putative species (molecular operational taxonomic units, MOTUs). Tea plants (Camellia sect. Thea) form a group of morphologically similar species with significant economic value, providing the raw material for tea, which is the most popular nonalcoholic caffeine-containing beverage in the world. Taxonomic challenges have arisen from vague species boundaries in this group.ResultsBased on the most comprehensive sampling of C. sect. Thea by far (165 individuals of 39 morphospecies), we applied three de novo species delimitation methods (ASAP, PTP, and mPTP) using plastome data to provide an independent evaluation of morphology-based species boundaries in tea plants. Comparing MOTU partitions with morphospecies, we particularly tested the congruence of MOTUs resulting from different methods. We recognized 28 consensus MOTUs within C. sect. Thea, while tentatively suggesting that 11 morphospecies be discarded. Ten of the 28 consensus MOTUs were uncovered as morphospecies complexes in need of further study integrating other evidence. Our results also showed a strong imbalance among the analyzed MOTUs in terms of the number of molecular diagnostic characters.ConclusionThis study serves as a solid step forward for recognizing the underlying species boundaries of tea plants, providing a needed evidence-based framework for the utilization and conservation of this economically important plant group.

A DNA barcode library of Austrian geometridae (Lepidoptera) reveals high potential for DNA-based species identification.

Situated in the Eastern section of the European Alps, Austria encompasses a great diversity of different habitat types, ranging from alpine to lowland Pannonian ecosystems, and a correspondingly high level of species diversity, some of which has been addressed in various DNA barcoding projects. Here, we report a DNA barcode library of all the 476 species of Geometridae (Lepidoptera) that have been recorded in Austria. As far as possible, species were sampled from different Austrian regions in order to capture intraspecific genetic variation. In total, 2500 DNA barcode sequences, representing 438 species, were generated in this study. For complete coverage of Austrian geometrid species in the subsequent analyses, the dataset was supplemented with DNA barcodes from specimens of non-Austrian origin. Species delimitations by ASAP, BIN and bPTP methods yielded 465, 510 and 948 molecular operational taxonomic units, respectively. Congruency of BIN and ASAP partitions with morphospecies assignments was reasonably high (85% of morphospecies in unique partitions), whereas bPTP appeared to overestimate the number of taxonomic units. The study furthermore identified taxonomically relevant cases of morphospecies splitting and sharing in the molecular partitions. We conclude that DNA barcoding and sequence analysis revealed a high potential for accurate DNA-based identification of the Austrian Geometridae species. Additionally, the study provides an updated checklist of the geometrid moths of Austria.

Environmental DNA time series analysis of a temperate stream reveals distinct seasonal community and functional shifts

AbstractEnvironmental DNA (eDNA) extracted from water is routinely used in river biodiversity research, and via metabarcoding eDNA can provide comprehensive taxa lists with little effort and cost. However, eDNA‐based species detection in streams and rivers may be influenced by sampling season and other key factors such as water temperature and discharge. Research linking these factors and also informing on the potential of eDNA metabarcoding to detect shifts in ecological signatures, such as species phenology and functional feeding groups across seasons, is missing. To address this gap, we collected water samples every 2 weeks for 15 months at a long‐term ecological research (LTER) site and at three different positions in the river's cross section, specifically the water surface, riverbed, and riverbank. For these 102 samples, we analyzed macroinvertebrate species and molecular operational taxonomic unit (OTU) richness and temporal community turnover across seasons based on cytochrome c oxidase subunit I (COI) metabarcoding data. Using Generalized Additive Models, we found a significant influence of sampling season on species richness. Community turnover followed a cyclic pattern, reflecting the continuous change of the macroinvertebrate community throughout the year (“seasonal clock”). Although water temperature had no influence on the inferred species richness, higher discharge reduced the number of Annelida and Ephemeroptera species detectable with eDNA. Most macroinvertebrate taxa showed the highest species richness in spring, in particular merolimnic species with univoltine life cycles. Further, we detected an increase in the proportion of shredders in winter and parasites in summer. Our results show the usefulness of highly resolved eDNA metabarcoding time series data for ecological research and biodiversity monitoring in streams and rivers.

Exploring the biological diversity and source species of medicinal horseflies through metabarcoding

Horseflies from the Tabanidae family play a significant role in traditional Chinese medicine to treat various health conditions, including coronary heart disease, stroke, headaches, liver cirrhosis, psoriasis, and hepatic carcinoma. There are 27 species of Tabaninae (Tabanidae) used as medicine, and they showed high morphological similarities with those for which medicinal properties have not been reported. Nonetheless, there have been reports suggesting that medicinal crude drugs sometimes contain irrelevant or false species, impacting the drug's efficacy. In this current study, we collected 14 batches, totaling 13,528 individuals, from various provinces in China. Instead of “classic” DNA barcoding strategy, we employed a high-throughput metabarcoding approach to assess the biological composition of crude drug mixtures derived from horseflies. Our analysis identified 40 Amplicon Sequence Variants (ASVs) with similarity percentages ranging from 92% to 100% with 12 previously reported species. Species delimitation methods revealed the presence of 11 Molecular Operational Taxonomic Units (MOTUs), with ten belonging to the Tabanus genus and one to Hybomitra. Tabanus sp6 displayed the highest relative abundance, and its ASVs showed close resemblance to Tabanus pleski. Our investigations revealed that the medicinal batches were biologically composed of 6 to 12 species. Some batches contained ASVs that closely resembled species previously associated with false Tabanus species. In conclusion, our findings offer valuable insights into the biological composition of crude drugs derived from horseflies and have the potential to enhance the quality of these traditional medicines.

Molecular characterisation of Amblyomma integrum circulating in southern India

Amblyomma integrum is a large gooseberry sized longirostrate tick (when fully repleted) found in India and Sri Lanka. In Kerala (India), this tick is commonly found in the forest and its fringe areas frequently infesting deer and hence it is locally known as “maan chellu / maanunny” (deer tick). In the present study, molecular characterisation and phylogenetic analysis of A. integrum collected from the area grazed by the sambar deer (Rusa unicolor) of Kerala, south India was performed using three molecular markers viz., the mitochondrial cytochrome c oxidase subunit 1 (COI), mitochondrial 16S ribosomal RNA, and nuclear 18S ribosomal RNA genes. Cytochrome c oxidase subunit 1 (COI) gene showed better resolving ability for elucidating the evolutionary relationship of A. integrum and identified two distinct clades, viz., A and B. The Tamil Nadu isolates of south India and Marayoor isolate 1 (from Idukki district of Kerala bordering with Tamil Nadu) belonged to clade A. Majority of Wayanad isolates from Kerala, occupied clade B. The intraspecific genetic distance among the A. integrum species ranged from 0.00 to 13.34%. Between clades A and B, the genetic distance observed was 11.49%. The clade B isolates were genetically close to A. geoemydae (GD: 1.22%). Morphological variations between the clades included darker exoskeletal coloration in clade A and distinct differences in the shape of basis capitulum. Further analysis using Assemble Species by Automatic Partitioning (ASAP) and Generalized Mixed Yule Coalescent (GMYC) provided additional insights. Assemble Species by Automatic Partitioning (ASAP) identified 26 Molecular Operational Taxonomic Units (MOTUs) at a threshold distance of 5.38%, supporting the species partition of A. integrum clade B. Generalized Mixed Yule Coalescent (GMYC) analysis retained the same species complex (A. integrum-geoemydae Complex) inferred from the ASAP analyses. It could be inferred from the present study that the A. integrum clades A and B could be two different putative pseudocryptic species.

Discovery of a new species of Synergus (Hymenoptera, Cynipidae, Synergini) based on morphology and molecular data.

A new species of gall inquiline, Synergusdilatatussp. nov., is described from Hubei Province, China. Morphological descriptions, photographs and biological information are provided. Mitochondrial cytochrome oxidase (COI) sequences of the new species were newly obtained and a molecular species delimitation analysis of 12 species of Synergus performed using the ASAP method recovered 16 molecular operational taxonomic units, providing support for recognition of the new species. The results also highlight a few conflicts between morphological and molecular species delimitations in Synergus.

Ciliate diversity in rodrigo de freitas lagoon (Rio de Janeiro, Brazil) from an integrative standpoint.

The Rodrigo de Freitas Lagoon is a highly eutrophic lacustrine system and has one of the longest histories of exploration and anthropic alteration in Brazil. Despite its relevance, limited studies explored the diversity of micro-eukaryotes in the lagoon. Ciliates (Alveolata, Ciliophora) are overlooked in environmental microbiology, especially in tropical and subtropical ecosystems, resulting in limited knowledge about their diversity and functional relevance in South American habitats, particularly in coastal lagoons. To fill this gap, here we investigated the diversity of ciliates in a brackish coastal lagoon in an urban area of Rio de Janeiro, Brazil, applying and comparing the performance of morphological and metabarcoding approaches. The metabarcoding analysis, based on high-throughput sequencing of the hipervariable region V4 of the 18S rRNA genes detected 37 molecular operational taxonomic units (MOTUs) assigned to Ciliophora, representing only about a half (56.9%) of the diversity detected by microscopy, which counted 65 ciliate morphotypes. The most representative classes in both approaches were Spirotrichea and Oligohymenophorea. The metabarcoding analysis revealed that 35.3% of the ciliate MOTUs had less than 97% similarity to available sequences in the NCBI database, indicating that more than one-third of these MOTUs potentially represents still not represented or undescribed ciliate species in current databases. Our findings indicate that metabarcoding techniques can significantly enhance the comprehension of ciliate diversity in tropical environments, but the scarcity of reference sequences of brackish ciliates in molecular databases represents a challenge to the taxonomic assignment of the MOTUs. This study provides new insights into the diversity of ciliates in a threatened coastal lagoon, revealing a vast array of still unknown and rare ciliate taxonomic units in tropical environments.

The metabarcoding of Grubs: Traditional herbal medicine of Scarabaeidae larvae

Grubs, called Qicao in China, have a long tradition as herbal medicine in East Asia. These larvae belong to the diverse family Scarabaeidae and are typically harvested from the wild during their immature stage based on morphological characteristics. However, rapid and accurate identification becomes challenging when relying solely on external morphological features, as the lack of clarity on biological sources raises safety concerns for clinical applications. The application of DNA metabarcoding provides a solution by enabling the determination of the biological source of a large sample. In the current study, we collected 19 batches of Grubs, consisting of 11,539 individuals, from the market and analyzed their biological composition through metabarcoding. We identified 49 Amplicon Sequence Variants (ASVs), 21 of which were Grubs. The 21 ASVs were classified into seven Molecular Operational Taxonomic Units (MOTUs) through species delimitation, which revealed that commercially available Grubs are predominantly sourced from Protaetia brevitarsis seulensis, while species of Rutelinae, Anomala, and Holotrichia were also abundant in some commercial batches. Among the identified ASVs, 28 belonged to non-Grub species and indicated adulteration from different animal families; high abundances of these ASVs were detected for Bombycidae, Tabanidae, and Viviparidae. Our findings underscore the complexity of Grubs' species composition and advocate for a deeper understanding of the wildlife sources contributing to herbal products. This research contributes valuable insights into the molecular identification of Grubs, paving the way for enhanced quality assurance in traditional medicine applications to provide safe and effective medicines for humanity.

The global genetic diversity of planktonic foraminifera reveals the structure of cryptic speciation in plankton.

The nature and extent of diversity in the plankton has fascinated scientists for over a century. Initially, the discovery of many new species in the remarkably uniform and unstructured pelagic environment appeared to challenge the concept of ecological niches. Later, it became obvious that only a fraction of plankton diversity had been formally described, because plankton assemblages are dominated by understudied eukaryotic lineages with small size that lack clearly distinguishable morphological features. The high diversity of the plankton has been confirmed by comprehensive metabarcoding surveys, but interpretation of the underlying molecular taxonomies is hindered by insufficient integration of genetic diversity with morphological taxonomy and ecological observations. Here we use planktonic foraminifera as a study model and reveal the full extent of their genetic diversity and investigate geographical and ecological patterns in their distribution. To this end, we assembled a global data set of ~7600 ribosomal DNA sequences obtained from morphologically characterised individual foraminifera, established a robust molecular taxonomic framework for the observed diversity, and used it to query a global metabarcoding data set covering ~1700 samples with ~2.48 billion reads. This allowed us to extract and assign 1 million reads, enabling characterisation of the structure of the genetic diversity of the group across ~1100 oceanic stations worldwide. Our sampling revealed the existence of, at most, 94 distinct molecular operational taxonomic units (MOTUs) at a level of divergence indicative of biological species. The genetic diversity only doubles the number of formally described species identified by morphological features. Furthermore, we observed that the allocation of genetic diversity to morphospecies is uneven. Only 16 morphospecies disguise evolutionarily significant genetic diversity, and the proportion of morphospecies that show genetic diversity increases poleward. Finally, we observe that MOTUs have a narrower geographic distribution than morphospecies and that in some cases the MOTUs belonging to the same morphospecies (cryptic species) have different environmental preferences. Overall, our analysis reveals that even in the light of global genetic sampling, planktonic foraminifera diversity is modest and finite. However, the extent and structure of the cryptic diversity reveals that genetic diversification is decoupled from morphological diversification, hinting at different mechanisms acting at different levels of divergence.

Integrative species delimitation and biogeography of the Rhinella margaritifera species group (Amphibia, Anura, Bufonidae) suggest an intense diversification throughout Amazonia during the last 10 million years

The accumulation of studies delimiting species in Amazonia has not only shed light on the patterns of its outstanding species richness but also allowed a better understanding of the processes of diversification within this immense region. Nevertheless, vast knowledge gaps remain even for prominent anuran species complexes, such as the Rhinella margaritifera species group. This clade of toads comprises 23 valid species-level taxa, mainly distributed in Amazonia but also in South America’s Dry Diagonal and Atlantic and trans-Andean rainforests. Species boundaries and taxonomy in this group are notoriously complex, with studies suggesting the existence of several unnamed species. Available phylogenetic information suggests an Andean-western Amazonian origin of the group with subsequent diversification within Amazonian lowlands during the last 10 Myr and secondary dispersals into other Neotropical regions. To further test this biogeographic scenario and improve knowledge on species diversity, we used an unprecedentedly large mtDNA sampling (>800 16S sequences) across the clade’s distribution and comprising all but one described species. We delimited 54 Molecular Operational Taxonomic Units, which we tested further based on patterns of variation of a nuclear locus and acoustic and morphological data. This approach confirmed the existence of at least 25 candidate species, 19 of which correspond to currently recognized taxa whereas 30 remained ‘unconfirmed’. Our results clarify the taxonomic status of some species but also suggest multiple introgression events that blur some mtDNA-based species boundaries. Lastly, to provide a temporal framework for the clade’s diversification, we generated a time-calibrated phylogenetic tree based on a mitogenomic matrix, which confirmed a Miocene (∼9 Ma) western Amazonian origin and six major clades in the group, each having initially diversified in different regions within Amazonia. Most of these clades have later dispersed throughout Amazonia during the establishment of the modern Amazonian hydrographic system, i.e., in the last 6 Myr.

Jump dispersal drives the relationship between micro- and macroevolutionary dynamics in the Sicydiinae (Gobiiformes: Oxudercidae) of Sundaland and Wallacea.

Insular biodiversity hotspots of Southeast Asia are remarkable for their biodiverse faunas. With a marine larval phase lasting up to several months, the freshwater fish subfamily Sicydiinae has colonized most islands of these hotspots. However, Sicydiinae diversity is still poorly understood in Southeast Asia. With the objective to estimate intraspecific genetic diversity and infer past demography, we conducted the molecular inventory of Sicydiinae species in Sundaland and Wallacea using 652bp of the mitochondrial cytochrome oxidase I gene, species delimitation methods and Bayesian Skyline plot reconstructions. In total, 24 Molecular Operational Taxonomic Units are delimited among the 603 sequences belonging to 27 species and five genera. Two cases of discordance between morphology and mitochondrial sequence are observed suggesting ongoing speciation and/or introgression in two genera. Multiple new occurrences are reported, either for a single biodiversity hotspot or both, some of which corresponding to observations of a few individuals far from the range distribution of their conspecifics. Among the ten species or species group whose intraspecific diversity was examined, high levels of genetic diversity and past population expansion are revealed by Tajima's D tests and Bayesian Skyline Plot reconstructions. Together these results indicate that long-distance dispersal is common and suggest that most endemic species originated through founder events followed by population expansion. Patterns of sexual dimorphism and males' coloration among diverging species pair seem to point to sexual selection as an important mechanism contributing to speciation in the Sicydiinae of Sundaland and Wallacea.

Trypanosomatid diversity in a bat community of an urban area in Campo Grande, Mato Grosso do Sul, Brazil

Bats have a long evolutionary history with trypanosomatids, but the role of these flying mammals on parasite transmission cycles in urban areas, especially for Trypanosoma and Leishmania species, remains poorly known. The objective of this study was to evaluate the species richness of trypanosomatids parasitizing a bat community in Campo Grande (CG), a state capital within the Cerrado of the Brazilian Midwest. We evaluated 237 bats of 13 species by means of hemoculture and molecular detection in spleen samples. The bat community of CG appears to participate in the transmission cycles of various species of trypanosomatids. We report an overall trypanosomatid detection rate of 34.2% (n = 81), involving 11 out of 13 sampled bat species. We identified six species of trypanosomatids from 61 bats by analyzing SSU rRNA and/or kDNA: Trypanosoma cruzi DTU TcI, T. c. marinkellei, T. dionisii, Leishmania infantum, L. amazonensis, and T. janseni, with this latter being detected by hemoculture for the first time in a bat species. We also detected a Molecular Operational Taxonomic Unit, Trypanosoma sp. DID, in the phyllostomids Glossophaga soricina and Platyrrhinus lineatus. The highest trypanosomatid richness was observed for Sturnira lilium, which hosted three species: L. infantum, T. dionisii and T. janseni. Given that visceral leishmaniasis is endemic in CG, special focus should be placed on L. infantum. Moreover, L. amazonensis and T. cruzi warrant attention, since these are zoonotic parasites responsible for human cases of tegumentary leishmaniasis and Chagas disease, respectively. In this respect, we discuss how bat communities may influence the Leishmania spp. transmission in endemic areas.