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Across the globe, anthropogenic environmental changes are threatening animal biodiversity and contributing to the emergence of vector‐borne and zoonotic pathogens through host range shifts. To combat these challenges, accurate and timely biodiversity assessments and molecular species monitoring efforts are critical. Here, we document how the implementation of a portable laboratory in combination with targeted long‐read nanopore sequencing can facilitate in situ genomic and systematic analyses across several animal taxa. Working at two ecologically divergent field sites in Guyana, South America, we collected small mammals and blood‐feeding insects, including bats, rodents, a marsupial, mosquitoes, and a phlebotomine sand fly. For each specimen sampled, genomic DNA was extracted in the field and used for the preparation of nanopore sequencing libraries. For field sequencing, we utilized a novel software‐based targeted sequencing approach—nanopore adaptive sampling (NAS)—that enabled the selective sequencing of mitochondrial reads using mitogenome assemblies of related taxa as enrichment targets. Basecalled reads from our field sequencing experiments were used to assemble complete mitogenomes and to generate mitochondrial biomarker consensus gene sequences for all nine small mammals and four blood‐feeding insects sequenced. Confirmatory molecular identifications were made with a combination of local nucleotide BLAST queries and maximum likelihood analyses using biomarker consensus sequences. Importantly, the mitogenome‐based targeted sequencing strategies outlined here are amplification‐free and allowed us to bypass time‐consuming and potentially troublesome PCR‐based methods in the field, streamlining library preparation, sequencing experiments, and on‐site analyses. Our findings describe targeted sequencing with NAS as an effective tool for implementation into portable laboratories to widely enhance field‐based biodiversity monitoring and rapid molecular species assessments across vertebrate and invertebrate hosts of consequential emerging pathogens.

Background Blood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources. Methods We used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes ( Aedes and Culex spp. ) and one deer fly ( Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method. Results Complete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans , Culex restuans , Culex territans and the deer fly, Chrysops niger . In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger , Culex restuans and Aedes trivittatus were human, house sparrow ( Passer domesticus ) and eastern cottontail rabbit ( Sylvilagus floridanus ), respectively. Conclusions Our findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel ‘phylogenetic capture’ methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens. Graphical Abstract

Hawthorn species ( L.; Rosaceae tribe Maleae) form a well-defined clade comprising five subgeneric groups readily distinguished using either molecular or morphological data. While multiple subsidiary groups (taxonomic sections, series) are recognized within some subgenera, the number of and relationships among species in these groups are subject to disagreement. Gametophytic apomixis and polyploidy are prevalent in the genus, and disagreement concerns whether and how apomictic genotypes should be recognized taxonomically. Recent studies suggest that many polyploids arise from hybridization between members of different infrageneric groups. We used target capture and high throughput sequencing to obtain nucleotide sequences for 257 nuclear loci and nearly complete chloroplast genomes from a sample of hawthorns representing all five currently recognized subgenera. Our sample is structured to include two examples of intersubgeneric hybrids and their putative diploid and tetraploid parents. We queried the alignment of nuclear loci directly for evidence of hybridization, and compared individual gene trees with each other, and with both the maximum likelihood plastome tree and the nuclear concatenated and multilocus coalescent-based trees. Tree comparisons provided a promising, if challenging (because of the number of comparisons involved) method for visualizing variation in tree topology. We found it useful to deploy comparisons based not only on tree-tree distances but also on a metric of tree-tree concordance that uses extrinsic information about the relatedness of the terminals in comparing tree topologies. We obtained well-supported phylogenies from plastome sequences and from a minimum of 244 low copy-number nuclear loci. These are consistent with a previous morphology-based subgeneric classification of the genus. Despite the high heterogeneity of individual gene trees, we corroborate earlier evidence for the importance of hybridization in the evolution of . Hybridization between subgenus and subgenus was documented for the origin of tetraploids, but not for a tetraploid species. This is also the first application of target capture probes designed with apple genome sequence. We successfully assembled 95% of 257 loci in , indicating their potential utility across the genera of the apple tribe.

Marine nematodes of the order Enoplida may represent the earliest lineage of nematodes and have a variety of fixed and movable feeding structures in their stomas. This study used an 18S ribosomal RNA phylogeny of the orders Enoplida and Triplonchida (subclass Enoplia) to explore the evolution of these feeding structures in light of previous hypotheses based solely on morphology. The Enoplida and Triplonchida were found to be paraphyletic, as several taxa currently classified as Triplonchida, such as Rhabdodemania, were found to be part of the Enoplida clade. The position of Rhabdodemania within Enoplida was unclear, but a close relation to Enoplidae and Thoracostomopsidae was not supported, making it unlikely that its movable odontia are homologous with the mandibles of these families. A member of Anticomidae was well-supported as the base of the clade containing Phanodermatidae, Enoplidae, and Thoracostomopsidae, suggesting that taxa with buccal rods and mandibles evolved from nematodes with unarmed stomas. The Phanodermatidae were shown to be more closely related to the Enoplidae and Thoracostomopsidae than were the Leptosomatidae, suggesting that the buccal rods of the phanoderms (rather than the mandibular ridge/odontia complex of the Leptosomatidae), may be the origin of the mandibles.

Onychophorans are carnivorous, terrestrial invertebrates that occur in tropical and temperate forests of the Southern Hemisphere and around the Equator. Together with tardigrades, onychophorans are regarded as one of the closest relatives of arthropods. One of the most peculiar features of onychophorans is their hunting and feeding behavior. These animals secrete a sticky slime, which is ejected via a pair of slime-papillae, to entangle the prey. After the prey has been immobilized, its cuticle is punctured using a pair of jaws located within the mouth. These jaws constitute internalized appendages of the second body segment and are innervated by the deutocerebrum; thus, they are homologous to the chelicerae of chelicerates, and to the (first) antennae of myriapods, crustaceans, and insects. The jaws are also serial homologs of the paired claws associated with each walking limb of the trunk. The structure of the jaws is similar in representatives of the two major onychophoran subgroups, the Peripatidae and Peripatopsidae. Each jaw is characterized by an outer and an inner blade; while the outer blade consists only of a large principal tooth and up to three accessory teeth, the inner blade bears numerous additional denticles. These denticles are separated from the remaining part of the inner jaw by a diastema and a soft membrane only in peripatids. The onychophoran jaws are associated with large apodemes and specialized muscles that enable their movement. In contrast to the mandibles of arthropods, the onychophoran jaws are moved along, rather than perpendicular to, the main axis of the body. Our elemental analysis reveals an increased incorporation of calcium at the tip of each blade, which might provide rigidity, whereas there is no evidence for incorporation of metal or prominent mineralization. Stability of the jaw might be further facilitated by the cone-incone organization of its cuticle, as each blade consists of several stacked, cuticular elements. In this work, we summarize current knowledge on the jaws of onychophorans, which are a characteristic feature of these animals.

The possibility for independently evolving entities to form and persist in the absence of sexual recombination in eukaryotes has been questioned; nevertheless, there are organisms that are known to be asexual and that have apparently diversified into multiple species as recognized by taxonomists. These organisms have therefore been identified as an evolutionary paradox. We explore three alternative hypotheses attempting to solve the apparent paradox, focusing on bdelloid rotifers, the most studied group of organisms in which all species are considered asexual: (1) they may have some hidden form of sex; (2) species do not represent biological entities but simply convenient names; and (3) sex may not be a necessary requirement for speciation. We provide ample evidence against the first two hypotheses, reporting several studies supporting (1) bdelloids asexuality from different approaches, and (2) the existence of species from genetics, jaw morphology, ecology, and physiology. Thus, we (3) explore the role of sex in speciation comparing bdelloid and monogonont rotifers, and conclude with some caveats that could still change our understanding of bdelloid species.

Predatory flatworms belonging to the taxon Kalyptorhynchia are characterized by an anterior muscular proboscis that they use to seize prey. In many cases, the proboscis is armed with hooks, derived either from the extracellular matrix that surrounds the muscles or from intracellular deposits in the epithelium covering the proboscis. Glands associated with the proboscis reportedly are venomous; however, there are few direct tests of this hypothesis. This article reviews the structure and current knowledge of the function of the proboscis in the Kalyptorhynchia, points to areas in which the current understanding of phylogenetic relationships within this taxon is incongruent with our hypothesis of how the proboscis evolved, and addresses areas in need of further research, especially as regards functional morphology and biomechanics.

The cuticular portion of the tardigrade feeding apparatus is a complex structure that can be schematically divided into four parts: a buccal ring, a buccal tube, a stylet system (formed by two piercing stylets, each within a stylet coat, and two stylet supports), and the lining of a myoepithelial sucking pharynx. To better understand the function and evolution of the feeding apparatus, the morpho-functional traits and chemical composition of the structures forming the feeding apparatuses of eight different species of tardigrades were analyzed. These eight species are representative of almost all main phylogenetic lineages of the phylum. The calcium and chitin in the feeding apparatus were examined by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, energy dispersive X-ray spectroscopy, and Raman microspectroscopy (Raman). In all species, the feeding apparatus had been subjected to biomineralization due to CaCO ₃ encrustations organized in the crystalline form of aragonite. Aragonite and chitin are present in different concentrations in the feeding apparatus according to the structures and species considered. Generally, where the structures are rigid there is more aragonite than chitin, and vice versa. The buccal tube and piercing stylets are rich in calcium, with the piercing stylets apparently composed exclusively of aragonite. In eutardigrades, chitin is in higher concentration in the structures subject to higher mechanical stresses, such as the crests of the buccal crown and the condyles of the stylet furca.

This study surveys animals that use soft tissues rather than rigid links to build jaw joints. Hard biting elements are useful; they are used in piercing or shearing during feeding and interactive behaviors and can directly impact survival and reproduction. The best understood biting systems include biting elements that are mounted on rigid jaw links that form a joint capable of transmitting the bite reaction forces. As such, jaws must incorporate joints that resist compression. Many jaw joints are “sliding joints”, in which jaw links come into direct contact and the shape of the sliding contact surfaces dictates possible motions. There are, however, organisms that have biting elements on jaws that are made of flexible muscle and connective tissues. If arranged as a muscular hydrostat, in which multiple orientations of the muscle fibers may co-contract to provide turgid skeletal support, the multifunctional joint not only (a) provides the force to move the biting elements, but also (b) creates repositionable pivots and (c) transmits bite reaction forces. Such flexible joints, termed “muscle articulations”, may be important to a number of soft-bodied animals. In this survey, we review the function of previously described muscle articulations: the joints found between inarticulate brachiopods’ valves, cephalopods’ beaks, the hooks of kalyptorhynch flatworms, and errant polychaetes’ jaws. We also review the morphology, physiology, and feeding behaviors of the hagfish as a putative muscle articulation in an effort to understand how this jawless craniate is capable of biting with surprising force, seemingly without the benefit of any obvious method of opposing the force of the dental plate that is used to remove portions of food. Initial analysis suggests that a muscle articulation may be a key feature in coordinating head and body movements to provide the leverage needed for strong “bites”.

Jaws have evolved numerous times in the animal kingdom and they display a wide variety of structural, compositional, and functional characteristics that reflect their polyphyletic origins. Among soft-bodied invertebrates, jaws are known from annelids, chaetognaths, flatworms, gnathostomulids, micrognathozoans, mollusks, rotifers, and several ecdysozoans. Depending on the taxon, jaws may function in the capture of prey (e.g., chaetognaths and flatworms), processing of prey (e.g., gnathostomulids and onychophorans), or both (e.g., rotifers). Although structural diversity among invertebrates’ jaws is becoming better characterized with the use of electron microscopy, many details remain poorly described, including neuromuscular control, elemental composition, and physical characteristics, such as hardness and resistance to wear. Unfortunately, absence of relevant data has impeded understanding of their functional diversity and evolutionary origins. With this symposium, we bring together researchers of disparately jawed taxa to draw structural and mechanistic comparisons among species to determine their commonalities. Additionally, we show that rotifers’ jaws, which are perhaps the best-characterized jaws among invertebrates, are still enigmatic with regard to their origins and mechanics. Nevertheless, technologies such as energy dispersive X-ray spectroscopy (EDX) and 3D modeling are being used to characterize their chemical composition and to develop physical models that allow exploration of their mechanical properties, respectively. We predict that these methods can also be used to develop biomimetic and bioinspired constructs based on the full range of the complexity of jaws, and that such constructs also can be developed from other invertebrate taxa. These approaches may also shed light on common developmental and physiological processes that facilitate the evolution of invertebrates’ jaws.