Rediscovery and systematics of the enigmatic genus Helicostoa reveals a new species of sessile freshwater snail with remarkable sexual dimorphism.

Helicostoa sinensis E. Lamy, 1926 is a unique freshwater gastropod species with a sessile habit. This enigmatic species was first found cemented on river limestones from China about 120 years ago and described together with the genus. It was never collected again and has been considered monotypic. Here, we report the rediscovery of Helicostoa from several rivers in China, and describe a second species of this genus based on a comprehensive study. In addition to the unique sessile habit of both species, the new Helicostoa species presents one of the most remarkable cases of sexual dimorphism within molluscs. Only the adult female is sessile and the original aperture of the female is sealed by shell matter or rock, while an opening on the body whorl takes the function of the original aperture. The male is vagile, with a normal aperture. Our results confirm the recently suggested placement of Helicostoa within the family Bithyniidae. The sessility of Helicostoa species is considered as an adaption to the limestone habitat in large rivers. The extreme sexual dimorphism and secondary aperture of females are considered as adaptations to overcome the obstacles for mating and feeding that come with a sessile life style.

A re-analysis of the data in Sharkey et al.'s (2021) minimalist revision reveals that BINs do not deserve names, but BOLD Systems needs a stronger commitment to open science.

Halting biodiversity decline is one of the most critical challenges for humanity, but monitoring biodiversity is hampered by taxonomic impediments. One impediment is the large number of undescribed species (here called "dark taxon impediment") whereas another is caused by the large number of superficial species descriptions, that can only be resolved by consulting type specimens ("superficial description impediment"). Recently, Sharkey et al. (2021) proposed to address the dark taxon impediment for Costa Rican braconid wasps by describing 403 species based on COI barcode clusters ("BINs") computed by BOLD Systems. More than 99% of the BINs (387 of 390) were converted into species by assigning binominal names (e.g. BIN "BOLD:ACM9419" becomes Bracon federicomatarritai) and adding a minimal diagnosis (consisting only of a consensus barcode for most species). We here show that many of Sharkey et al.'s species are unstable when the underlying data are analyzed using different species delimitation algorithms. Add the insufficiently informative diagnoses, and many of these species will become the next "superficial description impediment" for braconid taxonomy because they will have to be tested and redescribed after obtaining sufficient evidence for confidently delimiting species. We furthermore show that Sharkey et al.'s approach of using consensus barcodes as diagnoses is not functional because it cannot be applied consistently. Lastly, we reiterate that COI alone is not suitable for delimiting and describing species, and voice concerns over Sharkey et al.'s uncritical use of BINs because they are calculated by a proprietary algorithm (RESL) that uses a mixture of public and private data. We urge authors, reviewers and editors to maintain high standards in taxonomy by only publishing new species that are rigorously delimited with open-access tools and supported by publicly available evidence.

Miocene geologic dynamics of the Australian Sahul Shelf determined the biogeographic patterns of freshwater planorbid snails (Miratestinae) in the Indo-Australian Archipelago.

The complex geological and climatic processes that have shaped the Indo-Australian Archipelago since the Cenozoic likely also gave rise to its species-rich biota. Strictly freshwater organisms might be particularly suitable for understanding the influence of these abiotic factors on their biogeography in such a insular setting as their distribution may reflect past abiotic events at large and small geographical scales. We here investigate the historical biogeography of the Miratestinae, a subfamily of Planorbidae. These freshwater gastropods are widely distributed in the eastern IAA from Australia, New Guinea, the Moluccas, and Sulawesi to the Philippines. The first comprehensive molecular phylogeny of the Miratestinae was inferred based on two mitochondrial and two nuclear genetic markers using maximum likelihood and Bayesian inference. Four species delimitation methods were applied to identify molecular operational taxonomic units (MOTUs). Divergence times were inferred using an uncorrelated lognormal relaxed-clock model by applying a taxon- and marker-specific substitution rate. Ancestral geographic ranges were estimated based on the dated phylogeny using BioGeoBEARS. The species delimitation revealed a total of 23 MOTUs, 16 of which might represent species new to science. The BioGeoBEARS analyses suggest an Australian origin for the Miratestinae at c. 22 Ma and identified jump dispersal to be the main process of colonization. The first colonization events from Australia to the IAA occurred in the Middle-Late Miocene (12-13 Ma), whereas intra-island diversification took mainly place since the Late Miocene-Pliocene. Colonization and diversification events remarkably coincide with major geologic events that shaped the geography of the region. The increasing availability of landmasses along the Sahul Shelf likely promoted stepping-stone dispersal to New Guinea, Sulawesi and the Philippines as early as the islands emerged. Major geological and climatic events such as the amalgamation of the island Sulawesi, the regional aridification in Australia or the uplift of massive mountain ranges in New Guinea likely played a considerable role for intra-island diversification.

Global Diversification Dynamics Since the Jurassic: Low Dispersal and Habitat-Dependent Evolution Explain Hotspots of Diversity and Shell Disparity in River Snails (Viviparidae).

The Viviparidae, commonly known as River Snails, is a dominant group of freshwater snails with a nearly worldwide distribution that reaches its highest taxonomic and morphological diversity in Southeast Asia. The rich fossil record is indicative of a probable Middle Jurassic origin on the Laurasian supercontinent where the group started to diversify during the Cretaceous. However, it remains uncertain when and how the biodiversity hotspot in Southeast Asia was formed. Here, we used a comprehensive genetic data set containing both mitochondrial and nuclear markers and comprising species representing 24 out of 28 genera from throughout the range of the family. To reconstruct the spatiotemporal evolution of viviparids on a global scale, we reconstructed a fossil-calibrated phylogeny. We further assessed the roles of cladogenetic and anagenetic events in range evolution. Finally, we reconstructed the evolution of shell features by estimating ancestral character states to assess whether the appearance of sculptured shell morphologies was driven by major habitat shifts. The molecular phylogeny supports the monophyly of the three subfamilies, the Bellamyinae, Lioplacinae, and Viviparinae, but challenges the currently accepted genus-level classification in several cases. The almost global distribution of River Snails has been influenced both by comparatively ancient vicariance and more recent founder events. In Southeast Asia, Miocene dispersal was a main factor in shaping the modern species distributions. A recurrent theme across different viviparid taxa is that many species living in lentic waters exhibit sculptured shells, whereas only one strongly sculptured species is known from lotic environments. We show that such shell sculpture is habitat-dependent and indeed evolved several times independently in lentic River Snails. Considerably high transition rates between shell types in lentic habitats probably caused the co-occurrence of morphologically distinct shell types in several lakes. In contrast, directional evolution toward smooth shells in lotic habitats, as identified in the present analyses, explains why sculptured shells are rarely found in these habitats. However, the specific factors that promoted changes in shell morphology require further work. [biogeographical analyses; fossil-calibrated phylogeny; fossil-constrained analyses; Southeast Asia; stochastic character mapping.].

Lack of Signal for the Impact of Conotoxin Gene Diversity on Speciation Rates in Cone Snails.

Understanding why some groups of organisms are more diverse than others is a central goal in macroevolution. Evolvability, or the intrinsic capacity of lineages for evolutionary change, is thought to influence disparities in species diversity across taxa. Over macroevolutionary time scales, clades that exhibit high evolvability are expected to have higher speciation rates. Cone snails (family: Conidae, $>$900 spp.) provide a unique opportunity to test this prediction because their toxin genes can be used to characterize differences in evolvability between clades. Cone snails are carnivorous, use prey-specific venom (conotoxins) to capture prey, and the genes that encode venom are known and diversify through gene duplication. Theory predicts that higher gene diversity confers a greater potential to generate novel phenotypes for specialization and adaptation. Therefore, if conotoxin gene diversity gives rise to varying levels of evolvability, conotoxin gene diversity should be coupled with macroevolutionary speciation rates. We applied exon capture techniques to recover phylogenetic markers and conotoxin loci across 314 species, the largest venom discovery effort in a single study. We paired a reconstructed timetree using 12 fossil calibrations with species-specific estimates of conotoxin gene diversity and used trait-dependent diversification methods to test the impact of evolvability on diversification patterns. Surprisingly, we did not detect any signal for the relationship between conotoxin gene diversity and speciation rates, suggesting that venom evolution may not be the rate-limiting factor controlling diversification dynamics in Conidae. Comparative analyses showed some signal for the impact of diet and larval dispersal strategy on diversification patterns, though detection of a signal depended on the dataset and the method. If our results remain true with increased taxonomic sampling in future studies, they suggest that the rapid evolution of conid venom may cause other factors to become more critical to diversification, such as ecological opportunity or traits that promote isolation among lineages.

Novel Genes, Ancient Genes, and Gene Co-Option Contributed to the Genetic Basis of the Radula, a Molluscan Innovation.

The radula is the central foraging organ and apomorphy of the Mollusca. However, in contrast to other innovations, including the mollusk shell, genetic underpinnings of radula formation remain virtually unknown. Here, we present the first radula formative tissue transcriptome using the viviparous freshwater snail Tylomelania sarasinorum and compare it to foot tissue and the shell-building mantle of the same species. We combine differential expression, functional enrichment, and phylostratigraphic analyses to identify both specific and shared genetic underpinnings of the three tissues as well as their dominant functions and evolutionary origins. Gene expression of radula formative tissue is very distinct, but nevertheless more similar to mantle than to foot. Generally, the genetic bases of both radula and shell formation were shaped by novel orchestration of preexisting genes and continuous evolution of novel genes. A significantly increased proportion of radula-specific genes originated since the origin of stem-mollusks, indicating that novel genes were especially important for radula evolution. Genes with radula-specific expression in our study are frequently also expressed during the formation of other lophotrochozoan hard structures, like chaetae (hes1, arx), spicules (gbx), and shells of mollusks (gbx, heph) and brachiopods (heph), suggesting gene co-option for hard structure formation. Finally, a Lophotrochozoa-specific chitin synthase with a myosin motor domain (CS-MD), which is expressed during mollusk and brachiopod shell formation, had radula-specific expression in our study. CS-MD potentially facilitated the construction of complex chitinous structures and points at the potential of molecular novelties to promote the evolution of different morphological innovations.

Synchronous diversification of Sulawesi's iconic artiodactyls driven by recent geological events.

The high degree of endemism on Sulawesi has previously been suggested to have vicariant origins, dating back to 40 Ma. Recent studies, however, suggest that much of Sulawesi's fauna assembled over the last 15 Myr. Here, we test the hypothesis that more recent uplift of previously submerged portions of land on Sulawesi promoted diversification and that much of its faunal assemblage is much younger than the island itself. To do so, we combined palaeogeographical reconstructions with genetic and morphometric datasets derived from Sulawesi's three largest mammals: the babirusa, anoa and Sulawesi warty pig. Our results indicate that although these species most likely colonized the area that is now Sulawesi at different times (14 Ma to 2-3 Ma), they experienced an almost synchronous expansion from the central part of the island. Geological reconstructions indicate that this area was above sea level for most of the last 4 Myr, unlike most parts of the island. We conclude that emergence of land on Sulawesi (approx. 1-2 Myr) may have allowed species to expand synchronously. Altogether, our results indicate that the establishment of the highly endemic faunal assemblage on Sulawesi was driven by geological events over the last few million years.

Founder effects initiated rapid species radiation in Hawaiian cave planthoppers.

The Hawaiian Islands provide the venue of one of nature's grand experiments in evolution. Here, we present morphological, behavioral, genetic, and geologic data from a young subterranean insect lineage in lava tube caves on Hawai'i Island. The Oliarus polyphemus species complex has the potential to become a model for studying rapid speciation by stochastic events. All species in this lineage live in extremely similar environments but show strong differentiation in behavioral and morphometric characters, which are random with respect to cave age and geographic distribution. Our observation that phenotypic variability within populations decreases with increasing cave age challenges traditional views on founder effects. Furthermore, these cave populations are natural replicates that can be used to test the contradictory hypotheses. Moreover, Hawaiian cave planthoppers exhibit one of the highest speciation rates among animals and, thus, radically shift our perception on the evolutionary potential of obligate cavernicoles.

Borneo and Indochina are major evolutionary hotspots for Southeast Asian biodiversity.

Tropical Southeast (SE) Asia harbors extraordinary species richness and in its entirety comprises four of the Earth's 34 biodiversity hotspots. Here, we examine the assembly of the SE Asian biota through time and space. We conduct meta-analyses of geological, climatic, and biological (including 61 phylogenetic) data sets to test which areas have been the sources of long-term biological diversity in SE Asia, particularly in the pre-Miocene, Miocene, and Plio-Pleistocene, and whether the respective biota have been dominated by in situ diversification, immigration and/or emigration, or equilibrium dynamics. We identify Borneo and Indochina, in particular, as major "evolutionary hotspots" for a diverse range of fauna and flora. Although most of the region's biodiversity is a result of both the accumulation of immigrants and in situ diversification, within-area diversification and subsequent emigration have been the predominant signals characterizing Indochina and Borneo's biota since at least the early Miocene. In contrast, colonization events are comparatively rare from younger volcanically active emergent islands such as Java, which show increased levels of immigration events. Few dispersal events were observed across the major biogeographic barrier of Wallace's Line. Accelerated efforts to conserve Borneo's flora and fauna in particular, currently housing the highest levels of SE Asian plant and mammal species richness, are critically required.

Disjunct distributions of freshwater snails testify to a central role of the Congo system in shaping biogeographical patterns in Africa.

BACKGROUND: The formation of the East African Rift System has decisively influenced the distribution and evolution of tropical Africa's biota by altering climate conditions, by creating basins for large long-lived lakes, and by affecting the catchment and drainage directions of river systems. However, it remains unclear how rifting affected the biogeographical patterns of freshwater biota through time on a continental scale, which is further complicated by the scarcity of molecular data from the largest African river system, the Congo. RESULTS: We study these biogeographical patterns using a fossil-calibrated multi-locus phylogeny of the gastropod family Viviparidae. This group allows reconstructing drainage patterns exceptionally well because it disperses very poorly in the absence of existing freshwater connections. Our phylogeny covers localities from major drainage basins of tropical Africa and reveals highly disjunct sister-group relationships between (a) the endemic viviparids of Lake Malawi and populations from the Middle Congo as well as between (b) the Victoria region and the Okavango/Upper Zambezi area. CONCLUSIONS: The current study testifies to repeated disruptions of the distribution of the Viviparidae during the formation of the East African Rift System, and to a central role of the Congo River system for the distribution of the continent's freshwater fauna during the late Cenozoic. By integrating our results with previous findings on palaeohydrographical connections, we provide a spatially and temporarily explicit model of historical freshwater biogeography in tropical Africa. Finally, we review similarities and differences in patterns of vertebrate and invertebrate dispersal. Amongst others we argue that the closest relatives of present day viviparids in Lake Malawi are living in the Middle Congo River, thus shedding new light on the origin of the endemic fauna of this rift lake.

Paleo-drainage basin connectivity predicts evolutionary relationships across three Southeast Asian biodiversity hotspots.

Understanding factors driving diversity across biodiversity hotspots is critical for formulating conservation priorities in the face of ongoing and escalating environmental deterioration. While biodiversity hotspots encompass a small fraction of Earth's land surface, more than half the world's plants and two-thirds of terrestrial vertebrate species are endemic to these hotspots. Tropical Southeast (SE) Asia displays extraordinary species richness, encompassing four biodiversity hotspots, though disentangling multiple potential drivers of species richness is confounded by the region's dynamic geological and climatic history. Here, we use multilocus molecular genetic data from dense multispecies sampling of freshwater fishes across three biodiversity hotspots, to test the effect of Quaternary climate change and resulting drainage rearrangements on aquatic faunal diversification. While Cenozoic geological processes have clearly shaped evolutionary history in SE Asian halfbeak fishes, we show that paleo-drainage re-arrangements resulting from Quaternary climate change played a significant role in the spatiotemporal evolution of lowland aquatic taxa, and provide priorities for conservation efforts.

Mitogenomes do not substantially improve phylogenetic resolution in a young non-model adaptive radiation of freshwater gastropods.

BACKGROUND: Species flocks in ancient lakes, and particularly those arising from adaptive radiation, make up the bulk of overall taxonomic and morphological diversity in these insular ecosystems. For these mostly young species assemblages, classical mitochondrial barcoding markers have so far been key to disentangle interspecific relationships. However, with the rise and further development of next-generation sequencing (NGS) methods and mapping tools, genome-wide data have become an increasingly important source of information even for non-model groups. RESULTS: Here, we provide, for the first time, a comprehensive mitogenome dataset of freshwater gastropods endemic to Sulawesi and thus of an ancient lake invertebrate species flock in general. We applied low-coverage whole-genome sequencing for a total of 78 individuals including 27 out of the 28 Tylomelania morphospecies from the Malili lake system as well as selected representatives from Lake Poso and adjacent catchments. Our aim was to assess whether mitogenomes considerably contribute to the phylogenetic resolution within this young species flock. Interestingly, we identified a high number of variable and parsimony-informative sites across the other 'non-traditional' mitochondrial loci. However, although the overall support was very high, the topology obtained was largely congruent with previously published single-locus phylogenies. Several clades remained unresolved and a large number of species was recovered polyphyletic, indicative of both rapid diversification and mitochondrial introgression. CONCLUSIONS: This once again illustrates that, despite the higher number of characters available, mitogenomes behave like a single locus and thus can only make a limited contribution to resolving species boundaries, particularly when introgression events are involved.

Abundant, diverse, unknown: Extreme species richness and turnover despite drastic undersampling in two closely placed tropical Malaise traps.

Arthropods account for a large proportion of animal biomass and diversity in terrestrial systems, making them crucial organisms in our environments. However, still too little is known about the highly abundant and megadiverse groups that often make up the bulk of collected samples, especially in the tropics. With molecular identification techniques ever more evolving, analysis of arthropod communities has accelerated. In our study, which was conducted within the Global Malaise trap Program (GMP) framework, we operated two closely placed Malaise traps in Padang, Sumatra, for three months. We analyzed the samples by DNA barcoding and sequenced a total of more than 70,000 insect specimens. For sequence clustering, we applied three different delimitation techniques, namely RESL, ASAP, and SpeciesIdentifier, which gave similar results. Despite our (very) limited sampling in time and space, our efforts recovered more than 10,000 BINs, of which the majority are associated with "dark taxa". Further analysis indicates a drastic undersampling of both sampling sites, meaning that the true arthropod diversity at our sampling sites is even higher. Regardless of the close proximity of both Malaise traps (< 360 m), we discovered significantly distinct communities.

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

Drawn to the dark side: a molecular phylogeny of freshwater shrimps (Crustacea: Decapoda: Caridea: Atyidae) reveals frequent cave invasions and challenges current taxonomic hypotheses.

Atyid freshwater shrimps are globally distributed and form an important part of freshwater ecosystems, particularly in the tropics and subtropics. Despite their widespread distribution and ecological importance, their phylogenetic relationships are largely unresolved. Here we present the first comprehensive molecular phylogeny of the Atyidae investigating the evolutionary relationships among 32 of the 42 genera using mitochondrial and nuclear markers. Our data indicate that the established classification of the Atyidae is in need of substantial taxonomic revision at all taxonomic levels. We suggest a new suprageneric systematization of atyids and discuss problematic issues at the generic level, particularly in the most speciose genus, Caridina. Molecular clock based divergence time estimates for atyids vary widely, but invariably support the assumption that atyids are an ancient freshwater lineage with an origin in the mid-Cretaceous at the very latest. Atyid distribution patterns are the result of instances of both long-distance dispersal and vicariance, depending largely on the reproductive mode of taxa. From an evolutionary perspective, the high frequency of independent origin of both a complete (landlocked) freshwater life cycle and a cave-dwelling mode of life is remarkable and unparalleled among crustaceans.

Can fractal dimensions objectivize gastropod shell morphometrics? A case study from Lake Lugu (SW China).

Morphometrics are fundamental for the analysis of size and shape in fossils, particularly because soft parts or DNA are rarely preserved and hard parts such as shells are commonly the only source of information. Geometric morphometrics, that is, landmark analysis, is well established for the description of shape but it exhibits a couple of shortcomings resulting from subjective choices during landmarking (number and position of landmarks) and from difficulties in resolving shape at the level of micro-sculpture.With the aid of high-resolution 3D scanning technology and analyses of fractal dimensions, we test whether such shortcomings of linear and landmark morphometrics can be overcome. As a model group, we selected a clade of modern viviparid gastropods from Lake Lugu, with shells that show a high degree of sculptural variation. Linear and landmark analyses were applied to the same shells in order to establish the fractal dimensions. The genetic diversity of the gastropod clade was assessed.The genetic results suggest that the gastropod clade represents a single species. The results of all morphometric methods applied are in line with the genetic results, which is that no specific morphotype could be delimited. Apart from this overall agreement, landmark and fractal dimension analyses do not correspond to each other but represent data sets with different information. Generally, the fractal dimension values quantify the roughness of the shell surface, the resolution of the 3D scans determining the level. In our approach, we captured the micro-sculpture but not the first-order sculptural elements, which explains that fractal dimension and landmark data are not in phase.We can show that analyzing fractal dimensions of gastropod shells opens a window to more detailed information that can be considered in evolutionary and ecological contexts. We propose that using low-resolution 3D scans may successfully substitute landmark analyses because it overcomes the subjective landmarking. Analyses of 3D scans with higher resolution than used in this study will provide surface roughness information at the mineralogical level. We suggest that fractal dimension analyses of a combination of differently resolved 3D models will significantly improve the quality of shell morphometrics.

Evolutionary Divergence and Radula Diversification in Two Ecomorphs from an Adaptive Radiation of Freshwater Snails.

(1) Background: Adaptive diversification of complex traits plays a pivotal role in the evolution of organismal diversity. In the freshwater snail genus Tylomelania, adaptive radiations were likely promoted by trophic specialization via diversification of their key foraging organ, the radula. (2) Methods: To investigate the molecular basis of radula diversification and its contribution to lineage divergence, we used tissue-specific transcriptomes of two sympatric Tylomelania sarasinorum ecomorphs. (3) Results: We show that ecomorphs are genetically divergent lineages with habitat-correlated abundances. Sequence divergence and the proportion of highly differentially expressed genes are significantly higher between radula transcriptomes compared to the mantle and foot. However, the same is not true when all differentially expressed genes or only non-synonymous SNPs are considered. Finally, putative homologs of some candidate genes for radula diversification (hh, arx, gbb) were also found to contribute to trophic specialization in cichlids and Darwin's finches. (4) Conclusions: Our results are in line with diversifying selection on the radula driving Tylomelania ecomorph divergence and indicate that some molecular pathways may be especially prone to adaptive diversification, even across phylogenetically distant animal groups.

DiversityScanner: Robotic handling of small invertebrates with machine learning methods.

Invertebrate biodiversity remains poorly understood although it comprises much of the terrestrial animal biomass, most species and supplies many ecosystem services. The main obstacle is specimen-rich samples obtained with quantitative sampling techniques (e.g., Malaise trapping). Traditional sorting requires manual handling, while molecular techniques based on metabarcoding lose the association between individual specimens and sequences and thus struggle with obtaining precise abundance information. Here we present a sorting robot that prepares specimens from bulk samples for barcoding. It detects, images and measures individual specimens from a sample and then moves them into the wells of a 96-well microplate. We show that the images can be used to train convolutional neural networks (CNNs) that are capable of assigning the specimens to 14 insect taxa (usually families) that are particularly common in Malaise trap samples. The average assignment precision for all taxa is 91.4% (75%-100%). This ability of the robot to identify common taxa then allows for taxon-specific subsampling, because the robot can be instructed to only pick a prespecified number of specimens for abundant taxa. To obtain biomass information, the images are also used to measure specimen length and estimate body volume. We outline how the DiversityScanner can be a key component for tackling and monitoring invertebrate diversity by combining molecular and morphological tools: the images generated by the robot become training images for machine learning once they are labelled with taxonomic information from DNA barcodes. We suggest that a combination of automation, machine learning and DNA barcoding has the potential to tackle invertebrate diversity at an unprecedented scale.

DNA barcoding unveils a high diversity of caddisflies (Trichoptera) in the Mount Halimun Salak National Park (West Java; Indonesia).

Background: Trichoptera are one of the most diverse groups of freshwater insects worldwide and one of the main bioindicators for freshwater quality. However, in many areas, caddisflies remain understudied due to lack of taxonomic expertise. Meanwhile, globally increasing anthropogenic stress on freshwater streams also threatens Trichoptera diversity. Methods: To assess the Trichoptera diversity of the area within and around the Mount Halimun Salak National Park (MHSNP or Taman Nasional Gunung Halimun Salak) in West Java (Indonesia), we conducted a molecular-morphological study on Trichoptera diversity using larvae from a benthic survey and adults from hand-netting. In addition to morphological identification, we applied four different molecular taxon delimitation approaches (Generalized Mixed Yule Coalescent, Bayesian Poisson Tree Processes, Automatic Barcode Gap Discovery and Assemble Species by Automatic Partitioning) based on DNA barcoding of Cytochrome-C-Oxidase I (COI). Results: The molecular delimitation detected 72 to 81 Operational Taxonomic Units (OTU). Only five OTUs could be identified to species level by comparing sequences against the BOLD database using BLAST, and four more to the genus level. Adults and larvae could be successfully associated in 18 cases across six families. The high diversity of Trichoptera in this area highlights their potential as bioindicators for water quality assessment. Conclusions: This study provides an example of how molecular approaches can benefit the exploration of hidden diversity in unexplored areas and can be a valuable tool to link life stages. However, our study also highlights the need to improve DNA barcode reference libraries of Trichoptera for the Oriental region.

Phylogenetic relationships among the species of the Cameroonian endemic freshwater crab genus Louisea Cumberlidge, 1994 (Crustacea, Brachyura, Potamonautidae), with notes on intraspecific morphological variation within two threatened species.

Louisea Cumberlidge, 1994 (Crustacea, Brachyura, Potamonautidae) currently includes four endemic Cameroonian freshwater crab species whose phylogenetic relationships were previously unresolved. In the present study, phylogenetic analyses are carried out involving three mtDNA loci (COI, 12S rRNA, and 16S rRNA). The COI locus revealed divergence times of 5.6 million years ago (myr) for when L.balssi (Bott, 1959) diverged from L.edeaensis (Bott, 1969); 4.1 myr for when L.edeaensis diverged from L.yabassi Mvogo Ndongo, von Rintelen & Cumberlidge, 2019; and 2.48 myr for when the later species diverged from L.nkongsamba Mvogo Ndongo, von Rintelen & Cumberlidge, 2019. Three genetic lineages were found within L.nkongsamba that are supported by uncorrected p-distances and the haplotype network. Morphological variation in some taxonomically important characters was found within both L.nkongsamba and L.yabassi. No correlation, however, was found between the morphotypes within these species and the uncovered genetic lineages. Recognition of species boundaries and of subpopulations of species will prove valuable when making informed conservation decisions as part of the development of species action plans for these rare and threatened freshwater crabs.