Molecular tools have revolutionized the exploration of biodiversity, especially in organisms for which traditional taxonomy is difficult, such as for microscopic animals (meiofauna). Environmental (eDNA) metabarcode surveys of DNA extracted from sediment samples are increasingly popular for surveying biodiversity. Most eDNA surveys use the nuclear gene-encoding smallsubunit rDNA gene (18S) as a marker; however, different markers and metrics used for delimiting species have not yet been evaluated against each other or against morphologically defined species (morphospecies). We assessed more than 12,000 meiofaunal sequences of 18S and of the main alternatively used marker [Cytochrome c oxidase subunit I (COI) mtDNA] belonging to 55 datasets covering three taxonomic ranks. Our results show that 18S reduced diversity estimates by a factor of 0.4 relative to morphospecies, whereas COI increased diversity estimates by a factor of 7.6. Moreover, estimates of species richness using COI were robust among three of four commonly used delimitation metrics, whereas estimates using 18S varied widely with the different metrics. We show that meiofaunal diversity has been greatly underestimated by 18S eDNA surveys and that the use of COI provides a better estimate of diversity. The suitability of COI is supported by cross-mating experiments in the literature and evolutionary analyses of discreteness in patterns of genetic variation. Furthermore its splitting of morphospecies is expected from documented levels of cryptic taxa in exemplar meiofauna. We recommend against using 18S as a marker for biodiversity surveys and suggest that use of COI for eDNA surveys could provide more accurate estimates of species richness in the future.DNA barcodes | species delimitation | microinvertebrates | environmental DNA

Novel implications for the basal internal relationships of Gastrotricha revealed by an analysis of morphological characters. -Zoologica Scripta , 37 , 429-460. A cladistic analysis of Gastrotricha based on morphological characters is presented. Unlike previous morphological analyses, our study uses species rather than higher level taxa, for which the ground pattern is often unknown. The analysis comprises 79 ingroup taxa, 4 outgroup taxa and 135 binary and multistate characters in total. Character coding is based on a careful assessment of original species descriptions. Characters included cover general body organization, internal and external features as, for example, data on the adhesive tubes, digestive tract or cuticle armament. Character systems such as many ultrastructural findings, for which it was problematic to obtain data for a large set of the included taxa, were not considered. To minimize a priori assumptions, all characters were treated with equal weight and left unordered. The four outgroup representatives were chosen in accordance with the current sister group hypotheses for Gastrotricha. Two search strategies, a heuristic search (maximum parsimony) and a parsimony ratchet search, reveal a comparable scenario. Gastrotricha split into two sister taxa. One group comprises genus Neodasys only, the sister group N.N.1 (Eutubulata nom. nov.) consists of all remaining Gastrotricha. Within Eutubulata, monophyletic Macrodasyida s. str. and N.N.2 (Abursata nom. nov.) are sister taxa of highest rank. Abursata consists of the 'freshwater macrodasyids' Marinellina and Redudasys as sister group of monophyletic Paucitubulatina. Some traditional families are supported by this analysis. We evaluate possible apomorphies for the most basal stem lineages and track the evolution of selected organs. Our findings reveal that secondary character loss may play an important role in the stem lineage of Abursata and further in Paucitubulatina. Moreover, according to this analysis there might have been a single invasion of the freshwater environment in the stem lineage of Abursata followed by several independent returns to marine habitats within the monophylum Paucitubulatina.

Species of the marine meiofauna such as Gastrotricha are known to lack dispersal stages and are thus assumed to have low dispersal ability and low levels of gene flow between populations. Yet, most species are widely distributed, and this creates a paradox. To shed light on this apparent paradox, we test (i) whether such wide distribution may be due to misidentification and lumping of cryptic species with restricted distributions and (ii) whether spatial structures exist for the phylogeography of gastrotrichs. As a model, we used the genus Turbanella in NW Europe. DNA taxonomy using a mitochondrial and a nuclear marker supports distinctness of four traditional species (Turbanella ambronensis, T. bocqueti, T. mustela and T. cornuta) and provides evidence for two cryptic species within T. hyalina. An effect of geography on the within-species genetic structure is indeed present, with the potential for understanding colonization processes and for performing phylogeographic inference from microscopic animals. On the other hand, the occurrence of widely distributed haplotypes indicates long-distance dispersal as well, despite the assumed low dispersal ability of gastrotrichs.

Abstract. The reproductive anatomy of gastrotrichs is well known for several species, especially for the marine taxon Macrodasyida. However, there is little information on the reproductive organs and the modes of mating and sperm transfer in putative basal taxa, which is necessary for accurate reconstruction of the ground pattern of the Gastrotricha. We present the first detailed morphological investigation of the reproductive system of a putative basal gastrotrich, Dactylopodola typhle, using transmission and scanning electron microscopy, histology, and microscopic observations of living specimens. Dactylopodola typhle is a hermaphrodite that possesses paired female and male gonads, an unpaired uterus with an outlet channel that we call the cervix, and an additional accessory reproductive organ, the so-called caudal organ. We hypothesize that the hollow, secretory caudal organ serves for picking up autospermatozoa (self-sperm), for spermatophore formation, and finally for transferring the autospermatophore to a mating partner. The allospermatophore (foreign spermatophore) is stored within the uterus where fertilization occurs. We think that the mature and fertilized egg is released through the cervix and the dorsolateral female gonopore, and not by rupture of the body wall. Based on the morphology, we provide a plausible hypothesis for spermatophore formation and transfer in D. typhle. Preliminary phylogenetic considerations indicate that the stem species of Macrodasyida, perhaps that of all Gastrotricha, had paired ovaries and paired testes, an unpaired uterus, and only one accessory reproductive organ.