Grazing insects reduce algal biomass in a neotropical stream

The Darwinulidae are the only surviving post-Palaeozoic darwinulocopine family of an extensive radiation that reached its maximum in the Permian. Whereas at least some Palaeozoic darwinulids are known from sexual populations, the surviving lineages after the end-Permian mass extinction have abandoned sex since 200 million years ago. This makes the extant family Darwinulidae one of the few putative ancient asexual groups. Only about 30 species in 5 genera are presently known. The phylogeny of these taxa is here analysed using both morphological characters and molecular data. Twelve characters on valve morphology and 17 characters pertaining to appendages are used to construct the most parsimonious (unrooted) cladogram of 12 species in 5 genera. DNA sequences of one nuclear (ITS1) and a mitochondrial (COI) gene of 6 species in 5 genera are used to construct rooted maximum parsimony trees. Both molecular and morphological trees show a high degree of congruence, indicating that Alicenula and Vestalenula mostly cluster closely together, while Penthesilenula and Microdarwinula constitute a robust group. The position of the monospecific genus Darwinulais more variable, but is mostly closer to the two former genera. Congeneric species always cluster together in the morphological cladograms, and these results thus confirm that the five genera recognised by Rossetti & Martens (1998) are good phyletic units. An approximate molecular clock (calibrated with fossil data) indicates that the extant darwinulids share a common ancestor, which lived at least 100 million years ago.     

Evolution in the slow lane: molecular rates of evolution in sexual and asexual ostracods (Crustacea: Ostracoda)

Parthenogenetic lineages within non-marine ostracods can occur either in mixed (with sexual and asexual females) or exclusively asexual taxa. The former mode of reproduction is associated with a high intraspecific diversity at all levels (genetic, morphological, ecological) and, at least in the Cypridoidea, with geographical parthenogenesis. Obligate asexuality is restricted to the Darwinuloidea, the strongest candidate for an ancient asexual animal group after the bdelloid rotifers, and is characterized by low diversity. We have compared rates of molecular evolution for the nuclear ITS1 region and the mitochondrial COI gene amongst the three major lineages of non-marine ostracods with sexual, mixed and asexual reproduction. Absolute rates of molecular evolution are low for both regions in the darwinulids. The slow-down of evolution in ITS1 that has been observed for Darwinula stevensoni (Brady & Robertson) apparently does not occur in other darwinulid species. ITS1 evolves more slowly than COI within non-marine ostracod families, including the darwinulids, but not between superfamilies. The ancient asexuals might have a higher relative substitution rate in ITS1, as would be expected from hypotheses that predict the accumulation of mutations in asexuals. However, the speed-up of ITS could also be ancient, for example through the stochastic loss of most lineages within the superfamily after the Permian–Triassic mass extinction. In this case, the difference in rate would have occurred independently from any effects of asexual reproduction.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 93–100.     

Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda)

Background

Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied.

Methodology/Principal Findings

We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies.

Conclusions/Significance

Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.     

Genetic diversity of <Emphasis Type="Italic">Haemaphysalis qinghaiensis</Emphasis> (Acari: Ixodidae) in western China

Haemaphysalis qinghaiensis as an endemic species in China mainly infests domestic animals and causes great harm to animals and humans in Northwestern plateau. However, there is no information about genetic diversity within the recently established populations of this tick species. Therefore, the present study analyzed the fragments of mitochondrial 16S rDNA, COI and the nuclear gene ITS1 of 56 H. qinghaiensis ticks across four regions of China which are main endemic areas of this species. Analysis showed 98.1–100% (16S rDNA), 97.9–100% (COI), 99.7–100% (ITS1) identity within individuals. For these sequences, 9, 15 and 8 haplotypes were found for 16S rDNA, COI and ITS1, respectively. Ticks from Yongjing were the most variable group, followed by Lintan, Huangyuan, and Tianzhu. Based on parallel analysis of the mitochondrial and nuclear genetic diversity of H. qinghaiensis, our results indicated that mitochondrial markers (especially COI) were much more useful than nuclear ITS for intraspecific genetic variability analysis.     

Molecular phylogenetic analysis supports the synonymy of Prodontria modesta (Broun) and Prodontria bicolorata Given (Coleoptera: Scarabaeidae: Melolonthinae)

The validity of the synonymy of Prodontria modesta (Broun) and the nationally endangered Prodontria bicolorata Given, both flightless with very restricted distributions in Central Otago, New Zealand, was investigated using molecular phylogenetic analyses of a section of the mitochondrial gene region coding for cytochrome oxidase subunit 1 (COI) and nuclear rDNA internal transcribed spacer region (ITS1). Maximum likelihood analysis of the COI data found no separate, monophyletic lineages linked to colour form among six populations of the beetle and the genetic variation observed was less than that recorded within many other beetle species, including other melolonthines. The synonymisation of P. bicolorata with P. modesta should be considered valid and conservation efforts should focus on protecting habitats and populations, rather than on preserving colour forms.     

Ecological strategies in the ancient asexual animal group Darwinulidae (Crustacea, Ostracoda)

• 1 We investigated the relationship between geographical distribution and ecological tolerance within the ancient asexual family Darwinulidae. Distribution maps were compiled based on data from the literature, the Non‐marine Ostracod Distribution in Europe database and personal collections. Ecological tolerance was assessed experimentally by exposing individual ostracods to a combination of eight different salinities (range from 0 to 30 g L^?1) and three different temperatures (10, 20 and 30 °C).
• 2 The type species of the family, Darwinula stevensoni, is ubiquitous and cosmopolitan; the two species Penthesilenula brasiliensis and Microdarwinula zimmeri also have an intercontinental distribution. Two other darwinulid species tested here (Vestalenula molopoensis and P. aotearoa) are known only from their type localities. The latter is also true for most extant darwinulids.
• 3 Darwinula stevensoni and P. brasiliensis had a broad salinity tolerance, tolerating distilled water and also salinity up to 25–30 g L^?1, whereas the maximum salinity tolerance of V. molopoensis was 12 g L^?1 and of P. aotearoa, 20 g L^?1.
• 4 The results indicate that both ecological specialists and generalists, as well as intermediate forms, exist in the Darwinulidae and that taxa with the broadest ecological tolerance also have the widest distribution.

     

Integrative taxonomy in two free-living nematode species complexes

Integrative taxonomy considers species boundaries from multiple, complementary perspectives, with the main objective being to compare the observed data against the predictions of the methodologies used. In the present study we used three methods for delineating species boundaries within the cosmopolitan nematode species Rhabditis ( Pellioditis ) marina and Halomonhystera disjuncta . First, phylogenetic relationships among molecular sequences from the mitochondrial cytochrome oxidase c subunit 1 gene (COI), and from two nuclear regions, internal transcribed spacer (ITS) and D2D3, were analysed. Subsequently, multivariate morphometric analysis was used to investigate whether concordant molecular lineages were also morphologically distinct. When morphological differences were found, typological taxonomy was performed to identify fixed or non-overlapping characters between lineages. Interbreeding experiments were conducted between the two closest related lineages of R . ( P. ) marina to investigate potential reproductive isolation. This integrative approach confirmed the presence of several species within each nominal species: molecular lineages were concordant across two independent loci (COI and ITS), and were characterized by significant morphological divergence. Most lineages were also detectable in the D2D3 region, but were less resolved. The two lineages investigated in our study did not produce offspring. Our results highlight that classical taxonomy grossly underestimates species diversity within the phylum Nematoda.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 737–753.     

Evidence for cryptic hybridization between different evolutionary lineages of the invasive clam genus Corbicula (Veneroida,Bivalvia)

We studied two Corbicula morphotypes in a syntopic population in the Rhine River in order to reveal their taxonomic, reproductive and phylogenetic relationship, using morphometrics, DAF‐fingerprinting, mitochondrial COI and nuclear ITS1 sequence variation. Morphometric analysis showed that two statistically distinguishable morphotypes with few intermediates were present.Mitochondrial sequence analysis detected two divergent clades. DAF‐fingerprinting revealed three highly distinctive multilocus genotypes. Two of the multilocus genotypes were significantly associated with different morphotypes and mitochondrial lineages. The third genotype B, however, was found in both morphotypes, intermediates and mitochondrial lineages. Conclusive evidence for hybridization came from RFLP analysis of the nuclear ITS1 locus. We interpret the hybrids as F1 hybrids between different evolutionary lineages. Integration of Corbicula sequences from all over the world into Maximum Parsimony analysis suggested a simultaneous radiation resulting in several evolutionary lineages whose species status remained doubtful. An unequivocal taxonomic assignment of the two evolutionary lineages in the Rhine population was therefore not possible.     

Genetic diversity in Australian ancient asexual <Emphasis Type="Italic">Vestalenula</Emphasis> (Ostracoda,Darwinulidae): little variability down under

Darwinulid ostracods are putative ancient asexuals, and are thus assumed to be unable to purge deleterious mutations from their genomes. Some darwinulids species can be found both above (epigeic) and below ground (hypogeic). We hypothesize that surface populations carry more mutations than their below-ground counterparts, which are buffered from mutagens such as UV-B. Given the age of the investigated area, the Pilbara in Western Australia, we also expect geographic patterning of observed haplotypes. We have used DNA sequence data from the nuclear ITS and the mitochondrial COI region to investigate a (limited) data set on two Australian species, the endemic Vestalenula matildae and V. marmonieri from the Pilbara region. We do not find differences in genetic variability between specimens from subterranean habitats as compared to those from habitats above ground. There was also no congruence between hydrological basins and distribution patterns of the haplotypes identified. Although our data indicate that the two species may have split from each other ca. 70 myr ago, this has not resulted in any clear phylogeographic patterns among the analysed specimens across the regions of the Pilbara.     

Multilocus sequence evaluation for differentiating species of the trematode Family Gastrothylacidae,with a note on the utility of mitochondrial COI motifs in species identification

Amphistomiasis, a neglected trematode infectious disease of ruminants, is caused by numerous species of amphistomes belonging to six families under the Superfamily Paramphistomoidea. In the present study, four frequently used DNA markers, viz. nuclear ribosomal 28S (D1–D3 regions), 18S and ITS2 and mitochondrial COI genes, as well as sequence motifs from these genes were evaluated for their utility in species characterization of members of the amphistomes' Family Gastrothylacidae commonly prevailing in Northeast India. In sequence and phylogenetic analyses the COI gene turned out to be the most useful marker in identifying the gastrothylacid species, with the exception of Gastrothylax crumenifer, which showed a high degree of intraspecific variations among its isolates. The sequence analysis data also showed the ITS2 region to be effective for interspecies characterization, though the 28S and 18S genes were found unsuitable for the purpose. On the other hand, sequence motif analysis data revealed the motifs from the COI gene to be highly conserved and specific for their target species which allowed accurate in silico identification of the gastrothylacid species irrespective of their intraspecific differences. We propose the use of COI motifs generated in the study as a potential tool for identification of these species.     

A possible circulation of a dominant Dibothriocephalus nihonkaiensis haplotype in Japan revealed by molecular analysis of clinical tapeworm samples

Human diphyllobothriasis, caused by Dibothriocephalus nihonkaiensis, is prevalent globally, especially in regions where raw fish is consumed. Recent molecular diagnostic techniques have made species identification of tapeworm parasites and the determination of genetic variations among parasite populations possible. However, only a few studies done over a decade ago, have reported on the genetic variation among D. nihonkaiensis in Japan. The present study employed PCR-based mitochondrial DNA analysis to specifically detect D. nihonkaiensis from archived clinical samples, and to determine any genetic variation that may exist among the Japanese broad tapeworms from patients of Kanagawa Prefecture, Japan. Target genes were amplified from DNA extracted from the ethanol- or formaldehyde-fixed samples by PCR. Further sequencing and comparative phylogenetic analyses based on mitochondrial COI and ND1 sequences were also performed. In our results, all PCR-amplified and sequenced samples were identified as D. nihonkaiensis. Analysis of COI sequences revealed two haplotype lineages. However, clustering of almost all COI (and ND1) sample sequences into one of the two haplotype clades, together with reference sequences from different countries worldwide, revealed a common haplotype among D. nihonkaiensis samples in our study. Our results suggest a possible presence of a dominant D. nihonkaiensis haplotype, with a global distribution circulating in Japan. Results from this study have the potential to improve the management of clinical cases and establish robust control measures to reduce the burden of human diphyllobothriasis in Japan.     

DNA barcodes and phylogenetic affinities of the terrestrial slugs Arion gilvus and A. ponsi (Gastropoda,?Pulmonata,Arionidae)

The Iberian Peninsula is a region with a high endemicity of species of the terrestrial slug subgenus Mesarion. Many of these species have been described mainly on subtle differences in their proximal genitalia. It therefore remains to be investigated 1) whether these locally diverged taxa also represent different species under a phylogenetic species concept as has been shown for other Mesarion species outside the Iberian Peninsula, and 2) how these taxa are phylogenetically related. Here, we analysed DNA sequence data of two mitochondrial (COI and 16S) genes, and of the nuclear ITS1 region, to explore the phylogenetic affinities of two of these endemic taxa, viz. Arion gilvus Torres Mínguez, 1925 and A. ponsi Quintana Cardona, 2007. We also evaluated the use of these DNA sequence data as DNA barcodes for both species. Our results showed that ITS did not allow to differentiate among most of the Mesarion molecular operational taxonomic units (MOTUs) / morphospecies in Mesarion. Yet, the overall mean p-distance among the Mesarion MOTUs / morphospecies for both mtDNA fragments (16.7% for COI, 13% for 16S) was comparable to that between A. ponsi and its closest relative A. molinae (COI: 14.2%; 16S: 16.2%) and to that between A. gilvus and its closest relative A. urbiae (COI: 14.4%; 16S: 13.4%). Hence, with respect to mtDNA divergence, both A. ponsi and A. gilvus, behave as other Mesarion species or putative species-level MOTUs and thus are confirmed as distinct ‘species’.     

Desmophyllum dianthus (Esper, 1794) in the Scleractinian Phylogeny and Its Intraspecific Diversity

The cosmopolitan solitary deep-water scleractinian coral Desmophyllum dianthus (Esper, 1794) was selected as a representative model species of the polyphyletic Caryophylliidae family to (1) examine phylogenetic relationships with respect to the principal Scleractinia taxa, (2) check population structure, (3) test the widespread connectivity hypothesis and (4) assess the utility of different nuclear and mitochondrial markers currently in use. To carry out these goals, DNA sequence data from nuclear (ITS and 28S) and mitochondrial (16S and COI) markers were analyzed for several coral species and for Mediterranean populations of D. dianthus. Three phylogenetic methodologies (ML, MP and BI), based on data from the four molecular markers, all supported D. dianthus as clearly belonging to the “robust” clade, in which the species Lophelia pertusa and D. dianthus not only grouped together, but also shared haplotypes for some DNA markers. Molecular results also showed shared haplotypes among D. dianthus populations distributed in regions separated by several thousands of kilometers and by clear geographic barriers. These results could reflect limited molecular and morphological taxonomic resolution rather than real widespread connectivity. Additional studies are needed in order to find molecular markers and morphological features able to disentangle the complex phylogenetic relationship in the Order Scleractinia and to differentiate isolated populations, thus avoiding the homoplasy found in some morphological characters that are still considered in the literature.     

Genetic variation and population structure of the carpet shell clam <Emphasis Type="Italic">Ruditapes decussatus</Emphasis> along the Tunisian coast inferred from mtDNA and ITS1 sequence analysis

Surveys of allozyme polymorphisms in the carpet shell clam Ruditapes decussatus have revealed sharp genetic differentiation of populations. Analysis of population structure in this species has now been extended to include nuclear and mitochondrial genes. A partial sequence of a mitochondrial COI gene and of the internal transcribed spacer region (ITS-1) were used to study haplotype distribution, the pattern of gene flow, and population genetic structure of R. decussatus. The samples were collected from twelve populations from the eastern and western Mediterranean coasts of Tunisia, one from Concarneau and one from Thau. A total of twenty and twenty-one haplotypes were detected in the examined COI and ITS1 regions respectively. The study revealed higher levels of genetic diversity for ITS1 compared to COI. The analysis of haplotype frequency distribution and molecular variation indicated that the majority of the genetic variation was distributed within populations (93% and 86% for COI and ITS1 respectively). No significant differentiation was found among eastern and western groups on either side of the Siculo-Tunisian strait. However, distinct and significant clinal changes in haplotypes frequencies between eastern and western samples were found at the most frequent COI haplotype and at three out of five major ITS1 haplotypes. These results suggest the relative importance of historical processes and contemporary hydrodynamic features on the observed patterns of genetic structure.     

The genus Cyclops (Copepoda,Cyclopoida) in Europe

Although copepods of the genus Cyclops are among the most common and dominant plankton taxa of lakes in the northern temperate zone, their taxonomy is still unclear. We analysed an extensive array of Cyclops populations from Europe by means of molecular methods and evaluated morphological characters. Altogether, 68 populations of Cyclops species were sampled, assigned to morphospecies and sequenced for the 12S rRNA gene. Selected populations of each morphospecies were additionally sequenced for three mitochondrial (16S rRNA, cytochrome b, COI) and two nuclear genes (18S rRNA, ITS1) and analysed for micromorphological traits. Our analysis revealed fifteen lineages that can be regarded as separate species. Thirteen of these match currently accepted species, while the remaining two lineages were distinct from the other described species. Thus, their taxonomic status is open to further studies. Besides taxonomy, our study gives new insights into the ecology, distribution and phylogenetic relationships of these species. Finally, a set of morphological traits was selected to facilitate identification.     

Hidden Mediterranean biodiversity: molecular evidence for a cryptic species complex within the reef building vermetid gastropod Dendropoma petraeum (Mollusca: Caenogastropoda)

The reef building vermetid gastropod Dendropoma petraeum inhabits the warmest waters of the Mediterranean Sea and is considered a threatened marine species. The aim of this study was to characterize its genetic structure throughout its whole distribution range using mitochondrial and nuclear sequence data. Because of its sessile adult lifestyle and lack of a pelagic larval stage, we expected a markedly subdivided population structure with limited levels of gene flow. Fragments of the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S rRNA (16S), were sequenced, along with the nuclear ribosomal cluster (internal transcribed spacer; ITS) in specimens from 18 localities. Our analyses identified four highly distinct phylogroups separated by a mean divergence of > 14% according to the COI sequence data or > 9% according to 16S, but differing only slightly in morphology. The nuclear data (ITS) indicated a lower substitution rate (divergence among groups of around 1%). These large genetic distances among the four lineages clearly point to the existence of a cryptic species complex within D. petraeum comprising at least four species. Differences in the characteristics of intracapsular larval development and protoconch were also detected among these lineages. The allopatric distribution of these cryptic species supports a predominantly vicariant-based cladogenetic pattern for the genus Dendropoma in the Mediterranean.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 898–912.     

Differentiation of the dragonfly genus Davidius (Odonata: Gomphidae) in Japan inferred from mitochondrial and nuclear gene genealogies

To infer the differentiation of Japanese Davidius dragonflies, we investigated the genealogies of the mitochondrial cytochrome oxidase subunit I gene (COI) and the nuclear ribosomal RNA gene region encompassing 18S, ITS1, 5.8S, and ITS2 sequences for three species endemic to Japan--Davidius nanus, D. fujiama, and D. moiwanus--as well as D. lunatus from the Korean Peninsula. According to the mitochondrial and nuclear gene genealogies, D. nanus and D. moiwanus are closely related and are sister to the continental species D. lunatus, whereas D. fujiama differentiated from an ancestor of the other three species. Although the mitochondrial DNA data did not resolve the relationships between D. nanus and three D. moiwanus subspecies, the nuclear DNA data indicate the monophyly of D. moiwanus and its subspecies. The nuclear gene genealogy suggests that isolated wetlands used by larval D. moiwanus derive from the ancestral riverine habitats of D. nanus and other Davidius species. The COI sequence divergence among local populations was much greater in D. moiwanus than in D. nanus, which may be the result of differences in the dispersal ranges associated with the habitat types of these species.     

Reassessment of Morphological Diagnostic Characters and Species Boundaries Requires Taxonomical Changes for the Genus Orthopyxis L. Agassiz, 1862 (Campanulariidae,Hydrozoa) and Some Related Campanulariids

The genus Orthopyxis is widely known for its morphological variability, making species identification particularly difficult. A number of nominal species have been recorded in the southwestern Atlantic, although most of these records are doubtful. The goal of this study was to infer species boundaries in the genus Orthopyxis from the southwestern Atlantic using an integrative approach. Intergeneric limits were also tested using comparisons with specimens of the genus Campanularia. We performed DNA analyses using the mitochondrial genes 16S and COI and the nuclear ITS1 and ITS2 regions. Orthopyxis was monophyletic in maximum likelihood analyses using the combined dataset and in analyses with 16S alone. Four lineages of Orthopyxis were retrieved for all analyses, corresponding morphologically to the species Orthopyxis sargassicola (previously known in the area), Orthopyxis crenata (first recorded for the southwestern Atlantic), Orthopyxis caliculata (= Orthopyxis minuta Vannucci, 1949 and considered a synonym of O. integra by some authors), and Orthopyxis mianzani sp. nov. A re-evaluation of the traditional morphological diagnostic characters, guided by our molecular analyses, revealed that O. integra does not occur in the study area, and O. caliculata is the correct identification of one of the lineages occurring in this region, corroborating the validity of that species. Orthopyxis mianzani sp. nov. resembles O. caliculata with respect to gonothecae morphology and a smooth hydrothecae rim, although it shows significant differences for other characters, such as perisarc thickness, which has traditionally been thought to have wide intraspecific variation. The species O. sargassicola is morphologically similar to O. crenata, although they differ in gonothecae morphology, and these species can only be reliably identified when this structure is present.     

Cryptic species in tropic sands--interactive 3D anatomy, molecular phylogeny and evolution of meiofaunal Pseudunelidae (Gastropoda, Acochlidia)

Background

Towards realistic estimations of the diversity of marine animals, tiny meiofaunal species usually are underrepresented. Since the biological species concept is hardly applicable on exotic and elusive animals, it is even more important to apply a morphospecies concept on the best level of information possible, using accurate and efficient methodology such as 3D modelling from histological sections. Molecular approaches such as sequence analyses may reveal further, cryptic species. This is the first case study on meiofaunal gastropods to test diversity estimations from traditional taxonomy against results from modern microanatomical methodology and molecular systematics.

Results

The examined meiofaunal Pseudunela specimens from several Indo-Pacific islands cannot be distinguished by external features. Their 3D microanatomy shows differences in the organ systems and allows for taxonomic separation in some cases. Additional molecular analyses based on partial mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA markers revealed considerable genetic structure that is largely congruent with anatomical or geographical patterns. Two new species (Pseudunela viatoris and P. marteli spp. nov.) are formally described integrating morphological and genetic analyses. Phylogenetic analysis using partial 16S rRNA, COI and the nuclear 18S rRNA markers shows a clade of Pseudunelidae species as the sister group to limnic Acochlidiidae. Within Pseudunela, two subtypes of complex excretory systems occur. A complex kidney already evolved in the ancestor of Hedylopsacea. Several habitat shifts occurred during hedylopsacean evolution.

Conclusions

Cryptic species occur in tropical meiofaunal Pseudunela gastropods, and likely in other meiofaunal groups with poor dispersal abilities, boosting current diversity estimations. Only a combined 3D microanatomical and molecular approach revealed actual species diversity within Pseudunela reliably. Such integrative methods are recommended for all taxonomic approaches and biodiversity surveys on soft-bodied and small-sized invertebrates. With increasing taxon sampling and details studied, the evolution of acochlidian panpulmonates is even more complex than expected.