Describing novel mitochondrial genomes of Antarctic amphipods.

To date, only one mitogenome from an Antarctic amphipod has been published. Here, novel complete mitochondrial genomes (mitogenomes) of two morphospecies are assembled, namely, Charcotia amundseni and Eusirus giganteus. For the latter species, we have assembled two mitogenomes from different genetic clades of this species. The lengths of Eusirus and Charcotia mitogenomes range from 15,534 to 15,619 base pairs and their mitogenomes are composed of 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and 1 putative control region CR. Some tRNAs display aberrant structures suggesting that minimalization is also ongoing in amphipod mitogenomes. The novel mitogenomes of the two Antarctic species have features distinguishing them from other amphipod mitogenomes such as a lower AT-richness in the whole mitogenomes and a negative GC- skew in both strands of protein coding genes. The genetically most variable mitochondrial regions of amphipods are nad6 and atp8, while cox1 shows low nucleotide diversity among closely and more distantly related species. In comparison to the pancrustacean mitochondrial ground pattern, E. giganteus shows a translocation of the nad1 gene, while cytb and nad6 genes are translocated in C. amundseni. Phylogenetic analysis based on mitogenomes illustrates that Eusirus and Charcotia cluster together with other species belonging to the same amphipod superfamilies. In the absence of reference nuclear genomes, mitogenomes can be useful to develop markers for studying population genetics or evolutionary relationships at higher taxonomic levels.

Facilitating population genomics of non-model organisms through optimized experimental design for reduced representation sequencing.

BACKGROUND: Genome-wide data are invaluable to characterize differentiation and adaptation of natural populations. Reduced representation sequencing (RRS) subsamples a genome repeatedly across many individuals. However, RRS requires careful optimization and fine-tuning to deliver high marker density while being cost-efficient. The number of genomic fragments created through restriction enzyme digestion and the sequencing library setup must match to achieve sufficient sequencing coverage per locus. Here, we present a workflow based on published information and computational and experimental procedures to investigate and streamline the applicability of RRS. RESULTS: In an iterative process genome size estimates, restriction enzymes and size selection windows were tested and scaled in six classes of Antarctic animals (Ostracoda, Malacostraca, Bivalvia, Asteroidea, Actinopterygii, Aves). Achieving high marker density would be expensive in amphipods, the malacostracan target taxon, due to the large genome size. We propose alternative approaches such as mitogenome or target capture sequencing for this group. Pilot libraries were sequenced for all other target taxa. Ostracods, bivalves, sea stars, and fish showed overall good coverage and marker numbers for downstream population genomic analyses. In contrast, the bird test library produced low coverage and few polymorphic loci, likely due to degraded DNA. CONCLUSIONS: Prior testing and optimization are important to identify which groups are amenable for RRS and where alternative methods may currently offer better cost-benefit ratios. The steps outlined here are easy to follow for other non-model taxa with little genomic resources, thus stimulating efficient resource use for the many pressing research questions in molecular ecology.

A Survey of Transposon Landscapes in the Putative Ancient Asexual Ostracod Darwinula stevensoni.

How asexual reproduction shapes transposable element (TE) content and diversity in eukaryotic genomes remains debated. We performed an initial survey of TE load and diversity in the putative ancient asexual ostracod Darwinula stevensoni. We examined long contiguous stretches of DNA in clones from a genomic fosmid library, totaling about 2.5 Mb, and supplemented these data with results on TE abundance and diversity from an Illumina draft genome. In contrast to other TE studies in putatively ancient asexuals, which revealed relatively low TE content, we found that at least 19% of the fosmid dataset and 26% of the genome assembly corresponded to known transposons. We observed a high diversity of transposon families, including LINE, gypsy, PLE, mariner/Tc, hAT, CMC, Sola2, Ginger, Merlin, Harbinger, MITEs and helitrons, with the prevalence of DNA transposons. The predominantly low levels of sequence diversity indicate that many TEs are or have recently been active. In the fosmid data, no correlation was found between telomeric repeats and non-LTR retrotransposons, which are present near telomeres in other taxa. Most TEs in the fosmid data were located outside of introns and almost none were found in exons. We also report an N-terminal Myb/SANT-like DNA-binding domain in site-specific R4/Dong non-LTR retrotransposons. Although initial results on transposable loads need to be verified with high quality draft genomes, this study provides important first insights into TE dynamics in putative ancient asexual ostracods.

First annotated draft genomes of nonmarine ostracods (Ostracoda, Crustacea) with different reproductive modes.

Ostracods are one of the oldest crustacean groups with an excellent fossil record and high importance for phylogenetic analyses but genome resources for this class are still lacking. We have successfully assembled and annotated the first reference genomes for three species of nonmarine ostracods; two with obligate sexual reproduction (Cyprideis torosa and Notodromas monacha) and the putative ancient asexual Darwinula stevensoni. This kind of genomic research has so far been impeded by the small size of most ostracods and the absence of genetic resources such as linkage maps or BAC libraries that were available for other crustaceans. For genome assembly, we used an Illumina-based sequencing technology, resulting in assemblies of similar sizes for the three species (335-382 Mb) and with scaffold numbers and their N50 (19-56 kb) in the same orders of magnitude. Gene annotations were guided by transcriptome data from each species. The three assemblies are relatively complete with BUSCO scores of 92-96. The number of predicted genes (13,771-17,776) is in the same range as Branchiopoda genomes but lower than in most malacostracan genomes. These three reference genomes from nonmarine ostracods provide the urgently needed basis to further develop ostracods as models for evolutionary and ecological research.

Urbanization drives cross-taxon declines in abundance and diversity at multiple spatial scales.

The increasing urbanization process is hypothesized to drastically alter (semi-)natural environments with a concomitant major decline in species abundance and diversity. Yet, studies on this effect of urbanization, and the spatial scale at which it acts, are at present inconclusive due to the large heterogeneity in taxonomic groups and spatial scales at which this relationship has been investigated among studies. Comprehensive studies analysing this relationship across multiple animal groups and at multiple spatial scales are rare, hampering the assessment of how biodiversity generally responds to urbanization. We studied aquatic (cladocerans), limno-terrestrial (bdelloid rotifers) and terrestrial (butterflies, ground beetles, ground- and web spiders, macro-moths, orthopterans and snails) invertebrate groups using a hierarchical spatial design, wherein three local-scale (200 m × 200 m) urbanization levels were repeatedly sampled across three landscape-scale (3 km × 3 km) urbanization levels. We tested for local and landscape urbanization effects on abundance and species richness of each group, whereby total richness was partitioned into the average richness of local communities and the richness due to variation among local communities. Abundances of the terrestrial active dispersers declined in response to local urbanization, with reductions up to 85% for butterflies, while passive dispersers did not show any clear trend. Species richness also declined with increasing levels of urbanization, but responses were highly heterogeneous among the different groups with respect to the richness component and the spatial scale at which urbanization impacts richness. Depending on the group, species richness declined due to biotic homogenization and/or local species loss. This resulted in an overall decrease in total richness across groups in urban areas. These results provide strong support to the general negative impact of urbanization on abundance and species richness within habitat patches and highlight the importance of considering multiple spatial scales and taxa to assess the impacts of urbanization on biodiversity.

Novel Cardinium strains in non-marine ostracod (Crustacea) hosts from natural populations.

Endosymbiotic bacteria are known from many metazoan taxa, where they manipulate host biology and reproduction. Here, we used classic PCR amplification and direct DNA sequencing with universal primers for four different endosymbionts to test for their presence in more than 300 specimens of three recent non-marine ostracod superfamilies from different geographic areas and aquatic habitats. We verified these results with "high throughput" amplicon sequencing of 16S of nine selected specimens and evolutionary placement algorithms. The phylogenetic position of endosymbionts detected in ostracod hosts was compared to known endosymbionts from other metazoans. While Wolbachia, Spiroplasma and Rickettsia are absent, we find evidence for the general presence of Cardinium bacteria in natural populations of various non-marine ostracod species. Phylogenetic reconstructions based on Cardinium 16S data and estimates of genetic distances both indicate that Cardinium from ostracods are distantly related to Cardinium from Diptera and Nematoda but represent novel strains with a monophyletic origin. Cardinium bacteria from different ostracod hosts have genetic distances of up to 3.8%, providing evidence against recent and frequent horizontal transmissions amongst the three ostracod superfamilies. High throughput sequencing reveals more than 400 different 16S amplicon sequence variants in the investigated ostracods as well as the presence of different Cardinium strains within individual Eucypris virens and Heterocypris hosts. These results call for future, more in-depth investigations. Mapping Cardinium infections on COI trees of non-marine ostracod hosts shows that the occurrence of these endosymbionts is not linked to genetic species identity or phylogenetic host groups and, except for one ostracod morphospecies, prevalence never reaches 100%.

Body-size shifts in aquatic and terrestrial urban communities.

Body size is intrinsically linked to metabolic rate and life-history traits, and is a crucial determinant of food webs and community dynamics1,2. The increased temperatures associated with the urban-heat-island effect result in increased metabolic costs and are expected to drive shifts to smaller body sizes 3 . Urban environments are, however, also characterized by substantial habitat fragmentation 4 , which favours mobile species. Here, using a replicated, spatially nested sampling design across ten animal taxonomic groups, we show that urban communities generally consist of smaller species. In addition, although we show urban warming for three habitat types and associated reduced community-weighted mean body sizes for four taxa, three taxa display a shift to larger species along the urbanization gradients. Our results show that the general trend towards smaller-sized species is overruled by filtering for larger species when there is positive covariation between size and dispersal, a process that can mitigate the low connectivity of ecological resources in urban settings 5 . We thus demonstrate that the urban-heat-island effect and urban habitat fragmentation are associated with contrasting community-level shifts in body size that critically depend on the association between body size and dispersal. Because body size determines the structure and dynamics of ecological networks 1 , such shifts may affect urban ecosystem function.

Aquatic long-distance dispersal and vicariance shape the evolution of an ostracod species complex (Crustacea) in four major Brazilian floodplains.

Cladogenesis is often driven by the interplay of dispersal and vicariance. The importance of long-distance dispersal in biogeography and speciation is increasingly recognised, but still ill-understood. Here, we study faunal interconnectivity between four large Brazilian floodplains, namely the Amazon, Araguaia, Pantanal (on Paraguay River) and Upper Paraná River floodplains, investigating a species complex of the non-marine ostracod genus Strandesia. We use DNA sequence data from the mitochondrial COI and the nuclear Elongation Factor 1 alpha genes to construct molecular phylogenies and minimum spanning networks, to identify genetic species, analyse biogeographic histories and provide preliminary age estimates of this species complex. The Strandesia species complex includes five morphological and eleven genetic species, which doubles the known diversity in this lineage. The evolutionary history of this species complex appears to comprise sequences of dispersal and vicariance events. Faunal and genetic patterns of connectivity between floodplains in some genetic species are mirrored in modern hydrological connections. This could explain why we find evidence for (aquatic) long-distance dispersal between floodplains, thousands of kilometres apart. Our phylogenetic reconstructions seem to mostly indicate recent dispersal and vicariance events, but the evolution of the present Strandesia species complex could span up to 25 Myr, which by far exceeds the age of the floodplains and the rivers in their current forms.

Ostracod (Ostracoda, Crustacea) genomics - Promises and challenges.

Ostracods are well-suited model organisms for evolutionary research. Classic genetic techniques have mostly been used for phylogenetic studies on Ostracoda and were somewhat affected by the lack of large numbers of suitable markers. Genomic methods with their huge potential have so far rarely been applied to this group of crustaceans. We provide relevant examples of genomic studies on other organisms to propose future avenues of genomic ostracod research. At the same time, we suggest solutions to the potential problems in ostracods that the application of genomic techniques might present.

Redescriptions of six species of Ilyodromus Sars, 1894 (Crustacea, Ostracoda, Cyprididae) from New Zealand and Eastern Australia.

In this paper, we redescribe six species of the genus Ilyodromus Sars, 1894: I, stanleyanus (King, 1855), I. varrovillius (King, 1855), I. smaragdinus Sars, 1894, I. obtusus Sars, 1894, I. substriatus Sars, 1894 and I. viridulus (Brady, 1886) using materials stored in the Oslo museum (Norway) and (re-) described by G.O. Sars.  For each species examined, we have identified a number of additional diagnostic characters to those used by Sars and earlier authors. In particular, the length of setae, claws and segments of the antennule, antenna, sixth limb, and caudal ramus appear to be important for species delineation in the genus, as does the internal structure of the valves.

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.

Circumantarctic distribution in Southern Ocean benthos? A genetic test using the genus Macroscapha (Crustacea, Ostracoda) as a model.

The ostracod genus Macroscapha is used as a model to test theories predicting circumantarctic distribution of benthic species from the Southern Ocean. Earlier works on Antarctic Ostracoda reported five circumantarctic and/or eurybathic Macroscapha species. However, a recent taxonomic revision used a narrow morphological species definition and subdivided these five species into 20 morphospecies. Most of these narrowly defined species showed restricted depth and geographical distribution. Here, genetic markers are used to investigate the geographic and bathymetric distribution of seven species of the genus Macroscapha. The genetic results (especially COI, but partially also ITS) support more restricted geographical ranges and indicate more restricted depth distributions. Our results therefore corroborate the usefulness of a narrow morphological species definition. Our dataset also indicates that the 'genetic entities' of one species group (i.e. 'Mh. tensa-opaca') are not only geographically but also bathymetrically segregated. For that reason, a re-evaluation of the prevailing theories on the circumantarctic and eurybathic distribution of Southern Ocean benthic species is suggested.

A perspective on the importance of reproductive mode in astrobiology.

Reproduction is a vital characteristic of life, and sex is the most common reproductive mode in the eukaryotic world. Sex and reproduction are not necessarily linked mechanisms: Sexuality without reproduction exists, while several forms of asexual reproduction are known. The occurrence of sexuality itself is paradoxical, as it is very costly in evolutionary terms. Most of the hypotheses (more than 20) attempting to explain the prevalence of sex fall into two categories: Sex either creates good gene combinations for adaptation to environments or eliminates bad gene combinations counteracting the accumulation of mutations. In spite of this apparent wealth of beneficial effects of sex, asexuality is not rare. Most eukaryotic, asexual lineages are short-lived and can only persist through the presence of sexual roots, but at least two animal groups, bdelloid rotifers and darwinulid ostracods, seem to claim the status of ancient asexuals. Research on (a)sexuality is relevant to astrobiology in a number of ways. First, strong relationships between the origin and persistence of life in extreme environments and reproductive mode are known. Second, the "habitability" of nonterrestrial environments to life greatly depends on reproductive mode. Whereas asexuals can do equally well or better in harsh environments, they fail to adapt fast enough to changing abiotic and biotic environments. Third, it has been shown that plants reproduce mainly asexually in space, and sperm production and motility in some vertebrates are hampered. Both findings indicate that extraterrestrial life under conditions different from Earth might be dominated by asexual reproduction. Finally, for exchange of biological material between planets, the choice of reproductive mode will be important.

A general purpose genotype in an ancient asexual.

Many parthenogenetic species are geographically more widely distributed than their sexual relatives. Their success has been partly attributed to the existence of general purpose genotypes (GPGs). Darwinula stevensoni is an ostracod species, which has persisted for >25 million years without sex, and is both ubiquitous and cosmopolitan. Here, we test the hypothesis that this ancient asexual species may possess a highly generalised (or general purpose) genotype. The ecological tolerance of D. stevensoni was compared with asexual populations of Heterocypris incongruens, a common cypridinid species with mixed reproduction, as well as with that of another ancient asexual darwinulid species with a limited geographic and ecological distribution, Vestalenula molopoensis. The unusually wide tolerance range for both salinity (0-30 g/l) and temperature (10°C, 20°C and 30°C) of the freshwater species D. stevensoni, supports the hypothesis that this ancient asexual has indeed developed a GPG. This coincides with its wide geographic and ecological distribution and might explain its persistence as an obligate asexual in its long-term evolution. The more restricted salinity tolerance of V. molopoensis (maximum at 12 g/l) shows that not all species of the ancient asexual family Darwinulidae have a GPG. D. stevensoni has a much broader tolerance than the asexuals of H. incongruens. We argue why a GPG is most likely to develop in long-term asexuals.