A chromosome-level reference genome for the Versatile Fairy Shrimp, Branchinecta lindahli.

We present the novel reference genome of the Versatile Fairy Shrimp, Branchinecta lindahli. The Versatile Fairy Shrimp is a freshwater anostracan crustacean found across the western United States from Iowa to Oregon and from Alberta to Baja California. It is an ephemeral pool specialist, living in prairie potholes, irrigation ditches, tire treads, vernal pools, and other temporary freshwater wetlands. Anostracan fairy shrimp are facing global declines with 3 species in California on the Endangered Species list. This species was included in the California Conservation Genomics Project to provide an easily accessible reference genome, and to provide whole-genome resources for a generalist species, which may lead to new insights into Anostracan resiliency in the face of climate change. The final gapped genome comprises 15 chromosome-length scaffolds covering 98.63% of the 384.8 Mb sequence length, and an additional 55 unscaffolded contigs.

Comparative Mitogenomic Analyses and New Insights into the Phylogeny of Thamnocephalidae (Branchiopoda: Anostraca).

Thamnocephalidae, a family of Anostraca which is widely distributed on all continents of the world except Antarctica, currently consists of six genera and approximately 63 recognized species. The relationships among genera in Thamnocephalidae and the monophyly of Thamnocephalidae, determined using morphological characteristics or gene markers, remain controversial. In order to address the relationships within Thamnocephalidae, we sequenced Branchinella kugenumaensis mitogenomes and conducted a comparative analysis to reveal the divergence across mitogenomes of B. kugenumaensis. Using newly obtained mitogenomes together with available Anostracan genomic sequences, we present the most complete phylogenomic understanding of Anostraca to date. We observed high divergence across mitogenomes of B. kugenumaensis. Meanwhile, phylogenetic analyses based on both amino acids and nucleotides of the protein-coding genes (PCG) provide significant support for a non-monophyletic Thamnocephalidae within Anostraca, with Asian Branchinella more closely related to Streptocephalidae than Australian Branchinella. The phylogenetic relationships within Anostraca were recovered as follows: Branchinectidae + Chirocephalidae as the basal group of Anostraca and halophilic Artemiidae as a sister to the clade Thamnocephalidae + Streptocephalidae. Both Bayesian inference (BI)- and maximum likelihood (ML)-based analyses produced identical topologies.

Bacterial communities in Branchinella thailandensis and Streptocephalus sirindhornae.

The fairy shrimp is a freshwater crustacean found in both temporary and permanent freshwaters. In Thailand, fairy shrimp are farmed as live food for ornamental fish. This study aimed to investigate the bacterial compositions in two fairy shrimp species, Branchinella thailandensis and Streptocephalus sirindhornae. Both species were cultured, and total DNA was extracted. The V3-V4 region of the 16S rRNA gene was amplified and sequenced using Illumina Miseq. All data were analyzed by Illumina 16S Metagenomics (version 1.0.1) workflow in Base Space- Illumina. Each read was blasted against the Illumina-curated version of the Greengenes database to determine the operational taxonomic units (OTUs) corresponding to the 16S rRNA gene sequence. The results showed that the Shanon-Weiner diversity index of bacterial compositions in B. thailandensis and S. sirindhornae were 2.135 and 3.122, respectively. The evenness and genus-level richness of the bacterial composition in B. thailandensis were 0.364 and 354 genera, respectively. The dominant bacterium found in B. thailandensis was Nevskia. In S. sirindhornae, the evenness and genus-level richness of the bacterial composition were 0.521 and 400 genera, respectively. Azohydromonas was the dominant bacterium. Our results showed that the compositions and proportions of bacterial communities were specific to each species of fairy shrimp. This study will be useful for further experiments in aquaculture and ecological studies related to symbiotic interaction.

Pleistocene allopatric differentiation followed by recent range expansion explains the distribution and molecular diversity of two congeneric crustacean species in the Palaearctic.

Pleistocene glaciations had a tremendous impact on the biota across the Palaearctic, resulting in strong phylogeographic signals of range contraction and rapid postglacial recolonization of the deglaciated areas. Here, we explore the diversity patterns and history of two sibling species of passively dispersing taxa typical of temporary ponds, fairy shrimps (Anostraca). We combine mitochondrial (COI) and nuclear (ITS2 and 18S) markers to conduct a range-wide phylogeographic study including 56 populations of Branchinecta ferox and Branchinecta orientalis in the Palaearctic. Specifically, we investigate whether their largely overlapping ranges in Europe resulted from allopatric differentiation in separate glacial refugia followed by a secondary contact and reconstruct their postglacial recolonization from the inhabited refugia. Our results suggest the existence of distinct refugia for the two species, with genetic divergence among intraspecific lineages consistent with late Pleistocene glacial cycles. While B. ferox lineages originated from Mediterranean refugia, the origin of B. orientalis lineages was possibly located on the Pannonian Plain. We showed that most dispersal events predominantly happened within 100 km, coupled with several recent long-distance events (> 1000 km). Hence the regional habitat density of suitable habitats in Central Europe is possibly a key to the co-existence of the two species. Overall, our study illustrates how isolation in combination with stochastic effects linked to glacial periods are important drivers of the allopatric differentiation of Palaearctic taxa.

Environmental DNA sampling provides new management strategies for vernal pool branchiopods in California.

California's vernal pools are declining ecosystems that support valuable native plant and animal diversity. Vernal pool branchiopods are particularly at risk from vernal pool habitat loss and conservation efforts have targeted their long-term protection through the establishment of preserves and conservation banks. These conservation strategies require repeated, perpetual monitoring of preserved habitat, which is currently carried out through dip-net surveys and visual identification of specimens. Dip-netting may be destructive and frequently requires some sacrifice of protected species. Environmental DNA offers a new, modern method to monitor many protected freshwater organisms. We designed qPCR-based species-specific assays for four of California's vernal pool branchiopods: The Vernal Pool Fairy Shrimp Branchinecta lynchi (BRLY), the Midvalley Fairy Shrimp Branchinecta mesovallensis (BRME), and the Conservancy Fairy Shrimp Branchinecta conservatio (BRCO), and the Vernal Pool Tadpole Shrimp Lepidurus packardi (LEPA). We tested these assays using eDNA sampling protocols alongside traditional dip-net surveys to assess their viability as an alternative method to monitor vernal pool branchiopods. Based on occupancy modeling, each of our assays achieved a 95% or higher detection rate when using optimized sampling protocols.

Wnt repertoire and developmental expression patterns in the crustacean Thamnocephalus platyurus.

Wnt genes are a family of conserved glycoprotein ligands that play a role in a wide variety of cell and developmental processes, from cell proliferation to axis elongation. There are 13 Wnt subfamilies found among metazoans. Eleven of these appear conserved in arthropods with a pattern of loss during evolution of as many as six subfamilies among hexapods. Here we report on Wnt genes in the branchiopod crustacean, Thamnocephalus platyurus, including the first documentation of the expression of the complete Wnt gene family in a crustacean. Our results suggest fewer Wnt genes were retained in Thamnocephalus than in the related crustacean, Daphnia, although the Thamnocephalus Wnt repertoire is larger than that found in insects. We also find an intriguing pattern of staggered expression of Wnts-an anterior-posterior stagger within the posterior growth zone and a dorsal-ventral stagger in the developing segments-suggesting a potential for subfunctionalization of Wnts in these regions.

Morphometric and preliminary genetic characteristics of Branchinecta orientalis populations from Iran (Crustacea: Anostraca).

Branchinecta orientalis is a fairy shrimp endemic to the Palearctic region, from Mongolia to Spain. The patchy nature of its habitat is thought to result in a high degree of subdivision among populations, potentially promoting speciation. We combined morphometric characteristics with molecular phylogeny of cytochrome c oxidase I (COI) to test whether B. orientalis could be a species complex and whether there is any correlation between the genetic variation, morphometric characteristics and geographical variables. We studied six populations from Iran based on the comparison of morphometric and molecular datasets, we confirmed that the Aigher Goli (AIG) population is biometrically well separated from the Akh Gol, Hassar, Rashakan, Khaslou and Garagojanlou populations in northwestern Iran. The relatively high genetic divergence in the AIG from the other populations and its congruence with morphometric data were observed in B. orientalis populations. However, as these results were generated using a small sample size and on a limited sampling range, they should be considered as preliminary.

The complete mitogenome of the fairy shrimp Phallocryptus tserensodnomi (Crustacea: Anostraca: Thamnocephalidae).

The sequence of the mitochondrial genome (mitogenome) of the fairy shrimp Phallocryptus tserensodnomi Alonso & Ventura 2013 (Crustacea: Anostraca: Thamnocephalidae) has been determined. It is 16,493 bp with an AT-content of 65.4%, which encodes information of the typical 37 genes as all other metazoan mitogenomes. Both AT-content and putative control region of the genome show moderate values among all mitogenomes of the Branchiopoda sequenced to date. The mitochondrial gene order shows the same arrangement with the Artemiidae which is different from the pancrustacean ancestral pattern, due to translocation and inversion of two tRNA genes. Our results will provide important materials for not only phylogenetic but also biogeographic studies of the Anostraca.

The complete mitogenome of the freshwater fairy shrimp Streptocephalus sirindhornae (Crustacea: Anostraca: Streptocephalidae).

In this study, we amplified, sequenced and analyzed the complete mitogenome of the freshwater fairy shrimp Streptocephalus sirindhornae (Crustacea: Anostraca: Streptocephalidae). The full-length of the S. sirindhornae mitogenome is a circular molecule of 16,887 bp in size with an A + T content of 64.5%. It has the largest putative control region (2794 bp) with the lowest A + T content (62.6%) for all determined branchiopod mitogenomes. The genome consisted of 37 genes that are involved in the respiration chain as well as the mitochondrial translation system. The S. sirindhornae mitogenome exhibits an identical gene arrangement as the Artemia pattern, which shows translocation and inversion of two transfer-RNA genes compared to the pancrustacean ancestral pattern. This is by far the first determined mitogenome of a freshwater fairy shrimp. The results of our study will provide significant data for reconstructing the consensus Branchiopoda tree of life.

A new fairy shrimp Phallocryptus tserensodnomi (Branchiopoda: Anostraca) from Mongolia.

A new species of Phallocryptus Biraben 1951 (Branchiopoda, Anostraca) from Mongolia is described. Phallocryptus tserensodnomi sp. nov. is close to P. spinosa (Milne-Edwards 1840), but both morphological and molecular analyses (Cytochrome Oxidase I, COI) indicate that they represent separate species. Most relevant differential features of the new species include: (1) frontal appendage provided with small ventral conical outgrowths; (2) second antennamere evenly curved, sickle-shaped; (3) distal fleshy process on labrum evenly curved forwards and tapering; (4) short stout acute spine-like projections present at each side of basal portion of gonopods; (5) female second antennae shorter and wider than in P. spinosa, tapering. Based on morphological comparisons the new species appears to be a Mongolian endemic, although some genotypes of presumed P spinosa from Africa are similar to the new species, suggesting P. tserensodnomi might have a wider distribution.

Mitochondrial DNA regionalism and historical demography in the extant populations of Chirocephalus kerkyrensis (Branchiopoda: Anostraca).

BACKGROUND: Mediterranean temporary water bodies are important reservoirs of biodiversity and host a unique assemblage of diapausing aquatic invertebrates. These environments are currently vanishing because of increasing human pressure. Chirocephalus kerkyrensis is a fairy shrimp typical of temporary water bodies in Mediterranean plain forests and has undergone a substantial decline in number of populations in recent years due to habitat loss. We assessed patterns of genetic connectivity and phylogeographic history in the seven extant populations of the species from Albania, Corfu Is. (Greece), Southern and Central Italy. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed sequence variation at two mitochondrial DNA genes (Cytochrome Oxidase I and 16s rRNA) in all the known populations of C. kerkyrensis. We used multiple phylogenetic, phylogeographic and coalescence-based approaches to assess connectivity and historical demography across the whole distribution range of the species. C. kerkyrensis is genetically subdivided into three main mitochondrial lineages; two of them are geographically localized (Corfu Is. and Central Italy) and one encompasses a wide geographic area (Albania and Southern Italy). Most of the detected genetic variation (≈81%) is apportioned among the aforementioned lineages. CONCLUSIONS/SIGNIFICANCE: Multiple analyses of mismatch distributions consistently supported both past demographic and spatial expansions with the former predating the latter; demographic expansions were consistently placed during interglacial warm phases of the Pleistocene while spatial expansions were restricted to cold periods. Coalescence methods revealed a scenario of past isolation with low levels of gene flow in line with what is already known for other co-distributed fairy shrimps and suggest drift as the prevailing force in promoting local divergence. We recommend that these evolutionary trajectories should be taken in proper consideration in any effort aimed at protecting Mediterranean temporary water bodies.

Long distance dispersal of zooplankton endemic to isolated mountaintops--an example of an ecological process operating on an evolutionary time scale.

Recent findings suggest a convergence of time scales between ecological and evolutionary processes which is usually explained in terms of rapid micro evolution resulting in evolution on ecological time scales. A similar convergence, however, can also emerge when slow ecological processes take place on evolutionary time scales. A good example of such a slow ecological process is the colonization of remote aquatic habitats by passively dispersed zooplankton. Using variation at the protein coding mitochondrial COI gene, we investigated the balance between mutation and migration as drivers of genetic diversity in two Branchipodopsis fairy shrimp species (Crustacea, Anostraca) endemic to remote temporary rock pool clusters at the summit of isolated mountaintops in central South Africa. We showed that both species colonized the region almost simultaneously c. 0.8 My ago, but exhibit contrasting patterns of regional genetic diversity and demographic history. The haplotype network of the common B. cf. wolfi showed clear evidence of 11 long distance dispersal events (up to 140 km) with five haplotypes that are shared among distant inselbergs, as well as some more spatially isolated derivates. Similar patterns were not observed for B. drakensbergensis presumably since this rarer species experienced a genetic bottleneck. We conclude that the observed genetic patterns reflect rare historic colonization events rather than frequent ongoing gene flow. Moreover, the high regional haplotype diversity combined with a high degree of haplotype endemicity indicates that evolutionary- (mutation) and ecological (migration) processes in this system operate on similar time scales.

Early Distal-less expression in a developing crustacean limb bud becomes restricted to setal-forming cells.

Distal-less (Dll) plays a well-known role in patterning the distal limb in arthropods. However, in some taxa, its expression even during early limb development is not always limited to the distal limb. Here, I trace the expression of Distal-less in a crustacean (Thamnocephalus platyurus) from the early limb bud to later stages of limb development, a period that includes differentiation of juvenile and adult morphology. During early development, I find two distinct types of DLL expression: one correlated with proximal distal leg patterning and the other restricted to setal-forming cells. Later in development, all the DLL expression is restricted to setal-forming cells. Based on the particular cells expressing DLL, I hypothesize an ancestral role for Dll function in the formation accessory cells of sensilla.

Internal initiation of mRNA translation in insect cell mediated by an internal ribosome entry site (IRES) from shrimp white spot syndrome virus (WSSV).

Internal initiation of mRNA translation can be mediated by internal ribosome entry site (IRES) elements which are located mainly in RNA viruses as well as certain mammalian and insect mRNA molecules. Thus far, only one DNA virus has been discovered to contain IRES element. In this investigation, an IRES element from white spot syndrome virus (WSSV), a DNA virus of marine shrimp, was demonstrated to direct the efficient translation of dicistronic mRNA in Trichoplusia ni insect cells. The IRES was inserted between glutathione S-transferase (GST) and green fluorescent protein (GFP) genes to construct a dicistronic cassette (GST-IRES-GFP). After transfection of this dicistronic cassette in insect cell, the Northern blot indicated that only one transcript corresponding to the mRNA of GST-IRES-GFP could be detected. However, the GST and GFP genes were simultaneously translated as revealed by Western blot and fluorescent microscopy, respectively. Based on sequence orientation and deletion analyses, the IRES element was 180 nucleotides (nt) in length and orientation-dependent. By comparison with that of cap-dependent initiation, the translation efficiency mediated by IRES was 98.77%. This finding promises that the WSSV IRES could be very useful to co-express two or more proteins due to its shorter length and higher translation efficiency.

Phylogenetic analysis of anostracans (Branchiopoda: Anostraca) inferred from nuclear 18S ribosomal DNA (18S rDNA) sequences.

The nuclear small subunit ribosomal DNA (18S rDNA) of 27 anostracans (Branchiopoda: Anostraca) belonging to 14 genera and eight out of nine traditionally recognized families has been sequenced and used for phylogenetic analysis. The 18S rDNA phylogeny shows that the anostracans are monophyletic. The taxa under examination form two clades of subordinal level and eight clades of family level. Two families the Polyartemiidae and Linderiellidae are suppressed and merged with the Chirocephalidae, of which together they form a subfamily. In contrast, the Parartemiinae are removed from the Branchipodidae, raised to family level (Parartemiidae) and cluster as a sister group to the Artemiidae in a clade defined here as the Artemiina (new suborder). A number of morphological traits support this new suborder. The Branchipodidae are separated into two families, the Branchipodidae and Tanymastigidae (new family). The relationship between Dendrocephalus and Thamnocephalus requires further study and needs the addition of Branchinella sequences to decide whether the Thamnocephalidae are monophyletic. Surprisingly, Polyartemiella hazeni and Polyartemia forcipata ("Family" Polyartemiidae), with 17 and 19 thoracic segments and pairs of trunk limb as opposed to all other anostracans with only 11 pairs, do not cluster but are separated by Linderiella santarosae ("Family" Linderiellidae), which has 11 pairs of trunk limbs. All appear to be part of the Chirocephalidae and share one morphological character: double pre-epipodites on at least part of their legs. That Linderiella is part of the Polyartemiinae suggests that multiplication of the number of limbs occurred once, but was lost again in Linderiella. Within Chirocephalidae, we found two further clades, the Eubranchipus-Pristicephalus clade and the Chirocephalus clade. Pristicephalus is reinstated as a genus.

The origin and evolution of variable-region helices in V4 and V7 of the small-subunit ribosomal RNA of branchiopod crustaceans.

We sequenced the V4 and V7 regions of the small-subunit ribosomal RNA (SSU rRNA) from 38 species of branchiopod crustaceans (e.g., Artemia, Daphnia, Triops) representing all eight extant orders. Ancestral large-bodied taxa in the orders Anostraca, Notostraca, Laevicaudata, and Spinicaudata (limnadiids and cyzicids) possess the typical secondary structure in these regions, whereas the spinicaudatan Cyclestheria and all of the cladocerans (Anomopoda, Ctenopoda, Onychopoda, and Haplopoda) possess three unique helices. Although the lengths and primary sequences of the distal ends of these helices are extremely variable, their locations, secondary structures, and primary sequences at the proximal end are conserved, indicating that they are homologous. This evidence supports the classical view that Cladocera is a monophyletic group and that the cyclestheriids are transitional between spinicaudatans and cladocerans. The single origin and persistence since the Permian of the unique cladoceran helices suggests that births and deaths of variable region helices have been rare. The broad range of sequence divergences observed among the cladoceran helices permitted us to make inferences about their evolution. Although their proximal ends are very GC-biased, there is a significant negative correlation between length and GC content due to an increasing proportion of U at their distal ends. Slippage-like processes occurring at unpaired nucleotides or bulges, which are very U-biased, are associated with both helix origin and runaway length expansion. The overall GC contents and lengths of V4 and V7 are highly correlated. More surprisingly, the lengths of these SSU rRNA variable regions are also highly correlated with the length of the large-subunit rRNA expansion segment, D2, indicating that mechanisms affecting length variation do so both across single genes and across genes in the rRNA gene family.