The recent evolutionary origin of the phenotypically novel amphipod Hyalella montezuma offers an ecological explanation for morphological stasis in a closely allied species complex

Numerous molecular studies have identified morphologically cryptic, freshwater invertebrate species, but have not suggested possible mechanisms for their phenotypic stasis. The amphipod crustacean genus Hyalella contains numerous morphologically cryptic species in the H. azteca complex, as well as a small number of morphologically very divergent, narrowly endemic taxa. One such taxon, Hyalella montezuma, is the sole planktonic filter-feeder within the North American amphipod fauna, and is known only from Montezuma Well, a fishless travertine spring mound in Arizona, USA. In this study, we conduct a phylogenetic analysis of mtDNA sequence data using likelihood, Bayesian and cladistic approaches to determine both the relationship of H. montezuma to the H. 'azteca' species complex, and to ascertain if its morphological and ecological differentiation have been comparatively recent. The results show that H. montezuma has a very close phylogenetic affiliation with one lineage in the H. azteca complex, indicating that its origin has been recent. We present evidence suggesting that fish predation is an important ecological factor, which constrains morphological and ecological diversification within the genus Hyalella, and that Montezuma Well has provided a relaxation on this constraint.     

Biological identifications through DNA barcodes

Although much biological research depends upon species diagnoses, taxonomic expertise is collapsing. We are convinced that the sole prospect for a sustainable identification capability lies in the construction of systems that employ DNA sequences as taxon 'barcodes'. We establish that the mitochondrial gene cytochrome c oxidase I (COI) can serve as the core of a global bioidentification system for animals. First, we demonstrate that COI profiles, derived from the low-density sampling of higher taxonomic categories, ordinarily assign newly analysed taxa to the appropriate phylum or order. Second, we demonstrate that species-level assignments can be obtained by creating comprehensive COI profiles. A model COI profile, based upon the analysis of a single individual from each of 200 closely allied species of lepidopterans, was 100% successful in correctly identifying subsequent specimens. When fully developed, a COI identification system will provide a reliable, cost-effective and accessible solution to the current problem of species identification. Its assembly will also generate important new insights into the diversification of life and the rules of molecular evolution.     

GENETIC DIFFERENTIATION AT NUCLEAR AND MITOCHONDRIAL LOCI AMONG LARGE WHITE‐HEADED GULLS: SEX‐BIASED INTERSPECIFIC GENE FLOW?

We measured genetic differentiation among species of large white-headed gulls using mitochondrial (cytochrome b haplotypes) and nuclear (microsatellites) markers. Additional information was added using a previously published study of allozymes on the same species. Levels of differentiation among species at nuclear markers are much lower than would be expected for avian species and are not concordant with the level of differentiation in mitochondrial markers. This discrepancy is best explained by a combination of recent species origin and interspecific gene flow after speciation. The data also suggest that female-mediated gene flow is reduced compared to male-mediated gene flow, either due to behavioral bias or due to stronger counterselection of female hybrids in accordance with Haldane's rule for ZW species. Whatever the reasons for the low differentiation of the species' nuclear gene pools, the extensive similarity of their nuclear genome demonstrates that selection on a limited number of characters is an important factor in establishing and maintaining clear-cut phenotypic differences between these species and suggests that the number of loci involved in this process is quite low. This situation may not be exceptional in birds, indeed a number of studies have found similarly low level of differentiation in nuclear markers among congeneric bird species, although usually based on a single set of markers. Because hybridization is a widespread phenomenon in birds, many of these cases might be due to interspecific gene flow.     

The 3-D structure of microsomal glutathione transferase 1 at 6 A resolution as determined by electron crystallography of p22(1)2(1) crystals

Pure solubilised microsomal glutathione transferase 1 (MGST1) forms well-ordered two-dimensional (2-D) crystals of two different symmetries, one orthorhombic (p22(1)2(1)) and one hexagonal (p6), both diffracting electrons to a resolution beyond 3 A. A three-dimensional (3-D) map has previously been calculated to 6 A resolution from the hexagonal crystal form. From orthorhombic crystals we have now calculated a 6 A 3-D reconstruction displaying three repeats of four rod-like densities. These are inclined relative to the normal of the membrane plane and consistent with arising from a left-handed four-helix bundle fold. The rendered volume clearly displays the same structural features as the map previously calculated from the p6 crystal type including similar lengths and substructure of the helices, but several distinguishing features do exist. The helices are more tilted in the map calculated from the orthorhombic crystals indicating conformational flexibility. Density present on the cytosolic side is consistent with the location of the active site. In addition, the current map displays the noted similarity to subunit I of cytochrome c oxidase.     

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of ‘mature’ snRNPs, or the reclamation of unassembled snRNP components.     

In vivo kinetics of Cajal body components

Cajal bodies (CBs) are subnuclear domains implicated in small nuclear ribonucleoprotein (snRNP) biogenesis. In most cell types, CBs coincide with nuclear gems, which contain the survival of motor neurons (SMN) complex, an essential snRNP assembly factor. Here, we analyze the exchange kinetics of multiple components of CBs and gems in living cells using photobleaching microscopy. We demonstrate differences in dissociation kinetics of CB constituents and relate them to their functions. Coilin and SMN complex members exhibit relatively long CB residence times, whereas components of snRNPs, small nucleolar RNPs, and factors shared with the nucleolus have significantly shorter residence times. Comparison of the dissociation kinetics of these shared proteins from either the nucleolus or the CB suggests the existence of compartment-specific retention mechanisms. The dynamic properties of several CB components do not depend on their interaction with coilin because their dissociation kinetics are unaltered in residual nuclear bodies of coilin knockout cells. Photobleaching and fluorescence resonance energy transfer experiments demonstrate that coilin and SMN can interact within CBs, but their interaction is not the major determinant of their residence times. These results suggest that CBs and gems are kinetically independent structures.     

An amino acid triplet in the NH2 terminus of rat ROMK1 determines interaction with SUR2B

ATP-regulated (K(ATP)) channels are formed by an inward rectifier pore-forming subunit (Kir) and a sulfonylurea (glibenclamide)-binding protein, a member of the ATP binding cassette family (sulfonylurea receptor (SUR) or cystic fibrosis transmembrane conductance regulator). The latter is required to confer glibenclamide sensitivity to K(ATP) channels. In the mammalian kidney ROMK1-3 are components of K(ATP) channels that mediate K(+) secretion into urine. ROMK1 and ROMK3 splice variants share the core polypeptide of ROMK2 but also have distinct NH(2)-terminal extensions of 19 and 26 amino acids, respectively. The SUR2B is also expressed in rat kidney tubules and may combine with Kir.1 to form renal K(ATP) channels. Our previous studies showed that co-expression of ROMK2, but not ROMK1 or ROMK3, with rat SUR2B in oocytes generated glibenclamide-sensitive K(+) currents. These data suggest that the NH(2)-terminal extensions in both ROMK1 and ROMK3 block ROMK-SUR2B interaction. Seven amino acids in the NH(2)-terminal extensions of ROMK1 and ROMK3 are identical (amino acids 13-19 in ROMK1 and 20-26 in ROMK3) and may determine ROMK-SUR2B interaction. We constructed a series of hemagglutinin-tagged ROMK1 NH(2)-terminal deletion and substitution mutants and examined glibenclamide-sensitive K(+) currents in oocytes when co-expressed with SUR2B. These studies identified an amino acid triplet "IRA" within the conserved segment in the NH(2) terminus of ROMK1 and ROMK3 that blocks the ability of SUR2B to confer glibenclamide sensitivity to the expressed K(+) currents. The position of this triplet in the ROMK1 NH(2)-terminal extension is also important for the ROMK-SUR2B interactions. In vitro co-translation and immunoprecipitation studies with hemagglutinin-tagged ROMK mutants and SUR2B indicted that direct interaction between these two proteins is required for glibenclamide sensitivity of induced K(+) currents in oocytes. These results suggest that the IRA triplet in the NH(2)-terminal extensions of both ROMK1 and ROMK3 plays a key role in subunit assembly of the renal secretary K(ATP) channel.     

Regulation of ROMK1 channels by protein-tyrosine kinase and -tyrosine phosphatase

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.     

Self-association of coilin reveals a common theme in nuclear body localization

We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e. g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins.     

Stable baselines of temporal turnover underlie high beta diversity in tropical arthropod communities

While the high species diversity of tropical arthropod communities has often been linked to marked spatial heterogeneity, their temporal dynamics have received little attention. This study addresses this gap by examining spatio‐temporal variation in the arthropod communities of a tropical montane forest in Honduras. By employing DNA barcode analysis and Malaise trap sampling across 4 years and five sites, 51,596 specimens were assigned to 8,193 presumptive species. High beta diversity was linked more strongly to elevation than geographic distance, decreasing by 12% when only the dominant species were considered. When sampling effort was increased by deploying more traps at a site, beta diversity only decreased by 2%, but extending sampling across years decreased beta diversity by 27%. Species inconsistently detected among years, likely transients from other settings, drove the low similarity in species composition among traps only a few metres apart. The dominant, temporally persistent species substantially influenced the cyclic pattern of change in community composition among years. This pattern likely results from divergence–convergence dynamics, suggesting a stable baseline of temporal turnover in each community. The overall results establish that large sample sizes are necessary to reveal species richness, but are not essential for quantifying beta diversity. This study further highlights the need for standardized methods of sampling and species identification to generate the comparative data required to evaluate biodiversity change in space and time.