Comparative anatomy of slime glands in onychophora (velvet worms)

Onychophorans use a unique hunting and defense strategy, which involves the ejection of an adhesive slime secretion produced by a pair of specialized glands. So far, a comparative study on the anatomy of these glands has not been carried out among different species. In this article, we compare anatomical features of slime glands in representatives of two major onychophoran subgroups, the Peripatopsidae and the Peripatidae, from different parts of the world. Our data show that the musculature of the reservoir is conserved whereas the composition of the secretory duct displays taxon‐specific variation. Major differences concern the arrangement of glandular endpieces, which are distributed along the duct in Peripatopsidae but condensed in numerous repeated rosettes in Peripatidae. In addition, there are differences in the attachment pattern of slime glands to the inner surface of the body wall and to the outer surface of the gut between the two major onychophoran subgroups. A tube‐like structure with a putative valve‐like function is found at the transition of the secretory duct and the reservoir in the five Peripatopsidae species studied whereas it is absent in the two representatives of Peripatidae. Our findings suggest that the arrangement of musculature in the reservoir of the slime gland has remained unchanged since the divergence of Peripatidae and Peripatopsidae, while the composition of the secretory duct has been altered in one of these groups. However, the direction of evolutionary changes in duct composition cannot be determined unambiguously due to current uncertainty regarding the phylogenetic relationships of Onychophora. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Sperm ultrastructure of an oviparous and an ovoviviparous onychophoran species (Peripatopsidae) with some phylogenetic considerations

The spermatozoa of the Australian oviparous Ooperipatellus insignis and the South African ovoviviparous Opisthopatus cinctipes (both: Onychophora, Peripatopsidae) were studied and compared with the spermatozoal patterns already described in the taxon. The spermatozoa of both species conform with the general plan described for the Onychophora: they are filiform cells formed, in sequence, by an elongated, fully condensed nucleus capped by an acrosome and surrounded by several spiral ridges; by a mitochondrial midpiece characteristically interpolated between the nucleus and a characteristic flagellum. Major differences between the spermatozoa of both species concern their acrosome organization. The correlation between the acrosomal pattern and the size and structure of the ovarial eggs (oocytes) in onychophorans has been investigated. A parsimony analysis was performed on 21 spermatozoal characters of the species considered. Its results are congruent with those of the traditional systematics. A new set of autapomorphies characterising onychophoran sperm is suggested and some of the spermatological homologies proposed between Onychophora and Euclitellata spermatozoa are critically discussed. Our analysis suggests that spermatozoal characters are good phylogenetic markers among onychophorans, also at low taxonomic level.     

Variation in amine composition in plant species:How it integrates macroevolutionary and environmental signals

? Premise of the study: While plants show lineage-specific differences in metabolite composition, plant metabolites are also known to vary in response to the environment. The extent to which these different determinants of metabolite composition are mutually independent and recognizable is unknown. Moreover, the extent to which the metabolome can reconcile evolutionary constraint with the needs of the plant for rapid environmental response is unknown. We investigated these questions in plant species representing different phylogenetic lineages and growing in different subantarctic island environments. We studied their amines-metabolites involved in plant response to environmental conditions. ? Methods: Nine species were sampled under high salinity, water saturation, and altitude on the Kerguelen Islands. Their profiles of free aromatic, aliphatic, and acetyl-conjugated amines were determined by HPLC. We related amine composition to species and environment using generalized discriminant analyses. ? Key results: Amine composition differed significantly between species within the same environment, and the differences reflected phylogenetic positions. Moreover, across all species, amine metabolism differed between environments, and different lineages occupied different absolute positions in amine/environment space. Interestingly, all species had the same relative shifts in amine composition between environments. ? Conclusion: Our results indicate a similar response of amine composition to abiotic environments in distantly related angiosperms, suggesting environmental flexibility of species is maintained despite major differences in amine composition among lineages. These results aid understanding of how in nature the plant metabolome integrates ecology and evolution, thus providing primordial information on adaptive mechanisms of plant metabolism to climate change.     

Harnessing disorder: onychophorans use highly unstructured proteins,not silks,for prey capture

Onychophora are ancient, carnivorous soft-bodied invertebrates which capture their prey in slime that originates from dedicated glands located on either side of the head. While the biochemical composition of the slime is known, its unusual nature and the mechanism of ensnaring thread formation have remained elusive. We have examined gene expression in the slime gland from an Australian onychophoran, Euperipatoides rowelli, and matched expressed sequence tags to separated proteins from the slime. The analysis revealed three categories of protein present: unique high-molecular-weight proline-rich proteins, and smaller concentrations of lectins and small peptides, the latter two likely to act as protease inhibitors and antimicrobial agents. The predominant proline-rich proteins (200 kDa+) are composed of tandem repeated motifs and distinguished by an unusually high proline and charged residue content. Unlike the highly structured proteins such as silks used for prey capture by spiders and insects, these proteins lack ordered secondary structure over their entire length. We propose that on expulsion of slime from the gland onto prey, evaporative water loss triggers a glass transition change in the protein solution, resulting in adhesive and enmeshing thread formation, assisted by cross-linking of complementary charged and hydrophobic regions of the protein. Euperipatoides rowelli has developed an entirely new method of capturing prey by harnessing disordered proteins rather than structured, silk-like proteins.     

Neutral lipid fatty acid composition as trait and constraint in Collembola evolution

Functional traits determine the occurrence of species along environmental gradients and their coexistence with other species. Understanding how traits evolved among coexisting species helps to infer community assembly processes. We propose fatty acid composition in consumer tissue as a functional trait related to both food resources and physiological functions of species. We measured phylogenetic signal in fatty acid profiles of 13 field‐sampled Collembola (springtail) species and then combined the data with published fatty acid profiles of another 24 species. Collembola fatty acid profiles generally showed phylogenetic signal, with related species resembling each other. Long‐chain polyunsaturated fatty acids, related to physiological functions, demonstrated phylogenetic signal. In contrast, most food resource biomarker fatty acids and the ratios between bacterial, fungal, and plant biomarker fatty acids exhibited no phylogenetic signal. Presumably, fatty acids related to physiological functions have been constrained during Collembola evolutionary history: Species with close phylogenetic affinity experienced similar environments during divergence, while niche partitioning in food resources among closely related species favored species coexistence. Measuring phylogenetic signal in ecologically relevant traits of coexisting species provides an evolutionary perspective to contemporary assembly processes of ecological communities. Integrating phylogenetic comparative methods with community phylogenetic and trait‐based approaches may compensate for the limitations of each method when used alone and improve understanding of processes driving and maintaining assembly patterns.     

Breakdown of phylogenetic signal: a survey of microsatellite densities in 454 shotgun sequences from 154 non model eukaryote species

Microsatellites are ubiquitous in Eukaryotic genomes. A more complete understanding of their origin and spread can be gained from a comparison of their distribution within a phylogenetic context. Although information for model species is accumulating rapidly, it is insufficient due to a lack of species depth, thus intragroup variation is necessarily ignored. As such, apparent differences between groups may be overinflated and generalizations cannot be inferred until an analysis of the variation that exists within groups has been conducted. In this study, we examined microsatellite coverage and motif patterns from 454 shotgun sequences of 154 Eukaryote species from eight distantly related phyla (Cnidaria, Arthropoda, Onychophora, Bryozoa, Mollusca, Echinodermata, Chordata and Streptophyta) to test if a consistent phylogenetic pattern emerges from the microsatellite composition of these species. It is clear from our results that data from model species provide incomplete information regarding the existing microsatellite variability within the Eukaryotes. A very strong heterogeneity of microsatellite composition was found within most phyla, classes and even orders. Autocorrelation analyses indicated that while microsatellite contents of species within clades more recent than 200 Mya tend to be similar, the autocorrelation breaks down and becomes negative or non-significant with increasing divergence time. Therefore, the age of the taxon seems to be a primary factor in degrading the phylogenetic pattern present among related groups. The most recent classes or orders of Chordates still retain the pattern of their common ancestor. However, within older groups, such as classes of Arthropods, the phylogenetic pattern has been scrambled by the long independent evolution of the lineages.     

Monitoring of biomass composition from microbiological sources by means of FT‐IR spectroscopy

An FT‐IR spectroscopic method was developed for the simultaneous quantitative analysis of biomacromolecular components in biomass, originating from various microbiological sources. For the determination of protein, lipid and carbohydrate content, creatine phosphokinase, egg phosphatidyl choline and starch hydrolysate were chosen as external standards. This selection was based on spectral similarity and ease of availability. Protein content was based on the area under the amide II band profile around 1,545 cm^?1. Because of the heterogeneous lipid composition in the different species, lipid content was determined using integration over the C? H stretching vibrational population between 2,984 and 2,780 cm^?1. Carbohydrate content was determined using integration over a C? O and C? O? C stretching band area between 1,180 and 1,133 cm^?1. Linear regression analysis provided three calibration lines, according to which biomasses from ten species were analyzed. This approach showed good intra‐batch reproducibility. With this method we could demonstrate good reproducibility between batches of the same species with similar growth conditions while large differences in biomass composition were observed between the various species. Protein content as determined by FT‐IR spectroscopy compared well with the results obtained from elemental analysis. Biotechnol. Bioeng. 2009;103: 123–129. © 2008 Wiley Periodicals, Inc.     

Characterisation of the slime gland secretion from the peripatus, Euperipatoides kanangrensis (Onychophora: Peripatopsidae)

The Onychophora display a distinctive mechanism of feeding that involves the entanglement of prey in a sticky secretion. This secretion is produced in the slime glands and ejected as adhesive threads from a pair of oral papillae on either side of the head. Biochemical analyses of the secretion reveals it to be a composite material containing protein, sugar, lipid and a surfactant, nonylphenol. The identification of nonylphenol in the secretion is significant in that this is the first report of this compound from a natural source. The proteins are the principal component of the slime and the amino acid composition of the crude secretion suggests the presence of collagen or a ‘collagen-like’ domain. One or more of the high molecular weight proteins are O-glycosylated where the predominant modification is a single N-acetylgalactosamine (GalNAc). This study adds to our understanding of the chemical and biochemical composition of the unusual onycophoran slime gland secretion.     

Direct prediction of profiles of sequences compatible with a protein structure by neural networks with fragment‐based local and energy‐based nonlocal profiles

Locating sequences compatible with a protein structural fold is the well‐known inverse protein‐folding problem. While significant progress has been made, the success rate of protein design remains low. As a result, a library of designed sequences or profile of sequences is currently employed for guiding experimental screening or directed evolution. Sequence profiles can be computationally predicted by iterative mutations of a random sequence to produce energy‐optimized sequences, or by combining sequences of structurally similar fragments in a template library. The latter approach is computationally more efficient but yields less accurate profiles than the former because of lacking tertiary structural information. Here we present a method called SPIN that predicts Sequence Profiles by Integrated Neural network based on fragment‐derived sequence profiles and structure‐derived energy profiles. SPIN improves over the fragment‐derived profile by 6.7% (from 23.6 to 30.3%) in sequence identity between predicted and wild‐type sequences. The method also reduces the number of residues in low complex regions by 15.7% and has a significantly better balance of hydrophilic and hydrophobic residues at protein surface. The accuracy of sequence profiles obtained is comparable to those generated from the protein design program RosettaDesign 3.5. This highly efficient method for predicting sequence profiles from structures will be useful as a single‐body scoring term for improving scoring functions used in protein design and fold recognition. It also complements protein design programs in guiding experimental design of the sequence library for screening and directed evolution of designed sequences. The SPIN server is available at http://sparks‐lab.org . Proteins 2014; 82:2565–2573. © 2014 Wiley Periodicals, Inc.     

Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.     

Phylogenetic turnover during subtropical forest succession across environmental and phylogenetic scales

Although spatial and temporal patterns of phylogenetic community structure during succession are inherently interlinked and assembly processes vary with environmental and phylogenetic scales, successional studies of community assembly have yet to integrate spatial and temporal components of community structure, while accounting for scaling issues. To gain insight into the processes that generate biodiversity after disturbance, we combine analyses of spatial and temporal phylogenetic turnover across phylogenetic scales, accounting for covariation with environmental differences. We compared phylogenetic turnover, at the species‐ and individual‐level, within and between five successional stages, representing woody plant communities in a subtropical forest chronosequence. We decomposed turnover at different phylogenetic depths and assessed its covariation with between‐plot abiotic differences. Phylogenetic turnover between stages was low relative to species turnover and was not explained by abiotic differences. However, within the late‐successional stages, there was high presence‐/absence‐based turnover (clustering) that occurred deep in the phylogeny and covaried with environmental differentiation. Our results support a deterministic model of community assembly where (i) phylogenetic composition is constrained through successional time, but (ii) toward late succession, species sorting into preferred habitats according to niche traits that are conserved deep in phylogeny, becomes increasingly important.     

Carboniferous Onychophora from Montceau‐les‐Mines,France, and onychophoran terrestrialization

The geological age of the onychophoran crown‐group, and when the group came onto land, have been sources of debate. Although stem‐group Onychophora have been identified from as early as the Cambrian, the sparse record of terrestrial taxa from before the Cretaceous is subject to contradictory interpretations. A Late Carboniferous species from the Mazon Creek biota of the USA, Helenodora inopinata, originally interpreted as a crown‐group onychophoran, has recently been allied to early Cambrian stem‐group taxa. Here we describe a fossil species from the Late Carboniferous Montceau‐les‐Mines Lagerstätte, France, informally referred to as an onychophoran for more than 30 years. The onychophoran affinities of Antennipatus montceauensis gen. nov., sp. nov. are indicated by the form of the trunk plicae and the shape and spacing of their papillae, details of antennal annuli, and the presence of putative slime papillae. The poor preservation of several key systematic characters for extant Onychophora, however, prohibits the precise placement of the Carboniferous fossil in the stem or crown of the two extant families, or the onychophoran stem‐group as a whole. Nevertheless, A. montceauensis is the most compelling candidate to date for a terrestrial Paleozoic onychophoran.     

Allozyme evidence for extensive and ancient radiations in Australian Onychophora

Allozyme electrophoresis has been employed to examine genetic differentiation among eight described species, and representatives of an additional 15 taxa, of Australian peripatopsid Onychophora. The data reveal extremely high genetic differentiation among the described species and among the other taxa, each of which warrants specific recognition. Rapid protein evolution cannot account for the large genetic distances and it is proposed that these are a consequence of ancient divergence times. A method is presented for extracting phylogenetic information from allozyme data sets which are not amenable to conventional analysis.     

Integrating genomic and phenotypic data to evaluate alternative phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers

Although resolving phylogenetic relationships and establishing species limits are primary goals of systematics, these tasks remain challenging at both conceptual and analytical levels. Here, we integrated genomic and phenotypic data and employed a comprehensive suite of coalescent‐based analyses to develop and evaluate competing phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers (Chorthippus binotatus group) composed of two species and eight putative subspecies. To resolve the evolutionary relationships within this complex, we first evaluated alternative phylogenetic hypotheses arising from multiple schemes of genomic data processing and contrasted genetic‐based inferences with different sources of phenotypic information. Second, we examined the importance of number of loci, demographic priors, number and kind of phenotypic characters and sex‐based trait variation for developing alternative species delimitation hypotheses. The best‐supported topology was largely compatible with phenotypic data and showed the presence of two clades corresponding to the nominative species groups, one including three well‐resolved lineages and the other comprising a four‐lineage polytomy and a well‐differentiated sister taxon. Integrative species delimitation analyses indicated that the number of employed loci had little impact on the obtained inferences but revealed the higher power provided by an increasing number of phenotypic characters and the usefulness of assessing their phylogenetic information content and differences between sexes in among‐taxa trait variation. Overall, our study highlights the importance of integrating multiple sources of information to test competing phylogenetic hypotheses and elucidate the evolutionary history of species complexes representing early stages of divergence where conflicting inferences are more prone to appear.     

Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds

The use of nitrogen‐to‐protein conversion factors (N‐Prot factors) is the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of N‐Prot factors specific to individual species. Experimental data are needed to allow the use of this methodology with seaweeds. The present study was designed to characterize the amino acid composition and to establish specific N‐Prot factors for six green, four brown and nine red marine algae. Mean values for individual amino acids tended to be similar among the three groups, but some differences were found. Green algae tended to show lower percentages of both aspartic acid and glutamic acid than the other two groups of algae. The percentages of both lysine and arginine were higher in red algae, while brown algae tended to show more methionine than green and red algae. The actual protein content of the species, based on the sum of amino acid residues, varied from 10.8% (Chnoospora minima, brown algae) to 23.1% (Aglaothamnion uru‐guayense, red algae) of the dry weight. Nitrogen‐to‐protein conversion factors were established for the species studied, based on the ratio of amino acid residues to total nitrogen, with values ranging from 3.75 (Cryptonemia seminervis, red algae) to 5.72 (Padina gymnospora, brown algae). The relative importance of non‐protein nitrogen is greater in red algae, and consequently lower N‐Prot factors were calculated for these species (average value 4.59). Conversely, protein nitrogen content in both green and brown algae tends to be higher, and average N‐Prot factors were 5.13 and 5.38, respectively. An overall average N‐Prot factor for all species studied of 4.92 ± 0.59 (n = 57) was established. This study confirms that the use of the traditional factor 6.25 is unsuitable for seaweeds, and the use of the N‐Prot factors proposed here is recommended.     

Ectomycorrhizal fungal diversity and saprotrophic fungal diversity are linked to different tree community attributes in a field‐based tree experiment

Exploring the link between above‐ and belowground biodiversity has been a major theme of recent ecological research, due in large part to the increasingly well‐recognized role that soil microorganisms play in driving plant community processes. In this study, we utilized a field‐based tree experiment in Minnesota, USA, to assess the effect of changes in plant species richness and phylogenetic diversity on the richness and composition of both ectomycorrhizal and saprotrophic fungal communities. We found that ectomycorrhizal fungal species richness was significantly positively influenced by increasing plant phylogenetic diversity, while saprotrophic fungal species richness was significantly affected by plant leaf nitrogen content, specific root length and standing biomass. The increasing ectomycorrhizal fungal richness associated with increasing plant phylogenetic diversity was driven by the combined presence of ectomycorrhizal fungal specialists in plots with both gymnosperm and angiosperm hosts. Although the species composition of both the ectomycorrhizal and saprotrophic fungal communities changed significantly in response to changes in plant species composition, the effect was much greater for ectomycorrhizal fungi. In addition, ectomycorrhizal but not saprotrophic fungal species composition was significantly influenced by both plant phylum (angiosperm, gymnosperm, both) and origin (Europe, America, both). The phylum effect was caused by differences in ectomycorrhizal fungal community composition, while the origin effect was attributable to differences in community heterogeneity. Taken together, this study emphasizes that plant‐associated effects on soil fungal communities are largely guild‐specific and provides a mechanistic basis for the positive link between plant phylogenetic diversity and ectomycorrhizal fungal richness.     

A tree of life based on protein domain organizations

It is desirable to estimate a tree of life, a species tree including all available species in the 3 superkingdoms, Archaea, Bacteria, and Eukaryota, using not a limited number of genes but full-scale genome information. Here, we report a new method for constructing a tree of life based on protein domain organizations, that is, sequential order of domains in a protein, of all proteins detected in a genome of an organism. The new method is free from the identification of orthologous gene sets and therefore does not require the burdensome and error-prone computation. By pairwise comparisons of the repertoires of protein domain organizations of 17 archaeal, 136 bacterial, and 14 eukaryotic organisms, we computed evolutionary distances among them and constructed a tree of life. Our tree shows monophyly in Archaea, Bacteria, and Eukaryota and then monophyly in each of eukaryotic kingdoms and in most bacterial phyla. In addition, the branching pattern of the bacterial phyla in our tree is consistent with the widely accepted bacterial taxonomy and is very close to other genome-based trees. A couple of inconsistent aspects between the traditional trees and the genome-based trees including ours, however, would perhaps urge to revise the conventional view, particularly on the phylogenetic positions of hyperthermophiles.     

Cross-species cluster co-conservation: a new method for generating protein interaction networks

Co-conservation (phylogenetic profiles) is a well-established method for predicting functional relationships between proteins. Several publicly available databases use this method and additional clustering strategies to develop networks of protein interactions (cluster co-conservation (CCC)). CCC has previously been limited to interactions within a single target species. We have extended CCC to develop protein interaction networks based on co-conservation between protein pairs across multiple species, cross-species cluster co-conservation.     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.