Secondary Structure Model Research Articles

Customer Loyalty in Clusters: Perceived Value and Satisfaction as Antecedents

Purpose: This article studies, from the perspective of relationship marketing, the loyalty behavior of industrial customers in the context of an industrial cluster. Loyalty is a key variable for studying long-term relationships between firms. Research implications: Recent advances in consumer and services marketing consider that perceived value and satisfaction are central to explaining customer loyalty. However, very few business-to-business (B2B) studies explain the antecedents of customer loyalty, where perceived value acquires a multidimensional perspective. This study adopts the relationship marketing approach, and loyalty behavior is analyzed in a specific setting: an industrial cluster. Furthermore, the effect of the number of suppliers is analyzed as a possible moderator in the relationships of the model. Methodology approach: We have chosen the Spanish tile cluster to test a series of hypotheses. Questionnaires were elaborated from primary and secondary information and structural equation models (SEM) have been used for statistical treatment and confirmatory factor analysis (CFA). Findings: This study highlights the importance of the relationships among perceived value, satisfaction, and loyalty and the importance of the different dimensions of perceived value. Practical implications: The empirical study and the results provide important evidence for managers, specifically, the critical influence of the emotional and social perceived values by the customer on his or her level of satisfaction and on the achievement of final loyalty—the importance in the commercial training programs of this matters. Originality, value, and contribution: This study highlights the importance of the most intangibles dimensions of value for the industrial cluster relations between companies.

Dissecting the molecular mechanism of drosophila odorant receptors through activity modeling and comparative analysis

To gain insight into the molecular mechanism of odorant receptors (ORs) in Drosophila species, we developed a Quantitative Structure Activity Relationship (QSAR) model that predicts experimentally measured electrophysiological activities between 24 D. melanogaster ORs and 108 odorants. Although the model is limited by the tested odorants,analyzing the model allowed dissection of specific topological and chemical properties necessary for an odorant to elicit excitatory or inhibitory receptor response. Linear odorants with five to eight nonhydrogen atoms at the main chain and hydrogen-bond acceptor and/or hydrogen-bond donor at its ends were found to stimulate strong excitatory response. A comparative sequence analysis of 90 ORs in 15 orthologous groups identified 15 putative specificity-determining residues (SDRs) and 15 globally conserved residues that we postulate as functionally key residues. Mapping to a model of secondary structure resulted in 14 out of 30 key residues locating to the transmembrane (TM) domains. Twelve residues, including six SDRs and six conserved residues, are located at the extracellular halves of the TM domains. Combining the evidence from the QSAR modeling and the comparative sequence analysis, we hypothesize that the Drosophila ORs accept odorants into a binding pocket located on the extracellular halves of its TM domains. The QSAR modeling suggests that the binding pocket is around 15 A in depth and about 6 A in width. Twelve mainly polar or charged key residues, both SDRs and conserved, are located in this pocket and postulated to distinguish docked odorants via primarily geometry fitting and hydrogen-bond interaction.

An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models

BackgroundTunicates have been recently revealed to be the closest living relatives of vertebrates. Yet, with more than 2500 described species, details of their evolutionary history are still obscure. From a molecular point of view, tunicate phylogenetic relationships have been mostly studied based on analyses of 18S rRNA sequences, which indicate several major clades at odds with the traditional class-level arrangements. Nonetheless, substantial uncertainty remains about the phylogenetic relationships and taxonomic status of key groups such as the Aplousobranchia, Appendicularia, and Thaliacea.ResultsThirty new complete 18S rRNA sequences were acquired from previously unsampled tunicate species, with special focus on groups presenting high evolutionary rate. The updated 18S rRNA dataset has been aligned with respect to the constraint on homology imposed by the rRNA secondary structure. A probabilistic framework of phylogenetic reconstruction was adopted to accommodate the particular evolutionary dynamics of this ribosomal marker. Detailed Bayesian analyses were conducted under the non-parametric CAT mixture model accounting for site-specific heterogeneity of the evolutionary process, and under RNA-specific doublet models accommodating the occurrence of compensatory substitutions in stem regions. Our results support the division of tunicates into three major clades: 1) Phlebobranchia + Thaliacea + Aplousobranchia, 2) Appendicularia, and 3) Stolidobranchia, but the position of Appendicularia could not be firmly resolved. Our study additionally reveals that most Aplousobranchia evolve at extremely high rates involving changes in secondary structure of their 18S rRNA, with the exception of the family Clavelinidae, which appears to be slowly evolving. This extreme rate heterogeneity precluded resolving with certainty the exact phylogenetic placement of Aplousobranchia. Finally, the best fitting secondary-structure and CAT-mixture models suggest a sister-group relationship between Salpida and Pyrosomatida within Thaliacea.ConclusionAn updated phylogenetic framework for tunicates is provided based on phylogenetic analyses using the most realistic evolutionary models currently available for ribosomal molecules and an unprecedented taxonomic sampling. Detailed analyses of the 18S rRNA gene allowed a clear definition of the major tunicate groups and revealed contrasting evolutionary dynamics among major lineages. The resolving power of this gene nevertheless appears limited within the clades composed of Phlebobranchia + Thaliacea + Aplousobranchia and Pyuridae + Styelidae, which were delineated as spots of low resolution. These limitations underline the need to develop new nuclear markers in order to further resolve the phylogeny of this keystone group in chordate evolution.

Simultaneous alignment and folding of 28S rRNA sequences uncovers phylogenetic signal in structure variation

Secondary structure models of mitochondrial and nuclear (r)RNA sequences are frequently applied to aid the alignment of these molecules in phylogenetic analyses. Additionally, it is often speculated that structure variation of (r)RNA sequences might profitably be used as phylogenetic markers. The benefit of these approaches depends on the reliability of structure models. We used a recently developed approach to show that reliable inference of large (r)RNA secondary structures as a prerequisite of simultaneous sequence and structure alignment is feasible. The approach iteratively establishes local structure constraints of each sequence and infers fully folded individual structures by constrained MFE optimization. A comparison of structure edit distances of individual constraints and fully folded structures showed pronounced phylogenetic signal in fully folded structures. As model sequences we characterized secondary structures of 28S rRNA sequences of selected insects and examined their phylogenetic signal according to established phylogenetic hypotheses.

Cloning and Expression Analysis of an NBS-LRR Type Gene from Sugarcane

In the present study, a non-TIR-NBS-LRR type disease-related gene was cloned by rapid amplification of cDNA ends (RACE) using the high-resistant sugarcane variety NCo376. This gene was termed as SNLR, with the GenBank accession No. of EF155648. The full-length cDNA sequence of SNLR is 2 985 bp, including an open reading frame (ORF) of 2 661 bp and the typical 29 bp poly-A. The SNLR gene contained all the four typical conserved motifs of the NBS: P-loop (GMGGVGGKTT), Kinase-2 (LIVLDD), Kinase3a (GSR/KILVIIR) and hydrophobic region (GLPLAL), plus six putative LRR regions. It can be deduced from the hydrophobic character, secondary structure and 3D model analysis of the corresponding coding protein that the SNLR protein was alkalescent, with pI of 7.76 and without any obvious hydrophobic domain; coil and helices were the framework of secondary structure; no transmembrane region was found in its protein 3D model. The gene expression profile under the treat- ment of U. scitaminea, SA and H2O2 were investigated by Real-time qPCR. The results showed that expression of sugarcane SNLR gene was influenced by the fungus, SA and H2O2, with the expression patterns of "down-up", "down in the whole process" and "up-down", respectively. It was inferred that expression of SNLR gene occurs both via an H2O2- and SA-dependent pathway. At the same time, SNLR gene was found to be expressed highly in leaves, mildly in stalks and slightly in roots, which indicated its relation to resistance in this aspect.

Structure of the HIV‐1 Rev response element alone and in complex with regulator of virion (Rev) studied by atomic force microscopy

The interaction of multiple HIV-1 regulator of virion (Rev) proteins with the viral RNA target, the Rev response element (RRE), is critical for nuclear export of incompletely spliced and unspliced viral RNA, and for the onset of the late phase in the viral replication cycle. The heterogeneity of the Rev-RRE complex has made it difficult to study using conventional structural methods. In the present study, atomic force microscopy is applied to directly visualize the tertiary structure of the RRE RNA alone and in complex with Rev proteins. The appearance of the RRE is compatible with the earlier proposed RRE secondary structure in dimensions and overall shape, including a stalk and a head interpreted as stem I, and stem-loops II-V in the secondary structure model, respectively. Atomic force microscopy imaging of the Rev-RRE complex revealed an increased height of the structure both in the stalk and head regions, which is in accordance with a binding model in which Rev binding to a high affinity site in stem IIB triggers oligomerization of Rev proteins through cooperative binding along stem I in RRE. The present study demonstrates that atomic force microscopy comprises a useful technique to study complex biological structures of nucleic acids at high resolution.

Making ordered DNA and protein structures from computer‐printed transparency film cut‐outs

Instructions are given for building physical scale models of ordered structures of B-form DNA, protein α-helix, and parallel and antiparallel protein β-pleated sheets made from colored computer printouts designed for transparency film sheets. Cut-outs from these sheets are easily assembled. Conventional color coding for atoms are used for both types of biopolymers. Arrows facilitate following chain direction for the polypeptides. For DNA, the 5' to 3' direction is guided by a 5' phosphate group and a free hydroxyl group. Important chiral centers, for example, α-carbon, deoxyribose C1', are easily made. The main advantages of this version of DNA are the proportional major and minor grooves as in the actual molecule. More importantly, because of transparency of the film one can see successive base-pair stacking very clearly and also the sense of relative base-pair rotation. Because of the introduction of two central metal wire axes, the model of B-form DNA can be twisted to give a rather good representation of A-form and even a semblance of a left-handed helix. The models of secondary structure of protein allow a better insight into the axial alignment of side chains, the formation of hydrogen bond, the handedness of the α-helix, and the backbone connection between the β-strands. Students taught by these models understand 3D features of the biopolymers better than from textbook illustrations, computer graphic representations, and even common paper and plastic versions.

The interstrand amino acid pairs play a significant role in determining the parallel or antiparallel orientation of β-strands

It is widely considered that it is not appropriate to treat β-pairs in isolation, since other secondary structural models (such as helices, coils), protein topology and protein tertiary structures would limit β-strand pairing. However, to understand the underlying mechanisms of β-sheet formation, studies ought to be performed separately on more concrete aspects. In this study, we focus on the parallel or antiparallel orientation of β-strands. First, statistical analysis was performed on the relative frequencies of the interstrand amino acid pairs within parallel and antiparallel β-strands. Consequently, features were extracted by singular value decomposition from the statistical results. By using the support vector machine to distinguish the features extracted from the two types of β-strands, high accuracy was achieved (up to 99.4%). This suggests that the interstrand amino acid pairs play a significant role in determining the parallel or antiparallel orientation of β-strands. These results may provide useful information for developing other useful algorithms to examine to the β-strand folding pathways, and could eventually lead to protein structure predictions.

Mitochondrial DNA mutation m.3635G>A may be associated with Leber hereditary optic neuropathy in Chinese

Leber hereditary optic neuropathy (LHON) was the first disease to be linked to the presence of a mitochondrial DNA (mtDNA) mutation. Nowadays over 95% of LHON cases are known to be caused by one of three primary mutations (m.11778G>A, m.14484T>C, and m.3460G>A). Reports for other (rare) primary mutations in LHON patients are not infrequent. Among those is the mutation m.3635G>A in the MT-ND1 gene which was reported to be pathogenic in a Russian LHON family. In this study, we report on a Chinese family with clinical features of LHON but without any of the three well-known primary mutations. Analysis of the complete mitochondrial genome in the proband revealed the presence of m.3635G>A and m.6228C>T, along with a full array of other variants that suggest the haplogroup M7b1. Evolutionary analysis indicates that site 3635, but not 6228, is highly conserved in vertebrates. Protein secondary-structure modeling for the MT-ND1 protein harboring amino acid change S110N indicates that mutant m.3635G>A decreases the protein hydrophobicity. Our current observations provide further support for a pathogenic role of m.3635G>A in patients with LHON.

Secondary Structures for 5′ Regions of R2 Retrotransposon RNAs Reveal a Novel Conserved Pseudoknot and Regions that Evolve under Different Constraints

Sequences from the 5′ region of R2 retrotransposons of four species of silk moth are reported. In Bombyx mori, this region of the R2 messenger RNA contains a binding site for R2 protein and mediates interactions critical to R2 element insertion into the host genome. A model of secondary structure for a segment of this RNA is proposed on the basis of binding to oligonucleotide microarrays, chemical mapping, and comparative sequence analysis. Five conserved secondary structures are identified, including a novel pseudoknot. There is an apparent transition from an entirely RNA structure coding function in most of the 5′ segment to a protein coding function near the 3′ end. This suggests that local regions evolved under separate functional constraints (structural, coding, or both).

18S rRNA alignments derived from different secondary structure models can produce alternative phylogenies

Molecular sequence data are often aligned on the basis of secondary and/or tertiary structure models. However, these models are regularly updated and sometimes differ depending on the way in which they were constructed. We examined whether the choice of a particular 18S rRNA secondary structure model as alignment basis influences phylogeny inference. We therefore compared 18S rRNA phylogenies derived from alignments based on different models. We used: 1. Maximum parsimony; 2. The neighbour-joining method; 3. The maximum-likelihood approach; and 4. Evolutionary parsimony. This demonstrated that the secondary structure model on which an alignment is based may influence: 1. The tree topologies found by these four methods; 2. The numbers of most parsimonious trees found; and 3. The statistical values calculated by the evolutionary parsimony method.

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

The Na +-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 (∼ 70–100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated (∼ 68–90 kDa) with only 31–65% of WT l-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered (∼ 98% of WT activity). However, the N138Q/ N144Q double mutant displayed barely detectable membrane expression at ∼ 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 °C or 37 °C. Marker protein immunocytochemistry with N138Q/ N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.

A novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco

A novel Potato virus Y (PVY) isolate, L26, recovered from a Frontier potato line was initially typed as a PVY NTN strain using multiplex RT-PCR and serological assays. However, L26 induced mosaic and mild vein clearing symptoms in tobacco rather than vein necrosis characteristic of the PVY NTN strain. The whole genome sequence was determined for L26 and two other PVY NTN isolates, HR1 and N4, from Idaho that did induce vein necrosis in tobacco. The sequence of all three isolates was similar to typical European PVY NTN isolates that contain three recombination junctions in their genome. The sequence of the L26 genome was nearly identical to the genomes HR1, N4, and to a previously characterized PVY NTN isolate, 423-3, differing by only five nucleotides in the entire ca. 9.7-kb genome, only one resulting in a corresponding amino acid change, D-205 to G-205 in the central region of HC-Pro. Two “signature” amino acid residues, thought involved in induction of the vein necrosis syndrome in tobacco, K-400 and E-419, were present in the C-terminal region of HC-Pro of all three isolates. Multiple alignment of the whole genome sequences of L26 and other PVY NTN isolates whose phenotype in tobacco has been reported, suggests that a single nucleotide change (A-1,627 to G-1,627) resulting in the single amino acid change (D-205 to G-205) in the HC-Pro cistron of L26 correlates with the loss of the vein necrosis phenotype in tobacco. Secondary structure modeling of the HC-Pro protein predicts the G-205 residue, and the previously identified residues K-400 and E-419, would all be located on the exposed surface of the protein. Taken together, these data suggest that the vein necrosis genetic determinant of PVY in tobacco is complex and includes other element(s), in addition to the C-terminal fragment of HC-Pro.

Secondary structure and 3D homology modeling of swine leukocyte antigen class 2 (SLA-2) molecules

No information to date is available to elucidate the structure of swine leukocyte antigen class I (SLA-I) molecule which is comprised by a heavy chain of SLA-I non-covalently associated with a light chain, β 2-microglobulin (β 2m) proteins. Presently, one of SLA-I gene SLA-2 and β 2m gene were expressed as soluble maltose binding proteins (MBP-proteins) in a pMAL-p2X/ Escherichia coli TB1 system and identified by western blotting with anti-MBP polyclonal antibodies. The expressed proteins MBP-SLA-2 and MBP-β 2m were purified on amylose affinity columns followed by DEAE–Sepharose. The purified products were cleaved by Factor Xa, respectively, and the interest of proteins SLA-2 and β 2m were purified on amylose affinity columns followed by separation from MBP on DEAE–Sepharose. The secondary structures of SLA-2 and β 2m were analyzed by circular dichroism (CD) spectrophotometry. The three-dimensional (3D) structure of their peptide-binding domain (PBD) was modeled-based sequence homology. The content of the α-helix, β-sheet, turn, and random coil in the SLA-2 protein were 76, 95, 36, and 67 aa, respectively. In the 98 aa of β 2m, the contents of the α-helix, β-sheet, turn, and random coil were 0, 45, 8, and 45 aa, respectively. The SLA-2 protein displayed a typical α-helix structure while β 2m protein displayed a typical β-sheet structure. Homology modeling of the SLA-2 and β 2m proteins demonstrated similarities with the structure of human and mouse MHC (major histocompatibility complex) class I proteins.

Regulation of Translational Efficiency by Disparate 5' UTRs of PPARgamma Splice Variants.

The PPAR-γ gene encodes for at least 7 unique transcripts due to alternative splicing of five exons in the 5′-untranslated region (UTR). The translated region is encoded by exons 1–6, which are identical in all isoforms. This study investigated the role of the 5′-UTR in regulating the efficiency with which the message is translated to protein. A coupled in vitro transcription-translation assay demonstrated that PPAR-γ1, -γ2, and -γ5 are efficiently translated, whereas PPAR-γ4 and -γ7 are poorly translated. An in vivo reporter gene assay using each 5′-UTR upstream of the firefly luciferase gene showed that the 5′-UTRs for PPAR-γ1, -γ2, and -γ4 enhanced translation, whereas the 5′-UTRs for PPAR-γ5 and -γ7 inhibited translation. Models of RNA secondary structure, obtained by the mfold software, were used to explain the mechanism of regulation by each 5′-UTR. In general, it was found that the translational efficiency was inversely correlated with the stability of the mRNA secondary structure, the presence of base-pairing in the consensus Kozak sequence, the number of start codons in the 5′-UTR, and the length of the 5′-UTR. A better understanding of posttranscriptional regulation of translation will allow modulation of protein levels without altering transcription.

Mutational analysis of a signaling aptamer suggests a mechanism for ligand-triggered structure-switching

Structure-switching signaling aptamers are nucleic acids that change shape upon binding to a specific ligand. Previously, we applied a new in vitro selection strategy to isolate structure-switching RNA aptamers responsive to the aminoglycoside antibiotic tobramycin. Here, we report the results of mutational analysis, secondary structure modeling, and ligand-specificity studies that suggest a mechanism for tobramycin-triggered structure switching.

Ribosomal ITS Sequences Allow Resolution of Freshwater Sponge Phylogeny with Alignments Guided by Secondary Structure Prediction

Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatia muelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.

Molecular characterization of intervening sequences in 23S rRNA genes and 23S rRNA fragmentation in Taylorella equigenitalis

Using two primer pairs constructed in silico for the amplification of the intervening sequences (IVSs) of the 23S rRNA gene sequences of the genus Taylorella, none of the three representative T. equigenitalis strains NCTC11184(T), Kentucky 188 and EQ59 was shown to contain any IVSs in the first quarter region. In the central region, all three strains possessed one approximately 70 bp IVS (TeIVS2) different from any IVSs found in T. asinigenitalis. The predicted secondary structure model of the IVSs contained stem and loop structures. The central region of the IVS-stem structure contains an identical double-stranded consensus 15-bp sequence. The purified RNA fraction from the three strains contained 16S and 4-5S RNA species but no 23S rRNA species. Thus, the primary 23S rRNA transcripts from the three strains would be cleaved into approximately 1.2- and 1.6-kb rRNA fragments and approximately 70-bp IVS. In addition, 16 other T. equigenitalis isolates were found to carry a similar 70-bp IVS in the central region and to produce fragmented 23S rRNA.

Structural Domains of the 3′-Terminal Sequence of the Hepatitis C Virus Replicative Strand

Here we present the results of a structural analysis of the 3'-terminal region of the replicative strand of hepatitis C virus (HCV), IRES(-), by the Pb (2+)-induced cleavage approach and partial digestion with T1 ribonuclease. Oligoribonucleotides that represent selected domains of the earlier proposed in the literature secondary structure models of this region were also synthesized, their structures were analyzed in solution, and the results were compared to those obtained with the full-length molecule. Such "structural fingerprinting" gave better insight into the structure of the IRES(-) region. We showed that in the case of the IRES(-) fragment, which consists of 374 nucleotides, its three domains, D3 (nucleotides 1-104), DM (nucleotides 105-222), and D5 (nucleotides 223-374), independently fold on one another. However, when the IRES(-) molecule is extended by 25 nucleotides of the upstream viral sequence, domains D3 and DM fold autonomously, but a part of domain D5 interacts with that additional RNA stretch. Analysis in silico suggests that similar interactions involving the IRES(-) region and upstream sequences are also possible in other fragments of viral RNA, several hundreds of nucleotides in length. The results of experimental probing are supported by secondary structure predictions in silico and phylogenetic analysis.

Molecular phylogeny of the tribe Sphodrini (Coleoptera: Carabidae) based on mitochondrial and nuclear markers

A phylogenetic analysis of 6.4 kb of nucleotide sequence data from seven genes (mitochondrial cox1-cox2 and tRNA leu, and nuclear Ef-1α C0, Ef-1α C1, 28S, and 18S) was done to reconstruct the phylogenetic relationships of the ground-beetle tribe Sphodrini. Gene regions of variable nucleotide length were aligned using both a secondary structure model, Clustal W, and a combination of the two. Sensitivity analysis was performed in order to explore the effect of alignment methods. The ribosomal and protein-coding genes were largely congruent based on the ILD test and partitioned Bremer support measures. MtDNA analysis provided high resolution and high support for most clades. The tribe Sphodrini and the related tribes Platynini, Pterostichini and Zabrini made up monophyletic clades, but the relationship between them was weakly resolved and sensitive to alignment strategy. Previously suggested relationships between subtribes of Sphodrini were not corroborated, and only the subtribe Atranopsina revealed high support as the sister clade to the other subtribes. The analyses clearly demonstrated the importance of exploring effects of alignment methods that may become particularly important in resolving polytomies and nodes with low support.