Testing Alternative Hypotheses Research Articles

Evidence of ancient horizontal gene transfer between fungi and the terrestrial alga Trebouxia

Intimate and long-lasting relationships of fungi and algae have been known for centuries by scientists, and these ancient symbioses might have provided excellent opportunities for horizontal gene transfer (HGT) of protein encoding genes between the two organismal partners. In this study, we sequenced and assembled 451 Mbp of novel genomic DNA from Trebouxia decolorans (Trebouxiaceae, Chlorophyta), the green algal photobiont of the lichen Xanthoria parietina (Teloschistaceae, Lecanoromycetes, Ascomycota). This alga also occurs as a free-living terrestrial organism. The aim of our work was to search for candidate genes pointing to HGT between lichenized fungi and lichen algae. We found evidence for three putative HGT events of fungal genes into the Trebouxia genome, but these are likely more ancient (over 600 mya) than the origin of lichenization within the fungal Ascomycetes. The three transferred genes are part of gene groups that in other species encode a tellurite-resistance dicarboxylate transporter (TDT) family protein, a class-1 nitrilase/cyanide hydratase (CH), and an oxidoreductase/retinol dehydrogenase. In each case, our phylogenomic analyses show orthologs from Trebouxia as sister to orthologs from all fungi or basally placed within Ascomycetes, while the orthologs from green algae and land plants form separate, independent evolutionary lineages. Alternative hypothesis tests significantly support these HGT events. The presence of these genes in Trebouxia was validated by PCR amplification of separately isolated Trebouxia DNA. The ancient incorporation of fungal genes in the genomes of these particular green algae are intriguing and could be early evidence for symbiotic and co-evolutionary relationships among the major eukaryotic branches of algae and fungi present in early terrestrial life on Earth. These genes could have played a pre-disposition role for some fungi and algae in the origin of lichen symbiosis, but further studies are needed to evaluate this in detail.

Monte Carlo Comparison of Three Tests of Exponentiality Based on Kullback-Leibler Information in Ranked Set Sampling

This paper proposes three tests of exponentiality based on Kullback-Leibler information under ranked set sampling (RSS) scheme. The critical values of the proposed test statistics are obtained using different values of the significance level. Based on extensive Monte Carlo simulations, we calculate the power of the proposed tests for different sample sizes and alternative hypotheses. It is observed that, based on the same sample and window sizes, the suggested tests are more powerful than the other competitor tests based on simple random sampling (SRS) scheme.

House Prices, Wealth and Consumption: New Evidence from Australia and Canada

Over the past two decades, a number of countries have experienced appreciation in house prices at the same time that aggregate consumption has increased. This paper tests alternative hypotheses for this phenomenon by using repeated household surveys from Australia and Canada to identify the transmission mechanism that links consumption and household wealth. The empirical analysis suggests that neither a direct wealth effect nor a common causal factor likely accounts for the observed correlation between wealth and consumption in these two countries. Rather, indirect factors such as collateral effects arising from relaxation of credit constraints are a more likely explanation.

Evolutionary diversification of new Caledonian Araucaria.

New Caledonia is a global biodiversity hotspot. Hypotheses for its biotic richness suggest either that the island is a ‘museum’ for an old Gondwana biota or alternatively it has developed following relatively recent long distance dispersal and in situ radiation. The conifer genus Araucaria (Araucariaceae) comprises 19 species globally with 13 endemic to this island. With a typically Gondwanan distribution, Araucaria is particularly well suited to testing alternative biogeographic hypotheses concerning the origins of New Caledonian biota. We derived phylogenetic estimates using 11 plastid and rDNA ITS2 sequence data for a complete sampling of Araucaria (including multiple accessions of each of the 13 New Caledonian Araucaria species). In addition, we developed a dataset comprising 4 plastid regions for a wider taxon sample to facilitate fossil based molecular dating. Following statistical analyses to identify a credible and internally consistent set of fossil constraints, divergence times estimated using a Bayesian relaxed clock approach were contrasted with geological scenarios to explore the biogeographic history of Araucaria. The phylogenetic data resolve relationships within Araucariaceae and among the main lineages in Araucaria, but provide limited resolution within the monophyletic New Caledonian species group. Divergence time estimates suggest a Late Cretaceous-Cenozoic radiation of extant Araucaria and a Neogene radiation of the New Caledonian lineage. A molecular timescale for the evolution of Araucariaceae supports a relatively recent radiation, and suggests that earlier (pre-Cenozoic) fossil types assigned to Araucaria may have affinities elsewhere in Araucariaceae. While additional data will be required to adequately resolve relationships among the New Caledonian species, their recent origin is consistent with overwater dispersal following Eocene emersion of New Caledonia but is too old to support a single dispersal from Australia to Norfolk Island for the radiation of the Pacific Araucaria sect. Eutacta clade.

Bayesian tests for composite alternative hypotheses in cross-tabulated data

We present a methodology for constructing significance tests for “difficult” composite alternative hypotheses that have no natural test statistic. We apply our methodology to construct exact tests for cross-tabulated data, and our motivating example is constructing a test for discovering Simpson’s Paradox. Our tests are Bayesian extensions of the likelihood ratio test; they are optimal with respect to the prior distribution and are also closely related to Bayes factors and Bayesian FDR controlling testing procedures.

Integrative taxonomy of the stunt nematodes of the generaBitylenchusandTylenchorhynchus(Nematoda, Telotylenchidae) with description of two new species and a molecular phylogeny

Stunt nematodes are characterized by phenotypic plasticity, with overlapping morphology and morphometry leading to potential misidentification. Consequently, the application of integrative taxonomic approaches is useful to species delimitation based on a combination of different perspectives, e.g. morphology and DNA sequences. We conducted nematode surveys in cultivated and natural environments in Spain and the USA, from which we identified 18 known species of the family Telotylenchidae and two new taxa within the studied samples. These species were morphologically, morphometrically, and molecularly characterized. The results of light and scanning electron microscopic observations, and molecular and phylogenetic analysis also allowed two new species to be distinguished, described herein as Bitylenchus hispaniensis sp. nov. and Tylenchorhynchus mediterraneus sp. nov. The phylogenetic analysis was carried out using molecular data from nuclear ribosomal DNA genes [D2–D3 expansion segments of the large ribosomal subunit (28S), internal transcribed spacer (ITS), and partial small ribosomal subunit (18S)]. We also provide here a test of alternative hypotheses that confirms the monophyly of both Tylenchorhynchus and Bitylenchus sensu Siddiqi's classification but does not support Fortuner & Luc's conceptual view of Tylenchorhynchus as a large genus. Ancestral state reconstructions of several diagnostic morphological characters using a maximum parsimony approach showed congruence in morphological and molecular evolution for stylet knob inclination and tail tip annulation. Our analysis emphasizes some of the problems related to the taxonomy and phylogeny of nematodes of Telotylenchinae. © 2014 The Linnean Society of London

Mitochondrial phylogenomics of early land plants: mitigating the effects of saturation, compositional heterogeneity, and codon-usage bias.

Phylogenetic analyses using concatenation of genomic-scale data have been seen as the panacea for resolving the incongruences among inferences from few or single genes. However, phylogenomics may also suffer from systematic errors, due to the, perhaps cumulative, effects of saturation, among-taxa compositional (GC content) heterogeneity, or codon-usage bias plaguing the individual nucleotide loci that are concatenated. Here, we provide an example of how these factors affect the inferences of the phylogeny of early land plants based on mitochondrial genomic data. Mitochondrial sequences evolve slowly in plants and hence are thought to be suitable for resolving deep relationships. We newly assembled mitochondrial genomes from 20 bryophytes, complemented these with 40 other streptophytes (land plants plus algal outgroups), compiling a data matrix of 60 taxa and 41 mitochondrial genes. Homogeneous analyses of the concatenated nucleotide data resolve mosses as sister-group to the remaining land plants. However, the corresponding translated amino acid data support the liverwort lineage in this position. Both results receive weak to moderate support in maximum-likelihood analyses, but strong support in Bayesian inferences. Tests of alternative hypotheses using either nucleotide or amino acid data provide implicit support for their respective optimal topologies, and clearly reject the hypotheses that bryophytes are monophyletic, liverworts and mosses share a unique common ancestor, or hornworts are sister to the remaining land plants. We determined that land plant lineages differ in their nucleotide composition, and in their usage of synonymous codon variants. Composition heterogeneous Bayesian analyses employing a nonstationary model that accounts for variation in among-lineage composition, and inferences from degenerated nucleotide data that avoid the effects of synonymous substitutions that underlie codon-usage bias, again recovered liverworts being sister to the remaining land plants but without support. These analyses indicate that the inference of an early-branching moss lineage based on the nucleotide data is caused by convergent compositional biases. Accommodating among-site amino acid compositional heterogeneity (CAT-model) yields no support for the optimal resolution of liverwort as sister to the rest of land plants, suggesting that the robust inference of the liverwort position in homogeneous analyses may be due in part to compositional biases among sites. All analyses support a paraphyletic bryophytes with hornworts composing the sister-group to tracheophytes. We conclude that while genomic data may generate highly supported phylogenetic trees, these inferences may be artifacts. We suggest that phylogenomic analyses should assess the possible impact of potential biases through comparisons of protein-coding gene data and their amino acid translations by evaluating the impact of substitutional saturation, synonymous substitutions, and compositional biases through data deletion strategies and by analyzing the data using heterogeneous composition models. We caution against relying on any one presentation of the data (nucleotide or amino acid) or any one type of analysis even when analyzing large-scale data sets, no matter how well-supported, without fully exploring the effects of substitution models.

Mental structures and hierarchical brain processing: Comment on “Toward a computational framework for cognitive biology: Unifying approaches from cognitive neuroscience and comparative cognition” by W. Tecumseh Fitch

Fitch proposes an appealing hypothesis that humans are dendrophiles, who constantly build mental trees supported by analogous hierarchical brain processes [1]. Moreover, it is argued that, by comparison, nonhuman animals build flat or more compact behaviorally-relevant structures. Should we thus expect less impressive hierarchical brain processes in other animals? Not necessarily. Neuroscientific evidence is showing that the brains of humans and many animals are dendrophiles. The well-established notion of hierarchical brain processing was informed by human and nonhuman animal work [2,3]. Moreover, the form of processing is not just serial or parallel, because human, monkey and rodent brains have both local ‘small world’ connectivity patterns, as well as hubs that interconnect different ‘worlds’ [4]. Also, dendrograms as neuronal representations are prominent in a range of species: dendrograms can well describe monkey ventral frontal cortex neurons responding to vocalization sounds [5], and rodent hippocampal neurons can create multi-layered dendritic structures to encode several features associated with a memory [6]. In neither case is there any suggestion that different vocalizations or memory features are hierarchically organized by the animals for behavior. Also, hierarchically nested brain oscillations responding to speech are evident in humans [7], which is complemented by computational models of speech parsing [8]. Yet, nested oscillations are also evident in monkeys listening to natural sounds [9] and violations to a non-hierarchical artificial grammar that monkeys can learn appears to modulate nested oscillations in auditory cortex [10]. Much less clear, as Fitch suggests [1], is how to access the mental structures that humans and other animals sculpt. He suggests testing alternative hypotheses on the computations being conducted. For example, if one is evaluating strings generated by an AnBn grammar, different individuals might glean different structures depending on the mechanisms used, such as: (1) ‘push-down-stack’, where associations are computed and allocated to memory; or, (2) ‘count-and-compare’, where multiple counters keep track of the number of As and Bs (in the case of n = 3, 3 As and 3 Bs) and a comparison function evaluates whether they are equal. In the case of a ‘push-down-stack’, one might envision the human inferior frontal cortex associating different forms of input and holding the associations in memory. But it is unclear how this would differ or be informed by insights on hierarchically organized cognition in studies of neurons from monkey frontal cortex [11], or those on hierarchically organized memories in rodent fronto-hippocampal networks [6]. In the case of ‘count-and-compare’, this could be informed by numerosity encoding in monkey frontal neurons [12]. The danger, however, which Fitch does not appear to condone, is overlooking the insights that we can gain from nonhuman animals if most are viewed as having, at least by human standards, unsophisticated mental structures. I agree that a priority is for hypotheses to be better developed and tested on the computations subserving different behaviorally-relevant structures, which even in humans has remained largely theoretical. Each computational hypothesis supported or refuted clarifies the types of structures being built by an individual or their species. Neurobiological evidence can provide mechanistic insights. However, it is incorrect to assume that hierarchical neural processes subserve only hierarchical representations of the forms that humans uniquely can build. After all, it is already abundantly clear that the brains of most animals like building elaborate structures.

It is premature to regard the ego-depletion effect as "Too Incredible".

It is premature to regard the ego-depletion effect as "Too Incredible".

The iron K line profile of IRAS 18325–5926

IRAS 18325-5926 is an X-ray bright, Compton-thin, type-2 Seyfert galaxy and it was the first Seyfert 2 in which the presence of a broad Fe K-alpha emission line was claimed. However, although the structure of the Fe line appears broad, there is tentative evidence that it may comprise multiple lines. Nevertheless, previous analyses have only consisted of fitting standalone broad components to the Fe K band. Here, we have analyzed all available X-ray CCD data from Suzaku, XMM-Newton and ASCA to fully investigate the nature of the emission complex by testing broad-band physical models and alternative hypotheses. We find that both a model consisting of broad, blurred reflection from an ionized accretion disc and a model consisting of cold, neutral reflection plus narrow emission lines from highly-ionized photoionized gas (log \xi = 3.5) offer statistically comparable fits to the data although the true reality of the Fe line cannot currently be determined with existing data. However, it is hoped that better quality data and improved photon statistics in the Fe K band will allow a more robust distinction between models to be made.

Testing alternative hypotheses regarding the association between behavioral inhibition and language development in toddlerhood.

Studies have reported an inverse association between language development and behavioral inhibition or shyness across childhood, but the direction of this association remains unclear. This study tested alternative hypotheses regarding this association in a large sample of toddlers. Data on behavioral inhibition and expressive and receptive language abilities were collected from 816 twins at ages 14, 20, and 24months. Growth and regression models were fit to the data to assess the longitudinal associations between behavioral inhibition and language development from 14 to 24months. Overall, there were significant associations between behavioral inhibition and expressive language, and minimal associations with receptive language, indicating that the association is better explained by reticence to respond rather than deficient language development.

EPA-1403 – Testing alternative hypotheses for the increased rate of psychotic disorder in immigrants

Increased incidence rates of psychotic disorder have been consistently observed in immigrants with significant variability across ethnic groups. These findings have been attributed to cross-cultural biases, i.e. misinterpretation of culturally appropriate ideas leading to an overestimation of these disorders among ethnic minorities. Another alternative explanation constitutes the selection hypothesis positing that the increased rate is due to selective migration of predisposed people. We will present two studies that aimed at testing these hypotheses by examining (a) whether risk factors for psychosis are more prevalent among future emigrants, and (b) whether psychotic symptoms differ in severity and nature according to ethnic group. The first study was conducted among a cohort of 50 087 conscripts who were assessed at age 18 on cannabis use, IQ, psychiatric diagnosis, social adjustment, history of trauma and urbanicity of place of upbringing. Through data linkage we examined whether these exposures predicted emigration out of Sweden. The second study included 301 first episode psychosis (FEP) patients within a defined catchment area in Montreal, Canada. Patients were administered scales for the assessment of positive and negative symptoms, as well as general psychopathology. Symptom scores of the reference group were compared to those of patients with different ethnic backgrounds. The results of these studies will be discussed in light of candidate explanations for the increased risk of psychotic disorder among immigrants. Future avenues using data from the EUropean Gene-Environment Interaction (EU-GEI) project will be proposed.

An alternative hypothesis testing strategy for secondary phenotype data in case-control genetic association studies.

Motivated by the challenges associated with accounting for the ascertainment when analyzing secondary phenotypes that are correlated with case-control status, Lin and Zeng have proposed a method that properly reflects the case-control sampling (Lin and Zeng, 2009). The Lin and Zeng method has the advantage of accurately estimating effect sizes for secondary phenotypes that are normally distributed or dichotomous. This method can be computationally intensive in practice under the null hypothesis when the likelihood surface that needs to be maximized can be relatively flat. We propose an extension of the Lin and Zeng method for hypothesis testing that uses proportional odds logistic regression to circumvent these computational issues. Through simulation studies, we compare the power and type-1 error rate of our method to standard approaches and Lin and Zeng's approach.

Contrasting Alternative Hypotheses to Explain Rut‐Induced Hypophagia in Territorial Male Chamois

AbstractMale ungulates in temperate environments often show a severe reduction in time spent foraging during the mating season. Several hypotheses have been put forward to explain this phenomenon but, so far, no study investigated the proximate mechanisms underlying rut‐induced hypophagia in ungulates using alternative mating tactics (AMTs). Between the pre‐rut and post‐rut of 2011 and 2012, we collected data on activity budgets, parasite burden and androgen levels of territorial and non‐territorial male Alpine chamois Rupicapra r. rupicapra in the Gran Paradiso National Park (Italy). We aimed to investigate whether AMTs showed similar reduction in time spent foraging during the mating period and to test the predictions underlying alternative hypotheses that may explain rut‐induced hypophagia. Only territorial males showed a significant reduction in time spent foraging during the rut; the lack of correlation between proportion of time spent foraging and androgen metabolites or parasite burden did not fully support the physiological and the parasite hypotheses, while the foraging constraint, the energy‐saving and the physical rest hypotheses could not be discounted. Territorial males decreased the time spent lying down from the pre‐rut to the rut, but not their foraging‐to‐lying‐down ratio. During the mating period, we found negative correlations between time spent foraging or lying down and time spent rutting. Our data suggest that territorial males’ behaviour is more consistent with the foraging constraint hypothesis than with the energy‐saving hypothesis previously suggested. Yet, during the rut territorial males did not maximise their foraging time, and the optimisation of their energy balance could rather depend upon feeding on relatively high‐quality plants. This suggestion – possibly named ‘forage quality hypothesis’ – now requires further investigations. This work showed that alternative mating behaviours may underlie different patterns of foraging strategies: we suggest that tests of alternative hypotheses to explain rut‐induced hypophagia within ungulate populations should not ignore the occurrence of AMTs.

Explicit tests of palaeodrainage connections of southeastern North America and the historical biogeography of Orangethroat Darters (Percidae: Etheostoma:Ceasia)

The alteration in palaeodrainage river connections has shaped patterns of speciation, genetic diversity and the geographical distribution of the species-rich freshwater fauna of North America. The integration of ancestral range reconstruction methods and divergence time estimates provides an opportunity to infer palaeodrainage connectivity and test alternative palaeodrainage hypotheses. Members of the Orangethroat Darter clade, Ceasia, are endemic to southeastern North America and occur north and south of the Pleistocene glacial front, a distributional pattern that makes this clade of closely related species an ideal system to investigate the number and location of glacial refugia and compare alternative hypotheses regarding the proposed evolution of the Teays-Mahomet palaeodrainage. This study utilized time-calibrated mitochondrial and nuclear gene phylogenies and present-day geographical distributions to investigate hypothesized Teays-Mahomet River connections through time using a dispersal-extinction-cladogenesis (DEC) framework. Results of DEC ancestral area reconstructions indicate that the Teays-Mahomet River was a key dispersal route between disjunct highland regions connecting the Mississippi River tributaries to the Old-Ohio Drainage minimally at two separate occasions during the Pleistocene. There was a dynamic interplay between palaeodrainage connections through time and postglacial range expansion from three glacial refugia that shaped the current genetic structure and geographical distributions of the species that comprise Ceasia.

The sister-group relationships of the largest family of lichenized fungi, Parmeliaceae (Lecanorales, Ascomycota)

Parmeliaceae is the largest family of lichen-forming fungi. In spite of its importance for fungal diversity, its relationships with other families in Lecanorales remain poorly known. To better understand the evolutionary history of the diversification of lineages and species richness in Parmeliaceae it is important to know the phylogenetic relationships of the closest relatives of the family. A recent study based on two molecular loci suggested that either Protoparmelia s. str. or a group consisting of Gypsoplaca and Protoparmelia s. str. were the possible sister-group candidates of Parmeliaceae, but that study could not distinguish between these two alternatives. Here, we used a four-locus phylogeny (nuLSU, ITS, RPB1, MCM7) to reveal relationships of Parmeliaceae with other potential relatives in Lecanorales. Maximum likelihood and Bayesian analyses showed that Protoparmelia is polyphyletic, with Protoparmelia s. str. (including Protoparmelia badia and Protoparmelia picea) being most closely related to Parmeliaceae s. str., while the Protoparmelia atriseda-group formed the sister-group to Miriquidica. Gypsoplaca formed the sister-group to the Parmeliaceae s. str. + Protoparmelia s. str. clade. Monophyly of Protoparmelia as currently circumscribed, and Gypsoplaca as sister-group to Parmeliaceae s. str. were both significantly rejected by alternative hypothesis testing.

Patterns and processes in complex landscapes: testing alternative biogeographical hypotheses through integrated analysis of phylogeography and community ecology in Hawai'i

The Island of Hawai'i is a dynamic assemblage of five volcanoes with wet forest habitat currently existing in four distinct natural regions that vary in area, age and geographical isolation. In this complex landscape, alternative assumptions of the relative importance of specific habitat characteristics on evolutionary and ecological processes predict strikingly different general patterns of local diversity and regional similarity. In this study, we compare alternative a priori hypotheses against observed patterns within two distinct biological systems and scales: community composition of wet forest vascular plant species and mitochondrial and nuclear genes of Drosophila sproati, a wet-forest-restricted endemic. All observed patterns display strong and similar regional structuring, with the greatest local diversity found in Kohala and the windward side of Mauna Loa, the least in Ka'ū and Kona, and a distinctive pattern of regional similarity that probably reflects the historical development of this habitat on the island. These observations largely corroborate a biogeographical model that integrates multiple lines of evidence, including climatic reconstruction, over those relying on single measures, such as current habitat configuration or substrate age. This method of testing alternative hypotheses across biological systems and scales is an innovative approach for understanding complex landscapes and should prove valuable in diverse biogeographical systems.

A comprehensive molecular phylogeny of dalytyphloplanida (platyhelminthes: rhabdocoela) reveals multiple escapes from the marine environment and origins of symbiotic relationships.

In this study we elaborate the phylogeny of Dalytyphloplanida based on complete 18S rDNA (156 sequences) and partial 28S rDNA (125 sequences), using a Maximum Likelihood and a Bayesian Inference approach, in order to investigate the origin of a limnic or limnoterrestrial and of a symbiotic lifestyle in this large group of rhabditophoran flatworms. The results of our phylogenetic analyses and ancestral state reconstructions indicate that dalytyphloplanids have their origin in the marine environment and that there was one highly successful invasion of the freshwater environment, leading to a large radiation of limnic and limnoterrestrial dalytyphloplanids. This monophyletic freshwater clade, Limnotyphloplanida, comprises the taxa Dalyelliidae, Temnocephalida, and most Typhloplanidae. Temnocephalida can be considered ectosymbiotic Dalyelliidae as they are embedded within this group. Secondary returns to brackish water and marine environments occurred relatively frequently in several dalyeliid and typhloplanid taxa. Our phylogenies also show that, apart from the Limnotyphloplanida, there have been only few independent invasions of the limnic environment, and apparently these were not followed by spectacular speciation events. The distinct phylogenetic positions of the symbiotic taxa also suggest multiple origins of commensal and parasitic life strategies within Dalytyphloplanida. The previously established higher-level dalytyphloplanid clades are confirmed in our topologies, but many of the traditional families are not monophyletic. Alternative hypothesis testing constraining the monophyly of these families in the topologies and using the approximately unbiased test, also statistically rejects their monophyly.

Reinforcement in plants

A major goal of evolutionary biology is to understand how diverging populations become species. The evolution of reproductive isolation (RI) halts the genomic homogenization caused by gene flow and recombination, and enables differentiation and local adaptations to become fixed between newly forming species. Selection can favor the strengthening of RI through a process termed reinforcement. Reinforcement occurs when selection favors traits that decrease mating between two incipient species in response to costly mating or the production of maladapted hybrids. Although this process has been investigated more frequently in animals, there is also evidence of reinforcement in plants. There are three strategies for the investigation of the process of reinforcement: case studies of species or diverging taxa; experimental evolution studies; and comparative studies. Here, I discuss how all three strategies find evidence consistent with reinforcement occurring in plants. I focus largely on case studies, and use research on Phlox drummondii to illustrate the importance of testing alternative hypotheses. Although the existing evidence suggests that reinforcement can occur, further investigations, particularly using large-scale comparative studies, are needed to determine the importance of reinforcement in plant speciation.

Joint provision of external audit and non-audit services: empirical test of alternative hypotheses

Providing jointly external audit and non-audit services to the same client-company remains a major dilemma for the accounting profession. However, recent professional ethics reforms by the IFAC and the AICPA on networks of firms may have offered a new solution to the problem. However, in search for the best solution to the problem, we test the following three alternative hypotheses: 1) a same CPA firm provides jointly both services by using separate personnel teams; 2) a same CPA firm provides jointly both services by using two separate specialised divisions; 3) two legally distinct CPA firms, both members of the same network of CPA firms, provide one the external audit and the other the non-audit services. Dependent variables of interest are: 1) perceived audit firm’s independence; 2) perceived firm’s external audit quality, while the independent variable is the type of solution tested: solution 1, solution 2, or solution 3. The empirical results revealed, as hypothesised, the two-network firms arrangeme...