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© 2019 Palmer et al.
With the increased availability of genome sequences for bacteria, it has become routine practice to construct genome-based phylogenies. These phylogenies have formed the basis for various taxonomic decisions, especially for resolving problematic relationships between taxa. Despite the popularity of concatenating shared genes to obtain well-supported phylogenies, various issues regarding this combined-evidence approach have been raised. These include the introduction of phylogenetic error into datasets, as well as incongruence due to organism-level evolutionary processes, particularly horizontal gene transfer and incomplete lineage sorting. Because of the huge effect that this could have on phylogenies, we evaluated the impact of phylogenetic conflict caused by organism-level evolutionary processes on the established species phylogeny for Pantoea, a member of the Enterobacterales. We explored the presence and distribution of phylogenetic conflict at the gene partition and nucleotide levels, by identifying putative inter-lineage recombination events that might have contributed to such conflict. Furthermore, we determined whether smaller, randomly constructed datasets had sufficient signal to reconstruct the current species tree hypothesis or if they would be overshadowed by phylogenetic incongruence. We found that no individual gene tree was fully congruent with the species phylogeny of Pantoea, although many of the expected nodes were supported by various individual genes across the genome. Evidence of recombination was found across all lineages within Pantoea, and provides support for organism-level evolutionary processes as a potential source of phylogenetic conflict. The phylogenetic signal from at least 70 random genes recovered robust, well-supported phylogenies for the backbone and most species relationships of Pantoea, and was unaffected by phylogenetic conflict within the dataset. Furthermore, despite providing limited resolution among taxa at the level of single gene trees, concatenated analyses of genes that were identified as having no signal resulted in a phylogeny that resembled the species phylogeny of Pantoea. This distribution of signal and noise across the genome presents the ideal situation for phylogenetic inference, as the topology from a ≥70-gene concatenated species phylogeny is not driven by single genes, and our data suggests that this finding may also hold true for smaller datasets. We thus argue that, by using a concatenation-based approach in phylogenomics, one can obtain robust phylogenies due to the synergistic effect of the combined signal obtained from multiple genes.
Whole genome sequences are now routinely used for phylogenetic inference, particularly in bacteria (Abdul Rahman et al., 2016; Beukes et al., 2017; Callister et al., 2008; Gupta, Naushad & Baker, 2015; Meehan & Beiko, 2014; Palmer et al., 2017; Schwartz et al., 2015; Zhang et al., 2011). Many approaches for investigating evolutionary relationships across different taxonomic ranks have been developed (Daubin, Gouy & Perrière, 2001; Daubin, Gouy & Perriere, 2002; Jolley et al., 2012; Yokono, Satoh & Tanaka, 2018). These range from alignment-free approaches (Yokono, Satoh & Tanaka, 2018) to alignment-based analyses of a small number of highly conserved genes across large numbers of taxa (e.g., different bacterial phyla or orders (Abdul Rahman et al., 2016; Gupta, Naushad & Baker, 2015; Jolley et al., 2012)), to using hundreds or thousands of genes, obtained from whole genome sequences and shared by all members of smaller groups (e.g., species, genus or family (Meehan & Beiko, 2014; Palmer et al., 2017; Schwartz et al., 2015; Zhang et al., 2011)).
The use of large numbers of shared genes for phylogenetic inference, referred to here as phylogenomics (Daubin, Gouy & Perrière, 2001; Daubin, Gouy & Perriere, 2002; Eisen & Fraser, 2003; Kumar et al., 2011), have been argued to be the most reliable option for recovering a species topology reflective of vertical descent (Andam & Gogarten, 2011; Coenye et al., 2005; Daubin, Gouy & Perriere, 2002; Galtier & Daubin, 2008). This is because the massive numbers of characters sampled is thought to dilute phylogenetic conflict within the dataset, to levels where a single robust evolutionary hypothesis is obtainable (Andam & Gogarten, 2011; Coenye et al., 2005; Cohan, 2001; Daubin, Gouy & Perriere, 2002; Galtier & Daubin, 2008; Klenk & Göker, 2010). It has been suggested, particularly in bacteria, that an overall genomic core (the set of genes shared by all members of a group) exists between closely related taxa that remains evolutionarily cohesive (Coenye et al., 2005; Daubin, Gouy & Perriere, 2002). The signal found within these core genes would thus be the signal for inheritance and would be appropriate for inferring the ancestral relationships (Daubin, Gouy & Perriere, 2002).
Despite some evidence for a genomic core (Callister et al., 2008; Daubin, Gouy & Perriere, 2002; Grote et al., 2012; Sarkar & Guttman, 2004), numerous studies have shown that the evolutionary trajectory of genes within this subgenomic compartment may be incongruent (Bapteste et al., 2009; Dagan & Martin, 2006; Jeffroy et al., 2006; Rokas et al., 2003; Thiergart, Landan & Martin, 2014). Dagan & Martin (2006) captured this conflict in their “tree of one percent” concept. They referred to research by Ciccarelli and colleagues (2006), who used 31 protein sequences to recover a robust phylogenetic hypothesis across a diverse set of bacterial taxa. This was after the removal of sequences harbouring phylogenetic conflict from a conservative average bacterial genome of 3,000 genes. In other words, the resulting phylogenetic tree that was interpreted as the evolutionary history of the taxa, was based on roughly 1% of the average genome of these taxa (Dagan & Martin, 2006). Additionally, research has shown that species trees may in some cases be driven by only a handful of genes, particularly where contradictory species relationships are routinely observed from single gene trees (Salichos & Rokas, 2013; Shen, Hittinger & Rokas, 2017; Thiergart, Landan & Martin, 2014). It is thus still unclear whether employing genome data in a concatenation-based approach is truly an appropriate way of inferring evolutionary relationships, despite the popularity of this approach.
The incongruence often observed between gene and species trees can be attributed to two main factors: phylogenetic errors and organism-level evolutionary processes (Doyle, 1992; Wendel & Doyle, 1998). Phylogenetic errors (i.e., stochastic errors due to the use of too little information and systematic errors caused by non-phylogenetic signal) during tree inferences are mainly overcome by increased character and taxon sampling (Hedtke, Townsend & Hillis, 2006; Jeffroy et al., 2006; Palmer et al., 2017; Philippe et al., 2011; Pollock et al., 2002; Yokono, Satoh & Tanaka, 2018). Organism-level evolutionary processes can be difficult to account for if they result in different evolutionary histories for genes that cannot be integrated into a single bifurcating evolutionary hypothesis (Wendel & Doyle, 1998). When phylogenetic error is excluded, incomplete lineage sorting (ILS) and horizontal gene transfer (HGT) are frequently the primary organism-level processes responsible for phylogenetic incongruence (Galtier & Daubin, 2008; Mallet, Besansky & Hahn, 2016; Retchless & Lawrence, 2010). An ongoing debate in the scientific community is whether to concatenate and risk a well-supported but incorrect species tree that also captures all phylogenetic conflict in a dataset, or pool the phylogenetic signal from hundreds of gene trees in supertree or reconciliation approaches (Daubin, Gouy & Perrière, 2001; Galtier & Daubin, 2008; Ren, Tanaka & Yang, 2009; Retchless & Lawrence, 2010; Sanderson & Driskell, 2003; Szöllősi et al., 2012; Williams et al., 2017). Reconciliation approaches efficiently account for HGT because genome evolution is modelled and the data produced are used for quantifying gene transfer and for inferring species trees that accommodate this process (Szöllősi et al., 2012).
For this study, the bacterial genus Pantoea was used as a model to explore the impact of potentially conflicting signal caused by organism-level evolutionary processes on the current phylogenetic hypothesis for the group. This phylogeny was constructed previously using a concatenation-based approach that accounted for the majority of known phylogenetic errors through Maximum Likelihood analyses of partitioned datasets with appropriate evolutionary models (Palmer et al., 2017). Pantoea forms part of the family Erwiniaceae in the order Enterobacterales (Adeolu et al., 2016) and is closely related to the genera Erwinia and Tatumella (Adeolu et al., 2016; Brady et al., 2010b; Glaeser & Kämpfer, 2015; Palmer et al., 2017). This genus has been extensively studied and represents a diverse assemblage of organisms that employs an array of different and important lifestyles (Brady et al., 2010a; Brady et al., 2009; Brady et al., 2010b; Lim et al., 2014; Ma et al., 2016; Palmer et al., 2016; Palmer et al., 2017; Walterson & Stavrinides, 2015). Our three main objectives were to (i) determine whether or not the dataset used to infer hypotheses (based on concatenation and a multi-species coalescent approach) included phylogenetic conflict, and if so, how this conflict is distributed across the genome; (ii) to determine whether the observed conflicts could be ascribed to organism-level evolutionary processes, such as HGT and ILS; and (iii) to determine whether limited sets of genes contain enough phylogenetic signal to overshadow potential conflict within the dataset in order to obtain phylogenies resembling the species phylogenetic hypotheses for Pantoea. To achieve these objectives we investigated conflict at the level of gene partitions and at specific nucleotide sites to detect recombination between the different lineages of the Pantoea species phylogeny and also to compare regions that differed significantly in their nucleotide composition to the rest of the alignments.
Shared genes for the 27 taxa of interest (Table 1) were determined with the Efficient Database framework for comparative Genome Analyses using BLAST score Ratios (EDGAR) server (Blom et al., 2016). The nucleotide sequences for all shared genes were downloaded from the EDGAR server. Subsequently, the combined file of all sequences were split into individual gene files. Multiple sequence alignments of genes were generated with MUSCLE (Edgar, 2004) as part of CLC Main Workbench v 7.6 (CLC Bio, Aarhus, Denmark). This was followed by manual inspection and correction of alignments in BioEdit v. 7.0.9 (Hall, 2011) to ensure that the correct reading-frame was selected for all genes. Genes were then trimmed in BioEdit to eliminate gene length variation due to potential differences in gene prediction across the different genomes. To generate concatenated datasets, the respective nucleotide and protein sequences were combined with FASconCAT-G v. 1.02 (Kück & Longo, 2014).
Genome sequences utilised in this study.
aSuperscriptT indicates type strains for the species.
bAll numbers refer to GenBank assembly accession numbers (http://www.ncbi.nlm.nih.gov/; accessed 28/2/2017).
Approximate maximum likelihood (AML; Price, Dehal & Arkin, 2010) analyses were performed on all individual protein sequences, as well as, on the concatenated protein and nucleotide sequence data matrices. For individual gene trees, analyses were performed in a sequential manner, utilising an in-house python script (File S1). For computational efficiency, AML analyses were employed in this study instead of traditional maximum likelihood (ML) analyses in alternate software. Time estimates for the construction of a single gene tree based on ML is ca. 27 minutes/gene (RAxML v. 8.0.20 (Stamatakis, 2014)) versus ca. 4 minutes/gene for AML (FastTree v. 2.1), which is not surprising as up to 100 times speed increases were reported previously (Price, Dehal & Arkin, 2010). All AML phylogenies were constructed with FastTree v. 2.1 (Price, Dehal & Arkin, 2010) using default settings. When the relationships obtained from concatenated AML analyses were not robustly supported (SH-support >0.95), these relationships were verified using RAxML v. 8.0.20 (Stamatakis, 2014).
A multi-species coalescent (MSC) approach (Mirarab et al., 2014) was employed to construct a species tree from the individual gene phylogenies. This summary method was used to reconstruct a species tree, in the presence of potential ILS (Mirarab et al., 2014), by subjecting the unrooted AML phylogenies to an MSC analysis in ASTRAL v. 5.6.3 (Mirarab et al., 2014). Outputs were indicated with branch lengths in coalescent units and support values for the four clusters around a specific branch (quartet score; Sayyari & Mirarab, 2016). Additionally, three other approaches were used to infer the Pantoea species tree. The first involved inference of a Neighbour-Joining (NJ) tree using distances based on Average Nucleotide Identity (ANI; Richter & Rosselló-Móra, 2009) values. These were available from a previous study (Palmer et al., 2017) and used to generate a pairwise distance matrix in Microsoft Excel™ from which a NJ tree was inferred using MEGA v. 6.06 (Tamura et al., 2013). Note that this precluded resampling of the data for evaluating branch support. Secondly, a Neighbor-Net network was inferred from the concatenated nucleotide data using default settings in SplitsTree v. 4 (Huson & Bryant, 2005). Thirdly, this software was also used to construct a consensus network from the single gene AML phylogenies with a zero threshold (exclude no trees) and edge weights set to count.
To determine the degree of congruence and distribution of signal across individual gene genealogies relative to the Pantoea species phylogenies, individual phylograms were manually inspected. During this process, gene genealogies supporting specific backbone nodes (that were consistently recovered using multiple inference approaches) within the Pantoea species phylogenies were identified. This was done by evaluating a set of twelve query hypotheses (representing all of the internal backbone nodes in the Pantoea species trees) against each of the individual gene genealogies to determine whether they contained and/or supported the expected nodes. Each genealogy was then marked as (1) fully supporting, (2) supporting, but with other taxa nested, (3) not supporting or (4) lacking signal for the specific node depicted in the query hypothesis. The signal obtained from each of the different gene trees were then related back to the physical order of the shared genes as they appear in the genome of P. agglomerans (Lim et al., 2014), to determine whether specific signal patterns could be associated to areas of the genome.
As an indication of how phylogenetic conflicts were distributed across the concatenated nucleotide alignment, incongruent signals for the P. dispersa and P. ananatis lineages were investigated. For these purposes, the nucleotide sites causing incongruence in the Neighbor-Net network was noted and related back to the gene identifier. These data were visualised across the concatenated alignment using Circos v. 0.69 (Krzywinski et al., 2009).
To determine whether recombination, as an organism-level evolutionary process, could have contributed to phylogenetic conflict within the dataset, genes with possible signals for recombination were identified. This was done by subjecting the concatenated data matrix to the Recombination Detection Program (RDP) v. 4.84 (Martin et al., 2015), to test for recombination breakpoints using five genetic distance-based methods (RDP, GENECONV, MaxChi, Chimaera and 3Seq). RDP employs a sliding window to calculate pairwise distances between all unique taxon triplets for parsimony informative sites. Regions in contradiction to a UPGMA dendrogram, constructed from all sites, are identified as potentially recombinant (Martin & Rybicki, 2000). The GENECONV method entails pairwise comparisons of all polymorphic sites within the alignment to identify higher than expected similarity over unusually long regions compared to the rest of the alignment (Padidam, Sawyer & Fauquet, 1999). MaxChi identifies potential recombination breakpoints by examining differences in the proportions of variable polymorphic sites using a sliding window to calculate pairwise χ2 values (Smith, 1992). The Chimaera approach is in essence a modification of the MaxChi method, where triplets are screened using a sliding window for only polymorphic sites where recombinants match one of the parental sequences (Posada & Crandall, 2001). Lastly, the 3Seq method uses the same character set as Chimaera to query each sequence within each triplet combination to determine if it could be a possible recombinant of the other two sequences (Boni, Posada & Feldman, 2007). These data were also plotted on the concatenated alignment using Circos v. 0.69 (Krzywinski et al., 2009).
To determine whether limited sets of genes contained sufficient phylogenetic signal to overcome phylogenetic conflict within the dataset, randomised subsets of 20, 50, 60, 70, 80, 90, 100, 110 and 120 genes were constructed. For this purpose, genes were randomly identified in Microsoft Excel™ [=RANDBETWEEN(1,1357)] without resampling. The concatenation and phylogenetic analyses were conducted in the same manner as described above. In all cases, ten individual data subsets were constructed, followed by obtaining a strict and majority rule consensus tree of the ten phylograms of each gene set (i.e., 20, 50, 60, 70, 80, 90, 100, 110 or 120 genes).
Using the AML approach, a robust and well-supported evolutionary hypothesis regarding the species relationships in Pantoea was obtained. The AML phylogeny was based on 337,780 amino acid columns corresponding to the protein sequences of 1,357 genes (Fig. 1). This phylogeny was also congruent with the phylogeny obtained with a larger taxon set for Pantoea, Erwinia, Tatumella and outgroup taxa by Palmer et al. (2017), where ML inferences were performed with the appropriate evolutionary models for each gene partition. The only exceptions were the sister-grouping between P. agglomerans and P. vagans in the current tree, however due to their close relatedness this is not an uncommon problem, and the grouping of P. deleyi and P. anthophila. Similarly, a robust and equally well-supported phylogeny was obtained using the MSC approach where the species tree was inferred from the set of 1,357 individual gene trees (Fig. 1 and Fig. S1). Overall, the MSC topology was also congruent with the phylogeny obtained by Palmer et al. (2017). Exceptions were only observed at nodes at tips or leave nodes (i.e., the groupings observed in the P. agglomerans and P. dispersa lineages).
Figure 1: Comparison between the AML and MSCM phylogenies.
Blue branches indicate differences in topology and branches with support of lower than 1.0 are indicated with a star. (A) The approximate maximum likelihood (AML) phylogeny constructed from the concatenated data matrix of the protein sequences of 1,357 genes, consisting of 337,780 amino acid columns. The phylogeny was constructed with FastTree v. 2.1 (Price, Dehal & Arkin, 2010) with the JTT (Jones, Taylor & Thornton, 1992) evolutionary model with CAT approximation. Simodaira-Hasegawa branch support values from 1,000 replicates were used. (B) A species phylogeny using the multispecies coalescent model as implemented in ASTRAL v.5.6.3 (Mirarab et al., 2014) based on the individual phylogenies constructed from the protein sequences of 1,357 genes. The scale bar indicates one coalescent unit (Mirarab et al., 2014). Terminal branches are indicated as one coalescent unit, as branch lengths for taxa corresponding to species can only be calculated where multiple individuals per species are analysed. Shorter branches correspond to higher levels of incongruence and are generally associated with high levels of incomplete lineage sorting (ILS). Support values are determined based on Bayesian posterior probability values computed from the single gene tree quartet frequencies (Sayyari & Mirarab, 2016).
The AML and MSC topologies were highly congruent (Fig. 1). The only differences were those regarding relationships within the P. dispersa lineage and the P. agglomerans lineage (both topologies also lacked support for the relationships within this lineage). In terms of the MSC topology, comparison of the quartet scores for the main, the first alternative and second alternative topologies possible at each node (Sayyari & Mirarab, 2016), showed that the nodes where quartet scores between the topologies differed very little (where quartet scores for the three alternatives were almost equal) were generally those responsible for incongruence between the topologies inferred using different approaches (Fig. 1 and Fig. S1; particularly within the P. agglomerans and P. dispersa lineages). This suggests that none of these approaches are particularly robust when resolving closely related or undersampled lineages close to the leaves of the phylogenies.
The network approaches indicated a large amount of conflicting signal within the data. This was evident in the Neighbor-Net network (Fig. 2 and File S2) based on the concatenated nucleotide data matrix (1,010,946 bases), as well as the Consensus Network (Fig. S2 and File S3) of the individual gene trees (1,357 protein sequences). These conflicting signals were particularly prevalent at the deeper edges of the evolutionary hypotheses, e.g., the P. dispersa lineage compared to the P. ananatis lineage (denoted A and B in Fig. 1). However, despite the presence of this conflict, the evolutionary hypotheses obtained with the networks, overall, reflected the relationships obtained for the AML and MSC phylogenies (Figs. 1 and 2 and Fig. S2). Furthermore, the topology obtained for the ANI-based distances was mostly congruent to the lineages recovered from the various species tree inference approaches (Fig. S3). All backbone nodes that were consistently recovered in the other approaches, were also recovered with the ANI-based distances with the exception of P. eucrina grouping as sister to the singleton, P. septica.
Figure 2: Neighbor-Net network from the concatenated nucleotide data.
The Neighbor-Net network was constructed from p-distances with equal angles for the concatenated nucleotide dataset. Overall, the configuration of the network is congruent with the existing species phylogeny for the genus Pantoea. Clear separation between the P. agglomerans and P. ananatis lineages were obtained and were also clearly distinct from the P. rodasii and P. dispersa lineages. Point A denotes where signal in conflict to the grouping of the P. ananatis lineage was determined, while point B denotes where signal in conflict to the grouping of the P. dispersa lineage was determined (see text for details).
Figure 3: The summary of individual gene tree comparisons.
(A) The phylogenetic hypotheses evaluated during topology comparisons. Each number represents a specific hypothesis, where the monophyly of the group at each node was evaluated. An example is hypothesis 3, where the overall monophyly of Pantoea was evaluated. (B) A relative frequency histogram depicting the proportion of individual gene trees that support the phylogenetic hypotheses evaluated. Dark blue indicates genes that fully supported the monophyly of the corresponding hypothesis, while light blue indicates support for the monophyly of the hypothesis with additional taxa nested within the group. Red indicates gene trees that were incongruent with the corresponding hypotheses and yellow denotes gene trees with no signal (polytomies). (C) The signal obtained for each gene genealogy compared to the phylogenetic hypotheses were plotted against the position of the genes on the chromosome of Pantoea agglomerans. The same colour scheme is applied as in the frequency histogram. All genes were located on the chromosome of P. agglomerans R190 and was distributed across the chromosome consisting of two contigs. Signal for the respective nodes within the species phylogeny were distributed across the chromosome and no patterns of shared signal were detected for groups of adjacent genes.
To determine the degree of incongruence caused by phylogenetic conflict, comparisons of all individual gene trees were evaluated against a set of twelve query phylogenetic hypotheses (Fig. 3A). These query hypotheses were constructed to evaluate monophyly of lineages or groups across the backbone of the Pantoea species phylogenies, thus shallower nodes near the tips of the trees (leaves) were not considered. None of the 1,357 gene trees were fully congruent with the respective phylogenetic hypotheses of Pantoea. Of the individual gene trees, only six genes supported all the nodes in the backbone for the groupings observed previously (File S4). Additionally, seven gene genealogies produced polytomies of taxa and thus were marked as containing no signal for any of the nodes observed in the phylogenetic hypotheses of Pantoea (Fig. 4 and File S4). The remaining gene trees supported at least one of the nodes in the backbone observed in the Pantoea species trees. Exclusion from the concatenated analyses of either the six backbone-supporting genes or the seven genes providing no resolution among taxa, still provided the same overall topology, with the exception of the grouping of P. agglomerans, P. vagans and P. eucalypti (Figs. S4A and S4B) that lacked statistical support. The phylogenies constructed from the concatenated datasets with only the six backbone-supporting genes and only the seven genes showing no signal (confirmed with RAxML v. 8.0.20; Fig. 3H), also allowed the recovery of a mostly congruent phylogeny to that of the expected topology, but with very low or no support at a number of nodes and slight interspecies differences in the P. agglomerans lineage and the position of singleton taxa (Fig. 3H, Figs. S5A and S5B).
Figure 4: Summary of genes with limited to no signal.
Seven single gene phylogenies determined with approximate maximum likelihood (AML) analyses for genes identified as containing no signal (see File S4) and the maximum likelihood (ML) phylogeny inferred from the combined sequence of these seven genes. (A–G) The AML phylogeny constructed from the protein sequences for the 30S ribosomal protein S18, UDP-diphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferase, Prolyl-tRNA synthetase, Glutamate 5-kinase, Cold shock-like protein cspC, 30S ribosomal protein S10 and 30S ribosomal protein S12, respectively. Taxa are numbered according to taxon descriptors in H. (H) The concatenated ML phylogeny constructed using RAxML v. 8.0.20 with the appropriate amino acid model inferred using ProtTest v. 3.4 for each partition. All bootstrap support values above 70% are indicated at nodes with dots. The phylogeny resembles the known species phylogeny for Pantoea with the exception of some species relationships within the P. agglomerans and P. rodasii lineages and the grouping of singleton taxa.
Based on these topology comparisons, it appeared that the signal supporting different nodes across the Pantoea species phylogenies were supported by different genes. As a means to investigate the distribution of phylogenetic conflict at the gene partition-level across the genome, the signal for each gene was plotted against the genome of P. agglomerans (Fig. 3C). All shared genes were localized to the chromosome of P. agglomerans. This analysis also revealed that signal for all nodes were randomly distributed across the chromosome of P. agglomerans and no apparent patterns of shared signal were detected for adjacent genes (Fig. 3C).
To interrogate the distribution of conflict across the dataset at the nucleotide-level, nucleotide positions in phylogenetic conflict with relationships observed within the Pantoea species phylogeny were identified. Of the 1,010,946 bases within the nucleotide alignment, 493,834 bases (48.7%) were identical across all taxa, with 517,112 nucleotide positions being variable between taxa. For these analyses the P. ananatis lineage, with the least conflicting signal within the dataset (Fig. 2A), and the P. dispersa lineage, with the most conflicting signal within the dataset (Fig. 2B), were investigated as a best and worst case scenario, respectively. For the P. ananatis and P. dispersa lineages, a total of 1,764 and 3,856 nucleotide sites, respectively, supported relationships differing from the Pantoea species phylogenies (Fig. 2 points A and B; File S5). However, these sites were distributed across the concatenated alignment and were not localized to specific genomic regions (Fig. 5).
Figure 5: Conflicting signal and possible recombination breakpoints.
A circular diagram depicting the nucleotide concatenated alignment of all shared genes. The outer track indicates the gene boundaries within the alignment, with tick marks representing the length in nucleotides at 2,000 bp intervals. The second track indicates the nucleotide positions within genes supporting conflicting topologies for the P. dispersa lineage to species groupings observed in the concatenated species phylogeny. A total of 3,856 nucleotide positions supported conflicting topologies for the P. dispersa lineage. The third track indicates nucleotide positions supporting conflicting topologies for the P. ananatis lineage compared to species groupings observed in the concatenated species phylogeny. For this lineage 1,764 nucleotide positions supported conflicting topologies for the P. ananatis lineage. The inner track represents recombination breakpoints detected within gene boundaries for the concatenated alignment. These breakpoints were supported by at least three of the five methods employed (RDP, GENECONV, Chimaera, MaxChi and 3Seq) for detecting recombination. Stacked tiles reflect the number of methods that were successful in detecting recombination events at those regions, as well as multiple recombination events within the same region in various species (See File S6).
Using RDP, a total of 276 potential recombination events were detected (File S6), with 166 of these indicated as potentially caused by evolutionary processes other than recombination (Martin et al., 2015). This yielded 110 likely recombination events, occurring across 54 regions of the concatenated sequence, supported by at least three different analytical methods, of which 57 events were supported by all five methods (File S6). However, to avoid the inclusion of potentially artefactual recombination breakpoints associated with the concatenation process, we only considered recombination breakpoints occurring within the boundaries of single genes. This yielded a total of 15 recombination events, identified across 11 genes within the concatenated alignment (Fig. 5 and File S6). From these results, recombination break-points were detected in members of all lineages within Pantoea. None of these recombination breakpoints could, however, be linked to the nucleotide-level phylogenetic conflict identified (File S5).
The topology of the Pantoea species phylogenies could be recovered by some of the randomised subsets of 20, 50 and 60 genes, whereas all subsets containing the information for 70 or more genes recovered these nodes. Within each set of ten replicate data subsets, the length of individual alignments differed depending on the length of the specific genes used to construct them (Table 2 and File S7). For example, for the 20 gene subsets, the lengths of the alignments ranged from 5,560 to 7,152 amino acid columns, while the length of the alignments for the 120 gene subsets ranged from 36,614 to 42,793 amino acid columns (Table 2).
Summary of gene subset testsa.
bOne backbone node is not recovered in the phylogenies marked with an asterisk.
Overall, support for the backbone of the Pantoea species tree (Fig. 3A) deteriorated with a decrease in the number of genes concatenated and analysed (Table 2). When fewer genes were analysed in the replicates, various branches collapsed and branch support decreased in the strict consensus trees (Table 2, Fig. 6 and Fig. S6). Overall, strict consensus trees from the various replicates of 70 and more genes resulted in the recovery of a phylogeny congruent with the species phylogenies of Pantoea, however multiple individual replicates of the smaller datasets produced trees that were largely incongruent with these hypotheses. Only the trees from multi-gene subsets of 70 or more genes, consistently allowed robust and well-supported reconstruction of the expected Pantoea species trees, specifically with regards to branches in the backbone of the phylogeny (i.e., query hypotheses; Fig. 6).
Figure 6: The SH branch support for specific hypotheses in the trees constructed from the subset datasets.
Each hypothesis (Fig. 3A) was interrogated in each of the subset tree datasets, where 20, 50, 60, 70, 80, 90, 100, 110 and 120 genes were used to construct ten randomised datasets for each number of genes (File S7). The range indicated for each data point stretches from the lowest branch support (0 in the case where the nodes were not recovered) to the highest branch support (1 where the branch was fully supported) with the mean indicated with the data point. Regression analyses were performed in Microsoft ExcelTM to fit the best regression model to the data. (A) Support for the three hypotheses depicted represent the monophyly of the three genera Erwinia (hypothesis 1; green), Tatumella (hypothesis 2; red) and Pantoea (hypothesis 3; blue). All subsets datasets recovered the nodes representing the monophyly of the genera, but in the case of Pantoea, with less support in the replicates of the lower number of genes. (B) The support for the three test hypotheses (i) separating the remainder of Pantoea from Pantoea sp. A4 (hypothesis 4; green), (ii) grouping P. septica, and the P. agglomerans and P. ananatis lineages together (hypothesis 5; red) and (iii) the grouping of P. cypripedii and Pantoea sp. At-9b with the P. rodasii and P. dispersa lineages (hypothesis 9; blue). The node representing hypothesis 4 were not recovered in two repeats of the 20 gene subsets. (C) Hypothesis 6 depicts the sister grouping of the P. agglomerans and P. ananatis lineages with the support associated with the node depicted in green. The monophyletic grouping of the P. agglomerans lineage (hypothesis 7; red) were not recovered in one 50 gene repeat, but were further fully supported in all repeats. The grouping of the P. ananatis lineage was consistently recovered with full support (hypothesis 8; blue). (D) The support associated with the nodes depicting the sister relationship between the P. rodasii and P. dispersa lineages (hypothesis 10; green). This node was not recovered in one of the 20 gene repeats and one of the 50 gene repeats. hypothesis 11 represents the monophyletic grouping of the P. rodasii lineage, which was consistently recovered and well-supported with branch support >0.95 (red). Branch support associated with the monophyletic grouping of the P. dispersa lineage often ranged from very low (0.24) to fully supported (1) in the 20, 50 and 60 gene repeats (hypothesis 12; blue).
This study employed a novel approach to investigate phylogenetic conflict within concatenated datasets. We interrogated the distribution and effect of both phylogenetic signal and conflict at the gene partition and nucleotide levels. This entailed the use of various phylogenetic analyses coupled with manual inspection and evaluation of individual gene trees. These data revealed the effects of phylogenetic conflict and signal in concatenated datasets, which are the input typically used for phylogenomic reconstruction. Our findings support the idea that all genes, even if they appear to be phylogenetically uninformative when analysed alone, contribute signal toward a phylogenomic evolutionary hypothesis and that the obtained topology is not driven by single genes. This is reminiscent of Aristotle’s idea of synergism that “the whole is greater than the sum of its parts”. In other words, by concatenating single genes a synergistic effect is achieved, where the combined data seems to be superior to that of the proverbial sum of the signal.
As demonstrated previously (Palmer et al., 2017), the full set of shared genes allowed reconstruction of a robustly supported phylogenetic hypothesis for Pantoea using AML. In fact, it is quite common to obtain a robust, highly supported phylogeny through concatenation of all shared genes, despite the incongruent nature of individual gene trees (Hedtke, Townsend & Hillis, 2006; Jeffroy et al., 2006; Rokas et al., 2003; Salichos & Rokas, 2013; Thiergart, Landan & Martin, 2014). However, none of our single gene genealogies were fully congruent with the phylogenomic species tree of Pantoea, while only six genes allowed recovery of the backbone of the species phylogeny. This was not surprising as various previous studies showed that very few or no genes typically support a particular species phylogeny fully (Dagan & Martin, 2006; Hedtke, Townsend & Hillis, 2006; Jeffroy et al., 2006; Rokas et al., 2003; Salichos & Rokas, 2013; Thiergart, Landan & Martin, 2014). In contrast to conclusions drawn previously (Thiergart, Landan & Martin, 2014), most of the Pantoea gene trees supported at least some of the nodes within the species phylogeny. In other words, support for the respective nodes was not necessarily obtained from the same genes but rather scattered across different genes.
A fully resolved, well-supported phylogeny was obtained using the MSC approach, although it was not congruent with the AML tree regarding relationships at the tips of the trees. Concatenation is thought to be superior to MSC-based species trees if ILS is low, while MSC models perform better in the presence of moderate ILS (Mirarab et al., 2014). As our MSC and AML trees correspond perfectly regarding backbone nodes, these are strong hypotheses that may approach the real relationships among these taxa. However, because organism-level evolutionary processes were not quantified in this study, we cannot exclude the possibility that ILS were responsible for the incongruences observed. Our study therefore highlights that alternative approaches that model genome evolution, quantify organism-level evolutionary processes (Szöllősi et al., 2012; Williams et al., 2017), and that focusses on leave taxa are needed to fully resolve the species tree of a diverse assemblage such as Pantoea. This may be particularly true when taxa are very closely related or when the lineages in question are undersampled.
The random distribution of signal across the Pantoea genome is supported by the recovery of overall congruent subset phylogenies from random sub-samplings of gene sequences. Due to this random distribution, one should be able to obtain sufficient signal to reconstruct the species phylogeny by randomly sampling enough genes from the genome (Dutilh et al., 2004; Gadagkar, Rosenberg & Kumar, 2005). From our data, this idea was tested with consensus trees of 10 replicates with 20, 50, 60, 70, 80, 90, 100, 110 and 120 genes. We found that, with a decrease in the number of genes analysed, support for the backbone and the deeper branches decreased incrementally, as has also been observed previously (Rokas et al., 2003). Therefore, for these data and taxon set, it appear that at least 70 randomly selected genes from the genome is required to obtain a relatively robust, well-supported phylogeny, particularly to reconstruct the deeper relationships within and among the genera. Multi-gene phylogenies based on 70 genes may thus provide sufficiently robust hypotheses so that complete genome sequence data may not be required, our work suggests that sufficient data may be obtained from low level sequencing, although verification of this notion in other taxon sets is required.
The species tree hypotheses for Pantoea was generally also supported by the two network approaches employed here. Both accommodated non-vertical and non-phylogenetic signal (introduced through systematic error) as inferred from nucleotide data, as well as the individual gene trees (Bryant & Moulton, 2002; Holland & Moulton, 2003; Holland, Jermiin & Moulton, 2005). These methods produced networks in which the overall clustering patterns were generally congruent with that obtained through gene concatenation-based phylogenomic inferences. This would not have happened if insufficient signal (i.e., stochastic error Jeffroy et al., 2006; Philippe et al., 2011; Rosenberg & Kumar, 2003) or reconstruction artefacts (i.e., systematic error Hedtke, Townsend & Hillis, 2006; Hillis, 1998; Pollock et al., 2002; Zwickl & Hillis, 2002) were responsible for the observed relationships in the species trees. If conflict, particularly in the form of HGT and ILS, dominated the dataset, the splits graphs would not have such high overall congruence to the species trees (Bryant & Moulton, 2002), however, more in-depth future studies are required to fully elucidate the role and amount of organism-level evolutionary processes in the evolution of these taxa. Compared to previous analyses, often employing a limited gene set (Chen et al., 2013; Kennedy et al., 2005), more box-like structures were observed in Pantoea networks, particularly in deeper edges. However, these boxes were generally smaller, where the increased number of boxes indicate more alternate or conflicting relationships, while the shorter edges correlate to the particular relationships being observed less frequently. If one considers that these conflicts are visualized for the full shared gene set, the level of conflict appears to be relatively low and comparable to that seen in other bacteria (Retchless & Lawrence, 2010), but considerably lower than taxa undergoing extensive HGT (Doroghazi & Buckley, 2010). The generally low level of conflict thus supports the idea that sufficient phylogenetic signal is present within the concatenated dataset to overshadow the limited conflict present.
Overall, the relationships obtained using the ANI-based distance approach were mostly congruent to the species trees. The incongruences that were present can be ascribed to the fact that ANI is notoriously unreliable as an indicator of relatedness, especially among more distantly related taxa (Konstantinidis & Tiedje, 2007; Palmer et al., 2017; Qin et al., 2014; Rosselló-Mora, 2005). This phenomenon is the reason why many prokaryotic taxonomist would rather purport the use of Average Amino Acid Identity (AAI) values at this level (Konstantinidis & Tiedje, 2007; Qin et al., 2014; Rosselló-Mora, 2005), as substitution saturation and other factors resulting from endogenous evolutionary processes may be responsible for the decline in informativeness of this metric, the more distantly related the taxa become (Palmer et al., 2017).
We investigated the phylogenetic conflict within the Pantoea dataset for the two lineages in which we observed the least and most phylogenetic conflict. Respectively, these were the P. ananatis lineage, which includes plant pathogenic species, and the P. dispersa lineage, which includes generalists (Palmer et al., 2018; Palmer et al., 2017; Walterson & Stavrinides, 2015). The number of nucleotide sites supporting alternate topologies to the species trees were limited, with only 0.75% of variable nucleotide sites (3,856 sites out of 517,112) supporting conflicting topologies in the P. dispersa lineage. Also, the remaining variable sites did not necessarily support the species relationships observed in the species trees, because different genes and nucleotide positions supported different nodes within the species phylogenies. Moreover, the conflicting signal within the dataset was not localised to specific genomic regions or genes, but rather, was randomly distributed. Taken together, these results thus suggest that (i) the use of network approaches for constructing phylogenies can be extremely valuable for identifying phylogenetic conflicts in datasets (Bryant & Moulton, 2002; Holland & Moulton, 2003; Holland, Jermiin & Moulton, 2005), and (ii) organism-level evolutionary processes like HGT and/or ILS impacts different lineages and taxa to varying degrees.
Conflicting phylogenetic signal in the Pantoea dataset could potentially result from recombination events that led to gene conversions between species (Daubin, Moran & Ochman, 2003; Fraser, Hanage & Spratt, 2007; Holmes, Urwin & Maiden, 1999; Posada & Crandall, 2001; Posada & Crandall, 2002). We found evidence for at least 15 recombination events in 11 shared genes in the dataset. We attributed these to recent instances of recombination, because older organism-level evolutionary events, particularly ancient HGT and ILS (Knowles, 2009; Meng & Kubatko, 2009; Retchless & Lawrence, 2010), become difficult to detect due to deterioration of signals by endogenous evolutionary processes (Daubin, Moran & Ochman, 2003). It is also difficult to distinguish between these organism-level evolutionary processes as their signals may appear very similar (Knowles, 2009; Nosil, 2008; Wendel & Doyle, 1998) and future studies would be required to tease apart the roles of these processes in Pantoea. Nevertheless, identification of some of these organism-level evolutionary events in Pantoea provides possible mechanisms for how phylogenetic conflict could have been introduced into the data.
The use of all shared genomic information allowed for the recovery of robustly supported relationships, overcoming the weaknesses observed in individual gene datasets (Andam & Gogarten, 2011; Daubin, Gouy & Perriere, 2002; Gadagkar, Rosenberg & Kumar, 2005; Galtier & Daubin, 2008). In contrast to a previous similar study by Thiergart, Landan & Martin (2014), comparison of the single gene trees were specifically performed with backbone nodes (excluding taxa closer to the tips of the trees) with the aid of the query hypotheses, which allowed us to interrogate each node and its associated signal manually. Although Thiergart, Landan & Martin (2014) also compared nodes between concatenated trees and the single gene phylogenies, their comparisons were focussed only on the recovery of identical nodes, which likely overestimated the effect of finer differences between the trees, leading them to their conclusion that the signal associated with the backbone or deeper nodes of their concatenated phylogenies are not preserved in single gene trees. Based on our data, three of the query hypotheses evaluated (see Fig. 3B and Fig. S1) had a large proportion of individual gene trees that did not support the expected monophyly of the taxon groups specified. These were hypothesis 5 in which P. septica is a singleton taxon placed as sister to the P. agglomerans and P. ananatis lineages (48.9% trees), hypothesis 9 in which P. cypripedii and Pantoea sp. At-9b are placed as sister to the P. rodasii and P. dispersa lineages (40.4% trees) and hypothesis 10 in which the P. rodasii and P. dispersa lineages are placed as sister groups (35.2% trees). In these instances, limited species have been sampled for the respective lineages. This undersampling of the diversity may contribute to the lack in robust recovery of the lineages due to large systematic error in the smaller datasets. In future, increased taxon sampling may resolve these problematic relationships more accurately in smaller datasets like those employed for the single gene trees (Hedtke, Townsend & Hillis, 2006; Pollock et al., 2002).
Comparison of single gene trees with phylogenies obtained from concatenated datasets, presents both a philosophical and logical quandary. It is widely accepted that single gene phylogenies, often with very limited or no statistical support, cannot be equated to a species phylogeny (Degnan & Rosenberg, 2006; Doyle, 1992; Maddison, 1997; Pamilo & Nei, 1988; Rosenberg, 2002). Despite the common practice of evaluating the robustness of a species phylogeny constructed from thousands or millions of characters, by its topological congruence to single gene trees (Bapteste et al., 2009; Ciccarelli et al., 2006; Dagan & Martin, 2006), these phylogenies are clearly not directly comparable and no conclusions regarding species evolution should be drawn from raw tree topology comparisons. This rationale is like comparing single molecules with chemical compounds and being disappointed that they do not share the same characteristics. Based on our data, the signal required for reconstructing a species phylogeny is dispersed and the only appropriate comparison of single gene trees to species trees would be when focus is placed on the evolution of a particular gene or when species trees are inferred from single gene trees, as with the MSCM analyses.
Our findings confirm the robustness of phylogenies constructed from genomic data, based on the synergistic effect of combined genes, despite high levels of incongruence between individual gene trees. This is due to the phylogenetic signal for different nodes within the species phylogeny being distributed across the genome at higher levels than the randomly distributed conflicts within the dataset. These findings support previous conclusions suggested by several authors (Andam & Gogarten, 2011; Daubin, Gouy & Perriere, 2002; Galtier & Daubin, 2008; Retchless & Lawrence, 2010; Rokas et al., 2003), based on comparisons of single gene phylogenies with super trees and concatenated analyses using tree-to-tree distance approaches (Daubin, Gouy & Perriere, 2002; Retchless & Lawrence, 2010). Our results also suggest that the robustness of evolutionary hypotheses from whole genome data should be evaluated with phylogenetic network approaches that can depict conflicts, due to evolutionary processes or phylogenetic error, within the dataset (Bryant & Moulton, 2002; Holland & Moulton, 2003; Huson & Bryant, 2005). By employing such a total-evidence based approach, one would be able to recover a more realistic evolutionary hypothesis, particularly in terms of the deeper relationships, that also serves as an initial indication of the impact of organism-level evolutionary processes. Ultimately, such detailed evolutionary analyses would be invaluable for understanding the speciation process and for studying the development and distribution of important biological characteristics. Furthermore, our data also suggests that alternative approaches, focussing specifically on organism-level evolutionary processes, possibly at the population level, may be required to resolve relationships and elucidate the evolutionary history of younger taxa or leaves, where these processes may be rampant and phylogenetic incongruence highly prevalent.
We found that phylogenetic conflict, potentially caused by organism-level evolutionary processes, was present in our phylogenomic dataset at both the gene partition and nucleotide levels. Although this non-phylogenetic signal could result from organism-level evolutionary process, like HGT and ILS, more in-depth analyses are needed to differentiate between these processes and to quantify the overall impact of these processes on the evolutionary history of the taxa. From our results it appeared that both signal and noise are randomly distributed across the genome and that all genes included in a concatenation-based phylogenomic analysis contribute signal toward the possible species tree. In other words, for Pantoea at least, phylogenies constructed from concatenated datasets are not driven by single genes, but rather that the signal from individual genes work synergistically to provide robust phylogenies.
Raw Data: A nexus file for the NeighborNet Network constructed from the concatenated nucleotide data matrix (Fig. 2).
Raw data: A nexus file for the consensus network constructed from the individual gene trees (Fig. S2).
Datasets and trees for the individual gene trees marked as supporting the backbone and those with no signal.
Nucleotide positions with conflicting signal as determined from the NeighborNet.
Results obtained from the recombination detection program regarding potential recombination breakpoints.
Data pertaining to the randomised subset datasets constructed from 20, 50, 60, 70, 80, 90, 100, 110 and 120 randomly selected genes.
We would like to sincerely thank the reviewers, Dr. David Waite, Dr. Luis M. Rodriguez-R and an anonymous reviewer for valuable suggestions and significant contributions toward improving the manuscript.
Marike Palmer conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Stephanus N. Venter, Emma T. Steenkamp and Martin P.A. Coetzee conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.
Alistair R. McTaggart performed the experiments, analyzed the data, authored or reviewed drafts of the paper, approved the final draft.
Stephanie Van Wyk, Juanita R. Avontuur, Chrizelle W. Beukes, Gerda Fourie, Quentin C. Santana and Magriet A. Van Der Nest performed the experiments, analyzed the data, approved the final draft.
Jochen Blom performed the experiments, contributed reagents/materials/analysis tools, approved the final draft.
A simple Python script was written to construct the individual phylogenies with FastTree v. 2.1 for the 1,357 protein sequences shared by all taxa analysed.
Two nexus files for the SplitsTree analyses are available as Supplemental Files.
This work was supported by the National Research Foundation (South Africa), the DST-NRF Centre of Excellence in Tree Health Biotechnology (CTHB) and the University of Pretoria with regards to student funding. Informatics infrastructure was funded by the NRF National Bioinformatics Functional Genomics Grant (No: 93668). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Huson DH, Bryant D. 2005. Estimating phylogenetic trees and networks using SplitsTree 4.

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APPEAL from an order of the circuit court for Milwaukee County: JOHN A. FRANKE, Judge. Affirmed.
Before Wedemeyer, P.J., Fine and Curley, JJ.
¶ 1 John E. Isom appeals pro se from an order denying his petition for writ of habeas corpus attacking the sufficiency of the criminal complaint and his bindover on the charge of manufacturing and delivery of a controlled substance contrary to WIS. STAT. § 961.41(1)(cm) (2003-04). Because Isom has an adequate remedy at law in another pending appeal, the trial court was correct in denying the petition. We affirm.
All references to the Wisconsin Statutes are to the 2003-04 version unless otherwise noted.
¶ 2 On March 29, 2002, a jury convicted Isom of possession of a firearm by a felon and manufacturing and delivery of a controlled substance. Isom was sentenced to nineteen years of imprisonment, including seven years of initial confinement and twelve years of extended supervision. Isom did not appeal.
Isom was also charged with carrying a concealed weapon. The jury returned a hung verdict on this count and, on motion of the State, it was dismissed by the trial court.
The total term of imprisonment consisted of two consecutive sentences. Isom was sentenced to four years of confinement and six years of extended supervision for the firearm conviction. He was sentenced to three years of confinement and six years of extended supervision for the controlled substance conviction.
¶ 3 On March 21, 2003, Isom moved pro se for postconviction relief pursuant to WIS. STAT. § 974.06. The trial court denied this motion on March 24, 2003, without a hearing, stating that the claims set forth in the motion contained only conclusory allegations. The trial court's ruling was summarily affirmed by this court and the Wisconsin Supreme Court denied Isom's petition for review.
¶ 4 Isom subsequently filed a petition for writ of habeas corpus with this court, which was denied on January 14, 2005. The Wisconsin Supreme Court denied his petition for review on March 14, 2005.
¶ 5 On May 4, 2005, Isom filed another motion for postconviction relief under WIS. STAT. § 974.06, which the trial court denied on May 10, 2005. Isom appealed the trial court's decision, and that appeal is currently pending. In the pending appeal, Isom alleges ineffective assistance of postconviction counsel on several grounds.
This court affirmed the trial court on July 18, 2006. See State v. Isom , No. 05-1449, unpublished slip op. (WI App July 18, 2006). Isom filed a petition for review in the Wisconsin Supreme Court, which is still pending.
¶ 6 On August 19, 2005, while the appeal on his second WIS. STAT. § 974.06 motion was still pending, Isom filed another petition for writ of habeas corpus with the trial court. The petition challenges the sufficiency and validity of the original and "advanced" criminal complaints. Isom further challenges the use of only the information in the amended complaint as the basis to bind him over for trial on the charge for manufacturing and delivery of a controlled substance. The State filed a response opposing the petition on August 23, 2005.
¶ 7 On August 26, 2005, the trial court denied the petition, noting the pending appeal of the second WIS. STAT. § 974.06 motion, and concluding that Isom made no showing of why the issues set forth in the petition could not, or should not, have been raised in the motion. Isom subsequently filed a response to the State's reply. The trial court construed this response as a motion for reconsideration which it denied.
¶ 8 This appeal followed.
¶ 9 Isom claims that the trial court erroneously exercised its discretion in not granting his petition for writ of habeas corpus. We disagree.
¶ 10 Review of "[a] circuit court's order denying a petition for writ of habeas corpus presents a mixed question of fact and law." State v. Pozo , 2002 WI App 279, ¶ 6, 258 Wis. 2d 796, 654 N.W.2d 12. Factual determinations will be reversed only if they are clearly erroneous. Id. Whether a writ of habeas corpus is available to the petitioner is a question of law which is reviewed de novo. Id.
¶ 11 The writ of habeas corpus is an equitable remedy whose purpose is to provide relief when the authority restraining a person is constitutionally or judicially defective. State ex rel. Wohlfahrt v. Bodette , 95 Wis. 2d 130, 132, 289 N.W.2d 366 (Ct.App. 1980). The use of writ of habeas corpus may be employed to challenge the validity of a complaint or bindover. Id. A writ of habeas corpus is an extraordinary writ, available only where the petitioner demonstrates: (1) restraint of his or her liberty; (2) such restraint was imposed by a body without jurisdiction or that the restraint was contrary to constitutional protections; and (3) "that there is no other adequate remedy available" at law. State ex rel. Haas v. McReynolds , 2002 WI 43, ¶ 12, 252 Wis. 2d 133, 643 N.W.2d 771. "[T]he extraordinary writ of habeas corpus is not available to a petitioner when the petitioner has other adequate remedies available." Id. , ¶ 14.
¶ 12 Here, Isom's petition fails because the pending appeal of his second WIS. STAT. § 974.06 motion provides an adequate remedy at law. Because Isom did not challenge the sufficiency of the complaint or bindover at trial, he is limited to raising these challenges in the context of an ineffective assistance of counsel claim. Further, because Isom failed to challenge the effectiveness of trial counsel by filing a motion pursuant to § 974.06, he is limited to asserting that he received ineffective assistance from postconviction counsel. Isom's motion, currently on appeal, does just that.
¶ 13 Isom's brief in his pending appeal to the second WIS. STAT. § 974.06 motion asserts that postconviction counsel was ineffective for failing to file a motion pursuant to WIS. STAT. § 974.02 to preserve issues and claims of constitutional and jurisdictional violations. Even if this assertion could not be construed to address the same issues as his petition, Isom is not permitted to raise claims in the petition that were, or should have been, raised in postconviction proceedings. See Pozo , 258 Wis. 2d 796, ¶ 10. Thus, because the challenges to the complaint and bindover should have been included in the § 974.06 motion, and Isom has not demonstrated sufficient reason why they were not, he may not raise these claims by a petition for writ of habeas corpus. By the Court. — Order affirmed.


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Dylan, Bob. Bob Dylan. More Classic. New York. Music Sales. 2001. 4to. Soft cover. First edition. Fine copy.
Earl, Andy. Andy Earl. Vista. Photo available. London. Sanctuary. 2000. 4to. Soft cover. First edition. Illustrated in black, white and color. ir Important reference work. Fine copy.
Earle, Dan. The Art of Dan Earle. Inscribed by the author. Creative Exploration. Photo available. np. Self published. c. 2008. 24 pps. 4to. Decorated hard cover. First edition. Illustrated in black, white and color. Inscribed by the author "To John & Judy - Your love story is a work of art. I am honored to have "Flitht" in your collection. Love, Dan 3-14-08". Fine copy.
Earle, Susan (ed). An Errant Line: Ann Hamilton & Cynthia Schira. np. Spencer Museum of Art. 2013. Octavo. Soft cover. First edition. Illustrated in black, white and color. Fine copy.
Easley, Thomas. The Figure in Motion. A Visual Reference for the Artist. Photo available. New York. Watson-Guptill. 1986. 175 pps. 4to. Hardcover. Stated first edition. Illustrated with photographs by Mark Smith. Important reference work. Fine copy in fine dust jacket (in mylar).
Easson, Roger R. William Blake. Book Illustrator. A Bibliography and Catalogue of The Commercial Engravings. Volume I. Normal. Illinois State University. 1972. 4to. Soft cover. First edition. Illustrated. Fine copy.
Eastman, John. Who Lived Where. A Biographical Guide to Homes and Museums. Photo available. New York. Bonanza. 1987. 515 pps. 4to. Hardcover. Reprint. Illustrated. Important reference work. Fine (initials on edges, owner name on end paper) copy in fine dust jacket (in mylar).
Eaton, Jim. Dan Ostermiller. Inscribed by Ostermiller. A Sculptor of Animals. Photo available. Loveland. Actian Press. 1990. 4to. Hard cover. First edition. Illustrated in black, white and color. Inscribed by the artist. Fine copy in fine dust jacket (in mylar).
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(1) "Derivative proceeding" means a civil suit in the right of a domestic corporation or, to the extent provided by § 21.562, in the right of a foreign corporation.
(2) "Shareholder" includes a beneficial owner whose shares are held in a voting trust or by a nominee on the beneficial owner's behalf.
§ 21.552. STANDING TO BRING PROCEEDING.
(2) the shareholder fairly and adequately represents the interests of the corporation in enforcing the right of the corporation.
(a) A shareholder may not institute a derivative proceeding until the 91st day after the date a written demand is filed with the corporation stating with particularity the act, omission, or other matter that is the subject of the claim or challenge and requesting that the corporation take suitable action.
(3) irreparable injury to the corporation would result by waiting for the expiration of the 90-day period.
§ 21.554. DETERMINATION BY DIRECTORS OR INDEPENDENT PERSONS.
(3) a panel of one or more independent and disinterested persons appointed by the court on a motion by the corporation listing the names of the persons to be appointed and stating that, to the best of the corporation's knowledge, the persons to be appointed are disinterested and qualified to make the determinations contemplated by § 21.558.
(b) The court shall appoint a panel under Subsection (a)(3) if the court finds that the persons recommended by the corporation are independent and disinterested and are otherwise qualified with respect to expertise, experience, independent judgment, and other factors considered appropriate by the court under the circumstances to make the determinations. A person appointed by the court to a panel under this § may not be held liable to the corporation or the corporation's shareholders for an action taken or omission made by the person in that capacity, except for an act or omission constituting fraud or wilful misconduct.
§ 21.555. STAY OF PROCEEDING.
(a) If the domestic or foreign corporation that is the subject of a derivative proceeding commences an inquiry into the allegations made in a demand or petition and the person or group of persons described by § 21.554 is conducting an active review of the allegations in good faith, the court shall stay a derivative proceeding until the review is completed and a determination is made by the person or group regarding what further action, if any, should be taken.
(b) To obtain a stay, the domestic or foreign corporation shall provide the court with a written statement agreeing to advise the court and the shareholder making the demand of the determination promptly on the completion of the review of the matter. A stay, on application, may be reviewed every 60 days for the continued necessity of the stay.
(c) If the review and determination made by the person or group is not completed before the 61st day after the stay is ordered by the court, the stay may be renewed for one or more additional 60-day periods if the domestic or foreign corporation provides the court and the shareholder with a written statement of the status of the review and the reasons why a continued extension of the stay is necessary.
(3) the reasonableness of the procedures followed by the person or group in conducting the review.
(b) Discovery described by Subsection (a) may not be expanded to include a fact or substantive matter regarding the act, omission, or other matter that is the subject matter of the derivative proceeding. The scope of discovery may be expanded if the court determines after notice and hearing that a good faith review of the allegations for purposes of § 21.558 has not been made by an independent and disinterested person or group in accordance with that section.
§ 21.557. TOLLING OF STATUTE OF LIMITATIONS.
(2) the 31st day after the date the corporation advises the shareholder that the demand has been rejected or the review has been completed.
§ 21.558. DISMISSAL OF DERIVATIVE PROCEEDING.
(a) A court shall dismiss a derivative proceeding on a motion by the corporation if the person or group of persons described by § 21.554 determines in good faith, after conducting a reasonable inquiry and based on factors the person or group considers appropriate under the circumstances, that continuation of the derivative proceeding is not in the best interests of the corporation.
(2) the corporation in any other circumstance.
§ 21.559. PROCEEDING INSTITUTED AFTER DEMAND REJECTED.
If a derivative proceeding is instituted after a demand is rejected, the petition must allege with particularity facts that establish that the rejection was not made in accordance with the requirements of §§ 21.554 and 21.558.
§ 21.560. DISCONTINUANCE OR SETTLEMENT.
(a) A derivative proceeding may not be discontinued or settled without court approval.
(b) The court shall direct that notice be given to the affected shareholders if the court determines that a proposed discontinuance or settlement may substantially affect the interests of other shareholders.
§ 21.561. PAYMENT OF EXPENSES.
(3) expenses for which the domestic or foreign corporation or a corporate defendant may be required to indemnify another person.
(C) was interposed for an improper purpose, such as to harass, cause unnecessary delay, or cause a needless increase in the cost of litigation.
§ 21.562. APPLICATION TO FOREIGN CORPORATIONS.
(a) In a derivative proceeding brought in the right of a foreign corporation, the matters covered by this subchapter are governed by the laws of the jurisdiction of incorporation of the foreign corporation, except for §§ 21.555, 21.560, and 21.561, which are procedural provisions and do not relate to the internal affairs of the foreign corporation.
(b) In the case of matters relating to a foreign corporation under § 21.554, a reference to a person or group of persons described by that § refers to a person or group entitled under the laws of the jurisdiction of incorporation of the foreign corporation to review and dispose of a derivative proceeding. The standard of review of a decision made by the person or group to dismiss the derivative proceeding shall be governed by the laws of the jurisdiction of incorporation of the foreign corporation.
§ 21.563. CLOSELY HELD CORPORATION.
(2) no shares listed on a national securities exchange or regularly quoted in an over-the-counter market by one or more members of a national securities association.
(b) §§ 21.552-21.559 do not apply to a closely held corporation.
(2) a recovery in a direct or derivative proceeding by a shareholder may be paid directly to the plaintiff or to the corporation if necessary to protect the interests of creditors or other shareholders of the corporation.


82a-953a. Transfers from state general fund to state water plan fund; reductions. During each fiscal year, the director of accounts and reports shall transfer $6,000,000 from the state general fund to the state water plan fund created by K.S.A. 82a-951, and amendments thereto, one-half of such amount to be transferred on July 15 and one-half to be transferred on January 15, except that during the fiscal year ending June 30, 2018, the transfer shall not exceed $1,200,000. No moneys shall be transferred from the state general fund to the state water plan fund during the fiscal years ending June 30, 2019, and June 30, 2020.
History: L. 1989, ch. 186, § 33; L. 1990, ch. 28, § 19; L. 1990, ch. 350, § 12; L. 1993, ch. 292, § 76; L. 1994, ch. 297, § 11; L. 2000, ch. 130, § 107; L. 2002, ch. 204, § 165; L. 2003, ch. 138, § 131; L. 2004, ch. 165, § 3; L. 2005, ch. 206, § 88; L. 2006, ch. 142, § 164; L. 2007, ch. 167, § 201; L. 2009, ch. 2, § 93; L. 2009, ch. 124, § 146; L. 2010, ch. 165, § 158; L. 2011, ch. 118, § 190; L. 2012, ch. 175, § 168; L. 2013, ch. 136, § 281; L. 2015, ch. 104, § 250; L. 2017, ch. 104, § 243; July 1.


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There are various interventions on public games. Among the main we can mention: the disposal of gaming machines; the introduction of the register of distributors and game operators; the extension of the deadline after which clearance for AWP can no longer be issued; news on the taxation of bets on horse races; the mode of storage of the receipts played, and last, but not least, the news on bets and bingo tenders and the prolonged extension of the concessions. But let's see in detail the individual rules.
Paragraph 620 regulates calls for bets and bets and introduces the extended extension of the concessions. It is established, in fact, that in order to reconcile the principles according to which public concessions are attributed according to competitive selection procedures with the need to pursue, in the matter of concessions to collect bets on sporting events, even horse racing, and not sporting , including the simulated events, a proper distribution structure, also following the agreement ratified in the unified Conference, "the Customs and Monopolies Agency assigns by tender to be completed by September 30, 2018 the related concessions to the conditions already provided for in art. 1, paragraph 932, of the law of 28 December 2015 no. 208, with an income of at least 410 million euros ". To this end, the existing concessions, as well as the ownership of regular collection points pursuant to art. 1, paragraph 643, of the law December 23, 2014, n. 190, as well as the art. 1, paragraph 926, of the law of 28 December 2015, n. 208, are extended to December 31, 2018, against the payment of the annual sum of € 6,000 per law relating to the points of sale whose main activity is the marketing of public gaming products, including regularized collection points, and of € 3,500 for each right relating to points of sale having as an accessory activity the marketing of public gaming products. The methods of payment of the annual sum are not yet known.
Paragraph 621 of Article 1 provides that the regional laws must comply with the note agreed in the Unified Conference of 7 September u.s. The aforementioned paragraph states that: "In order to allow the selection procedures pursuant to paragraphs 619 and 620 to be carried out, the regions shall adapt their laws regarding the dislocation of public gaming points of sale to the agreement established in of the Unified Conference on 7 September 2017 ".
Paragraph 621-bis, always referring to the AWP, reads as follows: "In order to avoid possible illegal use of gaming equipment referred to in Article 110, paragraph 6, letter a), of the consolidated law of public security, in the Royal Decree of 18 June 1931, n. 773, which are dismissed from the market, with a provision of the Customs Agency and the monopolies, within 90 days from the date of entry into force of this law, a specific mandatory procedure for the disposal and destruction of the equipment is established ". The aforesaid paragraph therefore provides that a specific mandatory procedure for the disposal and destruction of gaming equipment (newslot) discontinued by the market is established by ADM provision, in order to avoid possible illicit use. The provision in question, in order to avoid possible illegal uses of gaming machines (new slot - AWP) requires a provision of ADM, the identification of a specific mandatory procedure for the disposal and destruction of the equipment.
Other changes come from the following paragraph 621-ter which introduces changes to the taxation of bets on horse races. In fact, within 90 days of the conversion of the Budget Law, the Mipaaf will start the procedure to select the Equestrian League. In addition, the taxation on the margin of bets on horses will be introduced with rates of 43% for those placed in the agency and 47% for those online. The amendment approved by the Commission provides that "the revenue earned remains destined for all fixed odds bets on horse races included in the Official Race Schedule (...) as well as for all fixed odds bets on horse races included in the program schedules. complementary measures, 33% for the single tax and 67% for the financing of prize money, equipment and images of races as well as provisions for the breeding of horses ". In the event that this revenue results in the twelve months of the previous financial year, higher than that envisaged under current legislation, the Minister of Economy and Finance may propose a regulatory intervention aimed at reducing the tax rates for the physical network and for gambling, in compliance with the public finance objectives (621-quater). However, the Minister will also have the power to tighten the rates if the revenue is below expectations. The Mef will still have - heard the Mipaaf - "homogeneous formulas of betting in totalizator on horse races, also establishing the unitary mail of the game, the minimum play, the introduction of any new betting formulas, and providing, where necessary , additional categories of winnings and the provision to be allocated to Jackpot ". In accordance with paragraph 621-quinquies, the ADM shall, within 90 days of the definitive approval of the Budget Law, have to regulate the ways in which the authorized networks offer their own complementary horse racing betting programs. Starting from January 1, 2018 the companies that manage the racetracks and bookmakers are authorized to collect all bets on horse races after signing a specific regulation with the Customs and Monopolies Agency (621-sexies). It is envisaged the obligation to conform the totalizator bets on the horse races to the model of distribution of the game mail adopted for totalizator bets. It is therefore postponed (paragraph 621-septies) to a decree of the Minister of Economy and Finance, to identify, in the absence of charges for public finance, the measures necessary to make homogeneous formulas of betting in totalizator on horse races.
Finally, paragraph 621-decies provides for the launch, by MIPAAF, of the reform of the sector through a public selection procedure to identify the body that will have the task of organizing horse racing events and the further extension of 6 months of the terms referred to in art. 15, paragraph 3, lett. b), as per law 28 July 2016, no .54.
Paragraph 622-bis establishes the register of distributors and exhibitors, inserting the art. 52 bis to the legislative decree 21 November 2007, n. 231, regarding anti-money laundering, which states that: "Art. 52-bis. - (Register of distributors and exhibitors). - 1. A computerized and confidential register of distributors and gaming operators is established at the Customs and Monopolies Agency ". The new regulation provides for the establishment in the ADM of a register of distributors and gaming operators in which the data for their identification as well as the type and mode of the gaming activity and also the measures for extinguishing the contractual relationship are noted. (for example in the case of serious infringements) and suspension issued by the Ministry of Economy and Finance. Access to the register is allowed to the MEF, to the Financial Police, to the DIA and to the FIU for the exercise of their respective competences in matters of supervision and prevention of money laundering and terrorist financing and to the National Anti-Mafia and Counter-Terrorism Directorate. Access is also granted to the Police Headquarters for the exercise of public security functions and for the purpose of issuing licenses and authorizing titles. It is expected that with a subsequent implementation decree will be established the technical methods of feeding and consultation of the register.
Paragraph 622-quater introduces an extension to the deadline after which no clearance can be issued for cash-winning devices, better known as new slots (Article 110, paragraph 6, letter a) of TULPS). The subsection modifies, therefore, the article 1, comma 943, of the law 28 December 2015, n. 208 replacing the words «after 31 December 2017» with the words «after 31 December 2018». Paragraph 943 provided, in fact, that: "The clearance for the appliances referred to in the aforementioned Article 110, paragraph 6, letter a), can no longer be issued after December 31, 2017; these appliances must be decommissioned by December 31, 2019. Starting from January 1, 2017, only permits can be issued for equipment that allow public clearance from a remote environment, providing for the proportional reduction, at a rate of no less than 30 per cent, of the number of the operating clearances relating to appliances operating as of July 31, 2015, referable to each concessionaire. The modalities of this reduction, also taking into account the territorial diffusion of the appliances, the cost of the new clearance and the methods, also in installments, of the relative payment are defined with the aforementioned ministerial decree ".
Paragraph 586, which provides for the establishment of the so-called Webtax, or the tax on digital transactions, has been partially reviewed in the Chamber. The paragraph specifies the taxable digital transactions such as those: "relating to the provision of services carried out by electronic means rendered to subjects resident in the territory of the State indicated in art. 23, paragraph 1 of the Decree of the President of the Republic, September 29, 1973, n. 600 ... and the permanent organizations of non-residents located in the same territory ". The rate has risen from 6% to 3%, and taxation is not extended to e-commerce. In order not to penalize small businesses in simplified accounting and start-ups, the obligation to pay taxes is triggered when at least 3,000 digital transactions have been made in a calendar year ".
In short, more operational interventions that follow the wave of prohibition with an attitude that reminds the gruff-maker of nineteenth-century novels, dull and obtuse.
These rooms, now loosened by the President, leave us a memory of bar Star Wars. The continuous reassurances on growth are not reflected in everyday reality. The only convergence that they found was on compressing (at very high cost) the public gaming sector, authorizing new and huge healthcare costs.
But as Vico teaches with its courses and historical appeals, it remains a hope that at the beginning of each new year it is legitimate and necessary to cultivate. Not so much for this sector, which has an emblematic value, but for the whole country.

matches:
art. 1
art. 1
art. 1
art. 15
art. 52
art. 23


Full Case Name: James and Maureen FRANCISCUS, as Parents and Natural Guardians of Femina Franciscus, Appellants v. Tolga SEVDIK, an Individual, Ashley Dailey, an Individual and John Steigerwald, an Individual t/d/b/a Fetch Pet Care of West Hills/South Hills, Appellee.
Summary: Five-year-old Femina asked the dog walker, Ms. Dailey, if she could pet Julius, the pit bull. When she bent over to do so, the dog jumped up and bit her on the chin. The Plaintiffs, Mr. and Mrs. Franciscus commenced this negligence action to recover damages for injuries sustained by their daughter, Femina. They filed the action against Mr. Sevdik, the owner of the dog, Ms. Dailey, the dog walker, and Mr. Steigerwald, the individual owner and operator of Fetch Pet Care of West Hills/South Hills. The Superior Court of Pennsylvania held that summary judgment granted by the trial court in favor of Ms. Dailey and Fetch Pet Care was improper. The Court reasoned that the dog was entrusted to these Defendants by Mr. Sevdik and the dog was in their control when the injury occurred. Since the Defendants knew the dog jumped on people, was to be muzzled when walked, and was not to be walked along routes where there were people, specifically children and other dogs, they had a duty to use reasonable care to protect others from harm while the dog was in their control. While the court stated it did not need to reach the issue of whether the trial court erred in refusing refusing to take judicial notice of dangerous propensities of pit bulls, it noted that Pennsylvania law does not recognize a presumption that pit bulls as a breed are dangerous or have dangerous propensities. The order was vacated and the case was remanded.
James and Maureen Franciscus (“Parents”) commenced the within negligence action to recover damages for injuries sustained by their minor daughter, Femina, when she was bitten by Tolga Sevdik's pit bull, Julius. At the time of incident, the dog was being walked by Ashley Dailey, an employee of Fetch Pet Care of West Hills/South Hills, which is owned and operated by John Steigerwald (collectively “Pet Care defendants”). The trial court granted summary judgment in favor of the Pet Care defendants, and the case against Mr. Sevdik was tried and an arbitration award in the amount of $4,000 was entered in favor of Parents. After thorough review, we vacate the summary judgment order and remand for further proceedings.
On December 18, 2009, five-year-old Femina was playing outside her home when she encountered Julius who was being walked by Ms. Dailey. The child asked if she could pet the dog, and when she bent over to do so, the dog jumped up and bit her on the chin. Femina was taken to Mercy Hospital for treatment.
Parents commenced this negligence action against Mr. Sevdik, the owner of the dog, Ms. Dailey, the dog walker, and Mr. Steigerwald, the individual owner and operator of Fetch Pet Care of West Hills/South Hills. After the close of the pleadings and discovery, all defendants filed motions for summary judgment. The trial court denied Mr. Sedvik's motion for summary judgment but granted summary judgment in favor of the Pet Care defendants, finding no evidence from which one could infer the latter should have been aware of the dog's dangerous propensities.
The parties agreed that the case against Mr. Sevdik would be transferred to the arbitration division and that the decision of the arbitrators would be final. The case was tried on September 17, 2014, and the board of arbitrators returned a verdict in favor of Parents in the amount of $4,000. On October 14, 2014, Parents filed the within appeal challenging the propriety of the trial court's grant of summary judgment in favor of the Pet Care defendants.
1. Whether the trial court properly considered evidence of record from which a conclusion could be drawn that the dog service had been put on notice of the dog's dangerous propensities?
2. Whether the lower court correctly found that based upon the record, the Defendants were entitled to summary judgment as a matter of law?
3. What evidence of record caused the trial court to find that summary judgment was proper for the Defendant dog service but not for the Defendant dog owner?
4. Whether it is an established fact that the pit bull breed has a dangerous propensity for inflicting serious injuries on people?
Parents' first three issues implicate the propriety of the trial court's grant of summary judgment and we will discuss them together. The following principles govern our review.
[S]ummary judgment is appropriate only in those cases where the record clearly demonstrates that there is no genuine issue of material fact and that the moving party is entitled to judgment as a matter of law. When considering a motion for summary judgment, the trial court must take all facts of record and reasonable inferences therefrom in a light most favorable to the non-moving party. In so doing, the trial court must resolve all doubts as to the existence of a genuine issue of material fact against the moving party, and, thus, may only grant summary judgment where the right to such judgment is clear and free from all doubt. On appellate review, then, an appellate court may reverse a grant of summary judgment if there has been an error of law or an abuse of discretion. But the issue as to whether there are no genuine issues as to any material fact presents a question of law, and therefore, on that question our standard of review is de novo. This means we need not defer to the determinations made by the lower tribunals. To the extent that this Court must resolve a question of law, we shall review the grant of summary judgment in the context of the entire record.
Summers v. Certainteed Corp., 606 Pa. 294, 997 A.2d 1152, 1159 (Pa.2010) (internal quotations and citations omitted).
(b) he is negligent in failing to prevent the harm.
Kinley v. Bierly, 876 A.2d 419, 422 (Pa.Super.2005) (quoting Restatement (Second) of Torts § 518: Liability for Harm Done by Domestic Animals That Are Not Abnormally Dangerous). A dog owner is subject to liability for negligence for injuries caused by his dog when he knows or has reason to know that the dog has dangerous propensities and yet fails to exercise reasonable care to secure the dog to prevent it from injuring another. Deardorff v. Burger, 414 Pa.Super. 45, 606 A.2d 489, 492 (Pa.Super.1992). The same liability extends to custodians and keepers of a dog with known dangerous propensities while the dog is in their custody and control.
Parents contend that, as a pet sitter, the Pet Care defendants were subject to the same liability as an owner while the dog was in their custody and control. They maintain that the record establishes that both Mr. Sevdik and Ms. Dailey knew that the sixty to seventy pound dog with a stocky body and a big head had an energetic nature and a tendency to jump on people. This awareness, according to Parents, implies actual or constructive knowledge of the dog's dangerous propensities. In support thereof, they rely upon Groner v. Hedrick, 403 Pa. 148, 169 A.2d 302, 303 (Pa.1961), for the proposition that “the law makes no distinction between an animal dangerous from viciousness and one merely mischievous or dangerous from playfulness,” and the animal's motivation or “the mood in which it inflicts harm is immaterial.” Therein, the Court cited the Restatement (Second) of Torts § 518(1), which defines a dangerous propensity as including the tendency of an animal to do any act that might endanger the safety of a person in a given situation. See also Rosenberry v. Evans, 48 A.3d 1255 (Pa.Super.2012) (whether dog's tic, which caused the animal to clench her teeth in a biting motion, may constitute a dangerous propensity, precluding grant of summary judgment).
In further support of their contention that Julius had dangerous propensities known to his owner and the Pet Care defendants, the Parents offered the following. Mr. Sevdik stated to Mrs. Franciscus that, “I always told her [Ms. Dailey] to make sure [the dog] was muzzled.” Plaintiffs' Answers to Interrogatories, No. 11; Maureen Franciscus Deposition, 2/27/13, at 5–7. He had a “Beware of Dog” sign on his front door. Finally, the Petsitting Work Order completed by Mr. Sevdik advised Pet Care that Julius should be walked for thirty minutes, “no dogs, children; Broadway—a lot of dogs/people –––avoid.” This evidence created reasonable inferences that Julius had dangerous propensities and that Mr. Sevdik and the Pet Care defendants knew of them.
The record reveals that, despite her acknowledged receipt of Mr. Sevdik's instructions, Ms. Dailey permitted the child to approach the unmuzzled dog. The dog jumped up, and, according to Ms. Dailey, the dog and the child bumped heads. She did not witness the dog biting the child but noticed the child bleeding. The child was treated for a five-centimeter Tshaped bite wound on the left side of her chin that left her with a onecentimeter scar.
The Pet Care defendants counter that the dog was loving and affectionate and never exhibited any type of vicious or violent behavior. They argue that the duty of a pet sitting service is akin to the duty of a landlord out of possession in Rosenberry, supra. Therein, this Court declined to impose liability for a pet bite unless the landlord had actual knowledge of a dangerous animal on its rental property and the right to control or remove the animal by retaking the premises.
Viewing the record in the light most favorable to the non-moving party, Parents herein, we find the following. The record evidences that Mr. Sevdik, the owner of the dog, had a sign on his residence warning visitors of the dog. Ms. Dailey was aware that the sixty to seventy pound dog had a tendency to jump on people when excited. Mr. Sevdik purportedly directed the Pet Care defendants to use a muzzle when walking the dog but the dog was unmuzzled on the day in question. The Petsitting Work Order completed by Mr. Sevdik directed the Pet Care defendants to avoid routes where there were people, specifically children, and dogs. Giving Parents the benefit of all reasonable inferences, as we must do, we find sufficient evidence of record that Julius had a dangerous propensity to jump on people and possibly bite, if unmuzzled.
Herein, the dog was entrusted to the Pet Care defendants and in their control when the injury occurred. Since the Pet Care defendants knew the dog jumped on people, was to be muzzled when walked, and was not to be walked along routes where there were people, specifically children, and other dogs, they had a duty to use reasonable care while the dog was in their charge to protect others from harm. This is far different from the situation in Rosenberry, supra, DISTINGUISHING where the issue was whether a landlord out of possession could be subject to liability to a third party injured by a tenant's dog on the tenant's premises. Therein, we held that the landlord had no duty unless he had both actual knowledge of the dog's dangerous propensities and the ability to control or remove the animal by retaking the premises. The duty herein flows from Pet Care's contractual undertaking to assume responsibility for the dog while it was in its custody and control and its knowledge of its dangerous propensities. We find the circumstances herein sufficient to subject the Pet Care defendants to liability for failure use reasonable care to prevent the dog from harming others while in their custody and control. Summary judgment was improper.
Due to our disposition, we need not reach the issue whether the trial court erred in refusing to take judicial notice of dangerous propensities of pit bulls generally. However, since we are remanding for further proceedings, we note that Pennsylvania law does not recognize a presumption that pit bulls as a breed are dangerous or have dangerous propensities. Our legislature, in crafting the Dog Law, did not define a pit bull or any other particular breed as a dangerous or vicious dog per se. See 3 P.S. § 459–502–A. Rather, that statute punishes dogs and owners only when a dog exhibits dangerous behavior. This is consistent with our tort approach to domesticated animals. Although the Restatement (Second) of Torts § 519, provides that, where a dog already has been determined to be dangerous based upon a prior incident, the owner or custodian of a dangerous dog is deemed to be carrying on an abnormally dangerous activity and is subject to strict liability for the harm that results, Pennsylvania has declined to adopt that rule. We have concluded instead that, “proof of negligence, in contrast to holding one absolutely liable, is the vehicle by which accountability for injury sustained because of a dog bite is to be established.” McCloud v. McLaughlin, 837 A.2d 541, 544 (Pa.Super.2003) (quoting Deardorff v. Burger, 414 Pa.Super. 45, 606 A.2d 489, 493 (Pa.Super.1992)).
Order vacated and case remanded for further proceedings consistent with this opinion. Jurisdiction relinquished.