A simple and widely used approach for detecting hybridization in phylogenies is to reconstruct gene trees from independent gene loci, and to look for gene tree incongruence. However, this approach may be confounded by factors such as poor taxon-sampling and/or incomplete lineage-sorting.
Using coalescent simulations, we investigated the potential of supernetwork methods to differentiate between gene tree incongruence arising from taxon sampling and incomplete lineage-sorting as opposed to hybridization. For few hybridization events, a large number of independent loci, and well-sampled taxa across these loci, we found that it was possible to distinguish incomplete lineage-sorting from hybridization using the filtered Z-closure and Q-imputation supernetwork methods. Moreover, we found that the choice of supernetwork method was less important than the choice of filtering, and that count-based filtering was the most effective filtering technique.
Filtered supernetworks provide a tool for detecting and identifying hybridization events in phylogenies, a tool that should become increasingly useful in light of current genome sequencing initiatives and the ease with which large numbers of independent gene loci can be determined using new generation sequencing technologies.
In recent years there has been growing interest in the problem of building explicit models of reticulate evolution [1–12]. This work has to a large part been motivated by biological research highlighting the potential importance of hybridization in the origin of biotic diversity, biological invasion and rapid adaptation [13–27].
One simple and widely used approach for detecting hybridization has been to compare gene trees built from independent gene loci, and to consider gene tree incongruence as evidence for hybridization [1, 28, 29]. However, hybridization is not the only possible cause of gene tree incongruence. Other explanations include phylogenetic error , unrecognised gene duplication and loss , incomplete lineage-sorting  and lateral gene transfer .
In light of current genome sequencing efforts and the ease of sequencing large numbers of independent gene loci using new generation sequencing technologies, it is important to find ways to differentiate between various explanations of gene tree incongruence. Here we focus on distinguishing hybridization from incomplete lineage-sorting. In this regard, a helpful concept might be "principal trees", which are the trees displayed by a hybridization network (see the subsection Simulations for a more formal definition of principal trees). If a phylogeny contains no hybridization or lateral gene transfer, then the expectation is for one principal or "species" tree. However, if hybridization has occurred, then there will be multiple principal trees. In the absence of incomplete lineage-sorting, each principle tree will represent the evolutionary history for a large collection of loci, but where incomplete lineage-sorting occurs gene trees may differ from their underlying principal tree.
Here we investigate the potential of filtered Z-closure  and Q-imputation  supernetworks to distinguish phylogenetic signals arising from principal trees from phylogenetic signals caused by a combination of incomplete taxon-sampling and incomplete lineage-sorting in evolutionary histories involving hybridization. We consider these methods since they are not only designed to cope with conflicting phylogenetic signals, but also with data that current genome sequencing efforts can fail to gather (for example, for multi-gene data sets, such as those generated from expressed sequence tag (EST) databases, gene sequences are often missing for species of interest). In particular, using gene trees generated under a coalescent process, we test whether these methods can be used to filter out phylogenetic signals that do not correspond to edges in principal trees, signals that have the potential of confounding efforts to reconstruct complex phylogenies.
Analogous to the supertree framework  our input is a set of trees on overlapping but not necessarily identical taxa. We refer to the complete taxa set as the union of the taxa sets of the input trees; complete splits are bipartitions of the complete taxa set and trivial splits are splits where one part consists of precisely one element. Furthermore partial trees and partial splits are trees and splits on a subset of the complete taxa set. We denote a split of the taxa set X as A|B where A and B are both subsets of X, A ∪ B = X, and A ∩ B is the empty set.
Our overall approach is to first generate a collection of partial trees along a hybridization network in the presence of incomplete lineage-sorting (modelled by the coalescent process), to then apply a supernetwork method to this collection of partial trees to obtain a collection of complete splits, and to then apply a filter to reduce the complexity of this collection. The reduced collection of complete splits is then compared to the splits associated with the hybridization network to determine if they have been accurately recovered. We use this approach to study two supernetwork methods – Z-closure  and Q-imputation , and two types of filter – one counting-based and one homoplasy-based .
We begin with a brief description of the two supernetwork methods that we considered. Supernetwork methods take as input a set of partial trees and produce a set of complete splits. Unlike the supertree framework, these splits need not be compatible, allowing possible conflict within the set of input trees to be represented. The first supernetwork method we consider, Z-closure, is underpinned by the Z-closure rule and is introduced in  and implemented in SplitsTree4 . The method begins with a collection of partial trees from which a list of partial splits is obtained – each partial split coming from an edge in some partial tree in the list (see e.g. [8, 34, 38]). The Z-closure rule takes a pair of partial splits A|B and C|D, and if A ∩ C, B ∩ C and B ∩ D are all non-empty and A ∩ D is empty, then it replaces the partial splits A|B and C|D with the extended splits A| B ∪ D and A ∪ C | D (c.f. Figure 1). To ensure that as many partial splits in the list as possible are extended to complete splits, the rule is iteratively applied to the partial splits in the list by taking pairs of partial splits and either overwriting them with two splits on a more inclusive taxa set, or, if the rule does not apply, returning the same two partial splits. When the Z-closure rule can no longer be applied the method returns the list of complete splits that have been generated.
Note that the output of Z-closure is dependent on the order of elements in the list of partial splits, and so we repeat the procedure for 10 random orderings keeping a cumulative count of how many times each complete split appears. (Simulations indicate that this order dependence is not strong , so there would be little benefit in performing a larger number of random orderings.) Also note that the Z-closure implementation used for this paper differs slightly from that in SplitsTree4 in that it keeps track of multiple copies of partial splits and complete splits, as this information is required by the counting filter that we apply later.
The other supernetwork method we consider, Q-imputation , also uses partial trees as input but uses an alternative approach to generating complete splits that is based on the four-taxon subtrees (quartets) of the partial trees. For each partial tree with missing taxa – that is, taxa that are in the complete taxa set but not in the taxa set for that tree – the missing taxa are inserted in the tree. This is done by processing the missing taxa in a fixed order and placing each taxon within the partial tree to maximise the number of quartets that contain the missing taxon and are also quartets of the other partial input trees. Once all the trees have been completed the list of complete splits displayed by the completed trees is returned. (In the special case where all the trees are on identical taxa sets, the Q-imputation method reduces to the consensus network method ).
We apply two different kinds of filter to the lists of complete splits obtained from the two supernetwork methods, a homoplasy-based filter and a counting-based filter. The homoplasy filter  requires two user-defined parameters x and y. The level of homoplasy for each complete split and partial tree is determined by recoding the split as a binary character, reducing it to the same taxa set as the partial tree, and evaluating the number of character state changes required to explain the character on the partial tree (i.e. the parsimony score). Splits that have a parsimony score greater than a given number x in more than a given number y of the partial trees are filtered out. The counting filter has one user-defined parameter n and keeps the n splits that appear most frequently in the list of complete splits (ties are broken arbitrarily). Note that for Q-imputation this is equivalent to the filter described in  for some choice of threshold.
The starting point for each simulation is a hybridization network such as the one shown in Figure 2a. Formally such networks are rooted, leaf-labelled, directed-acyclic-graphs in which the nodes are of one of four types: nodes with in-degree 2 and out-degree 1 correspond to hybridizations; nodes with in-degree 1 and out-degree 2 correspond to speciation events; nodes with in-degree 1 and out-degree 0 correspond to the extant species; and one special node of in-degree 0 and out-degree 2 is the root. Such a network can be thought of as a collection of rooted principal trees: These trees are obtained by starting from the tips of the hybridization network (these are the nodes with in-degree 1 and out-degree 0) and choosing one of the two possible paths at each hybridization node that is encountered on the way towards the root. The set of principal trees consists of the trees possible to obtain in this manner (Figure 2b). This leads to a natural definition of the collection of splits associated with a hybridization network as being the union of the splits associated with each of the principal trees of the network (Figure 2c and 2d). We will refer to such splits as the true splits of the hybridization network. The purpose of the simulations is to assess if filtered supernetworks can identify the splits present in the principal trees of the hybridization network. To be successful these splits need to be distinguishable from those arising from incomplete lineage-sorting under the coalescent process.
The main flow of our simulation is shown in Figure 3. Given a hybridization network, a collection of trees was created by sampling with replacement from the collection of principal trees (the same tree may appear multiple times). We used the software package COAL  to simulate trees according to the coalescent process given a principal tree with branch lengths specified in coalescent units (the number of generations divided by population size). We employed two different branch length settings. In each of the principal trees all branch lengths were either assigned coalescent units of 1 or all branch lengths were assigned coalescent units of 0.5. These choices for the branch length settings were also used in , a more sophisticated approach might be to assign branch lengths to the hybridization network itself and then have the principal trees inherit these branch lengths. We also simulated a situation where there were no lineage-sorting effects. In this case the only random aspect to the data generation is the choice of the principal trees. Each tree was then pruned of m taxa at random with the restriction that each taxon in the network must appear in at least one partial tree. These collections of partial trees were then used as input to each of the supernetwork methods. Note that, although COAL produces rooted trees, neither of the 2 supernetwork methods considered use any information about the root, so in effect they consider only the corresponding unrooted trees.
Accuracy of the supernetwork methods was determined by counting the number of false positive and false negative splits. A false positive is a split that is displayed by the supernetwork, but is not a true split of the hybridization network; a false negative is a split that is displayed by at least one of the principal trees of the hybridization network, but not displayed by the supernetwork. Note that these definitions differ from those used in  in that they measure accuracy with respect to an underlying hybridization network that has been used to generate the data.
We based our simulations on ten different hybridization networks each labelled by 8 taxa, and representing 0 to 3 hybridization events (Table 1). For each network the parameters in the simulation that varied were the number of trees (g = 5, 10, 15 or 20), the branch lengths used by COAL  (b = 8, 1, 0.5; where 8 represents the case where COAL was not used, i.e. there are no incomplete lineage-sorting effects) and the number of taxa missing from each tree (m = 0, 1, 2, or 3). For each parameter setting we made 100 replicate data sets, giving 6400 sets of partial trees for each of the ten hybridization networks.
We applied 4 different homoplasy filters to the lists of complete splits returned by Z-closure and Q-imputation:
• (HF1) keep only splits with no homoplasy (i.e. a parsimony score of 1) on all partial trees,
• (HF2) keep only splits with no homoplasy on 75% or more of the partial trees,
• (HF3) keep only splits with no homoplasy on 50% or more of the partial trees,
• (HF4) keep splits with a parsimony score of 2 or less on all partial trees.
We also applied the counting filter (CF) to both Z-closure and Q-imputation. For each hybridization network we selected n splits, where n was fixed to be the number of unique non-trivial splits associated with the principal trees of the network (Table 1).
Results and Discussion
Results were generated for each of the hybridization networks given in Table 1, but for brevity, in Figures 4, 5, 6, 7 and Table 2 we only show results for hybridization network 7 (the network shown in Figure 2a). Results for the other hybridization networks follow the same general trends [see Additional file 1].
Figure 4 shows the change in the average number of split false positives and false negatives with respect to the number of gene trees. The results are averaged over 100 repetitions and the 12 combinations of number of missing taxa m and coalescent branch lengths b.
As can be seen in Figure 4, the (HF1) filter is far too stringent in combination with either Z-closure or Q-imputation; it gives almost no false positives but false negatives increase with increasing g towards the maximum value of 8. The (HF4) filter is not stringent enough in combination with either Z-closure or Q-imputation; it gives almost no false negatives but false positives increase with increasing g. Moreover, (HF3) is ineffective in combination with Z-closure as the number of false positives increases with increasing number of partial trees, in combination with Q-imputation the average number of false positives stays close to 2 for all values of g. (HF2) is the most effective of the homoplasy filters, as for both Z-closure and Q-imputation both types of errors either decrease or stay reasonably constant with increasing g, a property that we would expect any filtered supernetwork method to satisfy. The counting filter also displays this property for both Z-closure and Q-imputation; both false positives and false negatives decrease with increasing number of input trees.
Figure 5 is similar to Figure 4 except that rather than averaging over all values of b and m we focus on the difficult case with the highest number of missing taxa and the most incongruence generated by incomplete lineage-sorting (m = 3, b = 0.5). While all the filtered supernetwork methods shown in Figure 5 control false negatives, false positives increase with increasing g for both Z-closure and Q-imputation using (HF3).
For the rest of this section, we restrict our attention to the best homoplasy filter (HF2) and the counting filter (CF).
Figure 6 shows the trends in the number of false positives and false negatives as the number of missing taxa per tree, m, increases from 0 to 3. Results are averaged over the 12 possible settings for number of partial trees g and coalescent branch lengths b. The (HF2) and (CF) filters exhibit very different behaviour. For (HF2) as m increases the number of false positives increases, in particular going from 2 to 3 missing taxa produces a dramatic increase. Conversely the number of false negatives decreases, presumably due to the fact that as the number of missing taxa gets large more splits meet the requirement of the filter. This effect was not observed for (CF) where the total number of splits is capped; here both false positives and negatives increase with growing m.
Recall that the parameter b affects the probability that the trees generated by COAL  will match the principal tree sampled from the hybridization network, b = 8 corresponds to trees that match exactly. Figure 7 shows the trends in the number of false positives and false negatives for different values of b. Results are averaged over the 16 possible settings for the number of partial trees g and the number of missing taxa m. As expected, both methods and filters perform better when b is large.
Table 2 shows the number of false positives for hybridization network 7 (Figure 2a), which has 2 hybridization events and 8 true splits, for m = 0, 1, 2 or 3, b = 0.5, 1 or 8, and g = 5, 10, 15 or 20 for both Z-closure and Q-imputation with (CF). If two out of three of the conditions (g, m, or b) are favourable (i.e. many input trees, few missing taxa, and the probability that the input trees are congruent with the principal trees is high) then both methods work well. However if two or three of the conditions are unfavourable then both methods start to break down.
Figures 8a and 8b show the average number of false positives and false negatives respectively (averaged over m, g, and b) versus the number of true splits for hybridization networks 1–9. Hybridization network 10 is not shown in the figures, as it is an outlier with 24 true splits, but results for this network follow the same trends as the other hybridization networks. For (CF) both types of errors increase slowly with increasing number of true splits. For (HF2) false positives appear fairly constant, but false negatives increase linearly with a slope close to one with increasing number of true splits.
We have evaluated the potential of Z-closure and Q-imputation filtered supernetworks to identify splits belonging to the sets of principal trees associated with hybridization networks. We have found that this approach can recover these splits when there are few hybridization events. However, our results imply that (1) if gene trees have many missing taxa then many gene trees are required; (2) if the gene trees are frequently incongruent with the principal trees of the hybridization network due to incomplete lineage-sorting then a large number of near complete gene trees is required; (3) and if there are few gene trees available they need to be both near complete and highly congruent with the principal trees.
In our simulations the counting filter picked the n best-supported splits, where n was chosen to be the known number of true non-trivial splits. Of course with real data n will not be known, although in practice n could be chosen by, for example, greedily introducing splits with highest support as long as the corresponding network does not become too complex to easily interpret. Approaches to do this are described in  for consensus networks. Note that by increasing n the risk of introducing false positive splits is increased, although the risk of failing to identify true positive splits is reduced.
Despite these limitations, with the potential now of obtaining large numbers of splits from independent gene loci using new generation sequencing technologies, our findings may nevertheless be applicable for tree-like phylogenies where some degree of hybridization is inferred . In such cases, filtered supernetworks can be used to identify the true splits of the underlying hybridization network. Once these are obtained, the method of  can be used to convert the split system into a hybridization scenario.
One of our most interesting findings is that the choice of whether to use Z-closure or Q-imputation seems to have much less impact on accuracy with regards to recovering the splits in the underlying hybridization network than the choice of filter. For both Q-imputation and Z-closure the counting filter (CF) has the desirable property that as the amount of data increases (more genes or more complete gene trees) the rate of both false positives and false negatives goes down. Several settings were tried for the homoplasy-based filter (HF1 – HF4). HF1 was too stringent, and HF3/HF4 tended to either suffer from increasing false positives or increasing false negatives as the number of gene trees increased. HF2 gave the best compromise between these extremes.
Using the HF2 filter, we found that Z-closure had a higher false positive rate than Q-imputation over a range of parameter combinations (Figure 8A). One explanation might be that Z-closure can potentially generate more splits than Q-imputation. For example, given g fully resolved gene trees on 8-m taxa (m = 1, 2, 3), Q-imputation can generate at most 5*g non-trivial complete splits, whereas Z-closure can produce at most 10*(5-m)*g non-trivial complete splits (where 10 is the number of random orderings). Hence the maximum number of splits that Z-closure could generate decreases as m grows, whereas the number of splits that Q-imputation could generate stays constant. What we observe for both methods and filters is that false positives increase with increasing m (Figure 6). Therefore, the maximum number of splits that Z-closure and Q-imputation could generate does not appear to explain the difference in false positive rates. We think a more likely explanation is that Q-imputation places missing taxa in such a way as to maximize agreement with the input trees, hence tending to produce multiple copies of the same splits. Conversely, Z-closure aims to find all possible complete splits that can be derived by extending partial splits using the Z-closure rule, a process that can yield many different splits. Hence we expect that Q-imputation would be likely to generate fewer false positives than Z-closure in general. This difference is not greatly reduced by HF2 as, in contrast to CF, it does not place a cap on the total number of splits.
We found that HF2 resulted in more false negatives than CF (Figure 8B). This may be due to the fact that this filter only selects splits that have no homoplasy when restricted to 75% of the input trees. Since the principal trees are obtained from a network, they can be different and in some cases may only agree on a small number of edges. Even a true split may have a high homoplasy score when restricted to a particular principal tree. In contrast, CF only selects splits that occur with high frequency, irrespective of whether they are in agreement with any of the input trees.
Although all the trees used in our simulations were fully resolved, both supernetwork methods considered here can be applied to partially resolved trees. Thus, when inferring gene trees to be used as input to a supernetwork method, it would probably be a reasonable approach to only retain those edges in the estimated gene trees that have high support (e.g. bootstrap support or posterior probability higher than some cut-off value).
In cases where there are many hybridization events, especially between individuals that are not closely related, there will be many principal trees and corresponding splits (as in hybridization network 10). Many of these splits will occur at low frequencies making them hard to distinguish from phylogenetic error. This means that phylogenetic inference will be limited, as gene-tree incongruence will be extensive. In such cases, rather than attempt to reconstruct a hybridization network, it may be more appropriate to formulate objective tests to better understand the complexity of the data and the extent to which hybridization contributes to this complexity. Joly, McLenachan and Lockhart (submitted manuscript) have recently proposed such a test.
An unexplored idea worthy of study is the investigation of model-based, rather than combinatorial, methods of filtering. One approach might be to consider posterior probability distributions on species trees . It will be interesting to investigate whether such posterior distributions can also be analysed for evidence of distinct principal trees in cases where evolutionary relationships are complex.
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All authors thank the Isaac Newton Institute for Mathematical Sciences, Cambridge, UK for hosting them in the context of their Phylogenetics Programme where part of the work presented in this paper was carried out. BRH and PJL thank Marsden Fund MAU0509 and MAU0510, VM thanks the Engineering and Physical Sciences Research Council (EPSRC), grant EP/D068800/1.
Authors and Affiliations
Allan Wilson Centre, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
Barbara R Holland & Steffi Benthin
Allan Wilson Centre, Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand
Peter J Lockhart
School of Computing Sciences, University of East Anglia, Norwich, UK
BRH developed and applied the simulation scheme, implemented the modified Z-closure method, homoplasy filter and counting filter, and contributed to writing the ms, especially the methods and results section. SB conducted initial simulations with Z-closure. PL ensured biological relevance, and contributed to writing the ms, especially the introduction and conclusions. VM ensured mathematical correctness and developed the overall concept. KH ensured mathematical correctness and developed the overall concept, contributed to writing the ms, especially the methods and results section.
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