Tuesday, October 17, 2017

Networks, not trees, identify "weak spots" in phylogenetic trees

A major application of networks in exploratory data analysis is to identify signal oddities and visualise ambiguity. Thus, they would be the natural choice when it comes to pinpointing weaknesses in phylogenetic trees. This is particularly so when the aim is to propose a relatively stable (and intuitive) ‘phylogenetic’ (identifying likely monophyla sensu Hennig) or ‘cladistic’ (clade-based) systematic framework for a group of organsims. In other words, whenever we try to translate branching patterns into monophyletic groups.

‘Weak spots’ in phylogenetic trees are relationships with either little or ambiguous support, or branching patterns strongly affected by sampling (taxa and characters). These are topological phenomena that are rather the rule than the exception when studying extinct groups of organisms (e.g. spermatophytes or ‘long-necks’).

One example appears to be probably one of the fiercest group of marine predators: the mosasaurs (mosasauroid squamates; Madzia & Cau 2017). I will discuss this example in this post.

Fig. 1. The tree-based systematic groups of mosasaurs (Mosasauroidae plus ancient relatives) when applying Madzia & Cau's nomenclature to their Bayesian-inferred majority-rule consensus tree. Most higher taxa (above genus) are "branch-based", except for the "node-based" Mosasauridae, Russellosaurina (wrong suffix, kept as rank-less taxon by the authors), Tethysaurinae, and Yaguarasaurinae. Genera represented by a single OTU in blue, 'non-monophyletic' genera in red. Thick branches received near unambiguous support (PP ≥ 0.95)

Madzia & Cau “re-examined a data set that results from modifications assembled in the course of the last 20 years and performed multiple parsimony analyses and Bayesian tip-dating analysis” in order to identify the ‘weak spots’ and take them into account when providing a revised cladistic nomenclature of “the ‘traditionally’ recognized mosasauroid clades” (Fig. 1). They define possibly monophyletic groups via recurring branching patterns in their various trees, along with the position of key taxa in those trees (see their chapter Phylogenetic [in fact: cladistic] nomenclature). This allows the groups to “self-destruct” when not forming a clade, and to be replaced.

Although the combination of unweighted and differentially weighted parsimony and Bayesian tip-dating analyses could be methodologically interesting (when examined in detail), it is hardly necessary in order to identify weaknesses and strengths of the data matrix used – going back to Bell 1997, and being emended since (see Introduction of Madzia & Cau) – to define possible monophyletic (or other) groups. A quick and simple neighbour-net splits graph would have done the trick, too.

The situation regarding tree inference, e.g. parsimony

The mosasaurid data matrix suffers from the typical problems: ambiguous, highly homoplasious signals, paired with a few missing data issues (typically lack of data overlap). Adding to this is the miscellaneous signal from taxa regarded as outgroups (here: ancient potential members of the mosasaurs): Adriosaurus suessi (which the authors used to root their trees), Dolichosaurus longicollis, and Ponto-saurus kornhuberi. Accordingly, standard parsimony analysis fails to provide a useful result for about half of the taxa, when documented in the traditional fashion (see my last post) — a strict consensus cladogram of all most parsimonious trees (MPTs) is shown in Fig. 2A.

Fig. 2 Strict consensus graphs based on 152 equally (most) parsimonious trees inferred from the matrix (all characters treated as unweighted and unordered) using PAUP*. Green, unambiguous placement/grouping; turquois, weakly 'rogue-ish', red, rogue taxa

But even the Adams consensus tree (Fig. 2B) is more informative, and the (near) strict consensus network (only showing splits that occur in more than a single MPT) highlights where the equally parsimonious solutions agree and disagree, and which taxa act more ‘rogueish’ than others (Fig. 2C). Weighting and Bayesian inference naturally produce more resolved trees; but the question remains whether the overall higher to unambiguous branch support sufficiently reflects the signal in the character matrix.

Data sets of extinct organisms need neighbour-nets, to start with

The consensus network of the most (equally) parsimonious trees (MPT; Fig. 2C) informs us about equally valid topological alternatives and ‘rogueness’. Using the branch-length averaging option, we can visualize character support to some degree for the alternatives. But there is a quicker and more comprehensive alternative, when it comes to (tree-)incompatible signal.

The neighbour-net (Fig. 3) directly identifies potentially strong signals and ‘weak spots’. First, we can see that the outgroup taxa are not clustered, which is never good. Obviously, they are not too useful to infer an ingroup root (Madzia & Cau discuss the outgroup sampling bias). Only one of the outgrops, Pontosaurus, is placed closed to the Aigialosauridae, which collects the earliest diverging Mosasauroideae lineage (see Fig. 1). Their signals are likely to mess-up any tree inference (Fig. 2).

Fig. 3 The neighbour-net based on simple (Hamming) mean distances inferred from Madzia & Cau's matrix. Colouring as in Fig. 1

Trivial (data-wise) lineages are e.g. the Tylosaurinae, supported by a very long narrow branch— this lineage is characterised by high group coherence and distinctness to any other taxon/taxon group and will inevitably have high support and placed close (phylogenetically and absolute) to the Plioplate-carpinae (Figs 2, 3). The Mosasaurinae are equally well circumscribed, with only one putative member, Dallasaurus, being substantially apart from the rest, and bridging Mosasaurinae and Halisaurinae, their putative sisters. Hence, trees will favour splits rejecting the "Natantia" group unless Dallasaurus is excluded from the inference.

Species of the same genera are conspicuously grouped; this differs from Madzia & Cau’s trees, where Mosasaurus or Prognathodon species are collected in the same subtrees, but are “non-monophyletic”, i.e. do not form an exclusive clade. Based on the neighbour-net, the main reason may be terminal noise and resulting flat likelihood surfaces (hence, low posterior probabilities). The placement of the older members of the mosasaurs (classified as Tethysaurinae and Yaguarasaurinae) to each other, and the slightly older outgroup taxa, is clearly difficult with this matrix, even though there is no ambiguity, e.g. in the MPT sample (Fig. 2). Hence, the branch-lengths do not reflect synapomorphies or rarely shared apomorphies in this subtree, but instead shared convergences — a perfect phylogeny always generates a perfectly tree-like distance matrix.

Oddly placed taxa in the neighbour-net? Probably unrepresentative distances; and the quick fix

In contrast to trees, the network in Fig. 3 fails to resolve a likely position for one Prognathodon species: P. currii, and the large associated box indicates a data issue. The pairwise distances of the oddly placed P. currii and the probably misplaced Dolichosaurus, are poorly defined: both have zero-distances to non-similar taxa, but also to each other. But whereas Dolichosaurus differs from other members of Prognathodon by mean morphological distances (MD) of 0.5–1.0 (1.0 means it differs in all defined characters!), P. currii is much more similar to its congeners (MD = 0.17–0.27 and 0.46). Their other affinities also lie with strongly different taxon sets.

Their position in the neighbour-net is the result of a missing data artefact. Being just a 2-dimensional graph, such severe signal ambiguity cannot be resolved. Unrepresentative distances are the major (only) obstacle for neighbour-nets in the context of extinct groups. Trees are more decisive in such cases, when the few covered characters fit well the preferred tree's topology. By removing the outgroup taxa and P. currii, we can generate a neighbour-net (Fig. 4) in-line, and going beyond the Bayesian-tree-based groups suggested by Madzia & Cau (Fig. 1).

Fig. 4 Same data and method as shown in Fig. 3; four OTUs were excluded, the non-Mosasauroidea (outgroup) and the misplaced Prognathodon currii

Using networks to define taxonomic groups

Just based on the neighbour-nets (Figs 3, 4), circumscription of genera and higher taxa can be discussed (assuming that morphology mirrors phylogeny). For instance, Mosasaurus can be kept as-is or can include Plotosaurus; whereas the Clidastes form a clearly distinct taxon (whether paraphyletic/ monophyletic or clade/grade may be impossible to decide, see Fig. 1). Including (all) Prognathodon in the Globidensini remains an option; Eremiasaurus may be included, too, or included in the likely sister clade, the Mosasaurini.  

Dallasaurus is not only the oldest possible but clearly the most unique (primitive?) member of the Mosasaurinae, and the Halisaurinae likely represent their early diverged sister lineage. Treating Tylosaurinae and Plioplatecarpinae as reciprocally monophyletic sister lineages makes sense with respect to the older taxa and the co-eval Mosasaurinae-Halisaurinae lineage. The ancient forms are generally more similar to Plioplatecarpinae (+ Tylosaurinae) than to the Mosasaurinae and Halisaurinae lineages; but whether they should be included in the same systematic group ("Russellosaurina") cannot be judged based on the data matrix or the inferred trees (see also Figs 1, 2). Their topological attraction may be due to more shared primitive features (Hennig's ‘symplesiomorphies’), and the "Russellosaurina" could be a paraphyletic clade.

An interesting pronounced central edge bundle in the network in Fig. 4, which agrees well with Madzia & Cau's Bayesian consensus tree (Fig. 1), is the one separating all oldest, potentially more primitive taxa/lineages (> 90 Ma) from the later more diversified lineages (Mosasaurinae, Halisaurinae, Plioplatecarpinae, and Tylosaurinae). Regarding primitiveness vs. derivedness, an option to map characters on networks and extract alternative trees directly from the network would be handy (see also David’s 500th post).

Fig. 5 Bootstrap (BS) support network based on 10,000 BS (pseudo)replicates optimised under parsimony. Splits are shown that occurred only in at least 20% of the BS replicates; trivial splits are collapsed. Some taxa have low, but unchallenged support, in other cases no preference at all is found (e.g. for the highest level bracketing taxa) or two alternatives compete with each other.

Also in the case of the mosasaurs: when we want to use phylogenetic trees as the sole (or main) basis for classification, rather than neighbour-nets (see my last post) and common sense backed up by EDA (e.g. Fig. 4; Bomfleur et al. 2017), the method of choice would be the support consensus networks based on parsimony (example provided in Fig. 5), least-squares, and/or likelihood bootstrapping pseudoreplicate samples. in addition to or instead of the Bayesian-inferred topologies sample. The posterior probabilities in Madzia & Cau’s tip-dated tree and Bayesian majority-rule consensus tree include values << 1.0, which already can be an indication of very strong signal conflict or just lack of discriminating signal (flat likelihood surfaces).

We should not be over-confident in PP, when the underlying data are not tree-like at all, as they too easily tilt towards one alternative (see also Zander 2004). The same holds for post-analysis character weighting, designed to eliminate (down-weigh) conflicting signals. While parsimony and distance methods are more easily affected by branching artefacts, probabilistic methods may struggle with flat likelihood surfaces. Thus, bootstrap support networks should be the first choice for ‘phylogenetic’ (by identifying Hennigian monophyla) or ‘cladistic’ (clade-based) classification as they show the robustness of the signal for the preferred and other topological alternatives, and can be generated under different optimality criteria. Having a certain support for a clade is nice, but one should always consider the support for alternatives, and consider how many characters support or oppose an alternative.

Morphological matrices need to be analysed using network approaches

Madzia & Cau’s study is methodologically interesting by providing a tip-dated Bayesian tree for an extinct group of organisms. A one-to-one comparison of their parsimony-BS support using different character and weighting schemes vs. Bayesian PP may be interesting, too — note the difference between the tip-dated tree and the majority rule consensus trees for several critical branches. However, following the current standard practice, no BS pseudoreplicate and Bayesian saved topologies samples were provided. Regarding the main objective, the identification of ‘weak spots’ to propose enhanced systematic groups, networks (Figs 2–5) would have been more informative and straightforward.

No matter what classification philosophy is applied, when we deal with morphological matrices of extinct groups of organisms, the first step should always be to explore the primary signal in the data before we infer trees using (highly) sophisticated methods, and interpret them — the latter may actually obscure ‘weak spots’ rather than identifying them. The quickest analyses are neighbour-nets, but watch out for odd pairwise distance patterns (easily visualised using heat maps)!

The second step is producing support consensus networks, for the fine-tuning and to decide on the most probable trees to explain the data. Regarding classification, we should ask ourselves whether we really want inevitably unstable clade-based classification systems (when dealing with extinct organisms), or robust ones that reflect the general data situation and include potentially or likely paraphyletic taxa (see e.g. Clidastes in Figs 2–5 and Madzia & Cau's trees, and their elaborate discussion of higher level taxa, which – to a good degree – could become superfluous when allowing paraphyletic taxa).


All graphics, and some primary data files, are publicly available from figshare. An archive including all re-analysis files can be downloaded at www.palaeogrimm.org.


Bell GL (1997) A phylogenetic revision of North American and Adriatic Mosasauroidea. In: Callaway JM, and Nicholls EL, eds. Ancient Marine Reptiles. San Diego: Academic Press, pp. 293–332 [cited from Madzia & Cau 2017]

Bomfleur B, Grimm GW, McLoughlin S. 2017. The fossil Osmundales (Royal Ferns)—a phylogenetic network analysis, revised taxonomy, and evolutionary classification of anatomically preserved trunks and rhizomes. PeerJ 5:e3433. https://peerj.com/articles/3433/.

Madzia D, Cau A (2017) Inferring 'weak spots' in phylogenetic trees: application to mosasauroid nomenclature. PeerJ 5: e3782. https://peerj.com/articles/3782/.

Zander RH (2004) Minimal values of reliability of Bootstrap and Jackknife proportions, Decay index, and Bayesian posterior probability. PhyloInformatics 2: 1–13.

Tuesday, October 10, 2017

Where to retire in the USA

Some weeks ago I published a post on recommended countries for Where to retire. Not everyone wants to leave their homeland, however, and so for many of our readers it may therefore be relevant to consider which states in the USA might be recommended as most desirable for retirees.

In this regard, the Bankrate web site has recently considered Where are the best and worst states to retire? They collated data (from various sources) for each of the 50 states for the following eight characteristics:
  • Cost of living
  • Healthcare quality
  • Crime rate
  • Cultural and social vitality
  • Weather
  • Taxes (income and sales taxes)
  • Senior citizens' overall well-being
  • The prevalence of other seniors
For 2017, the states were then ranked from 1–50 for each of these characteristics separately. These rankings were then weighted according to a survey of the reported relative importance of each of these characteristics — they are listed above in the order of decreasing importance. From the weighted data, Bankrate produced an overall ranking of the states for their desirability to retirees, which you can check out on their web site.

However, this ranking is overly simplistic, because it suggests that there is only one main dimension to retirement desirability, from best to worst. Clearly, retirement is multi-dimensional — there is no reason to expect the eight characteristics to be highly correlated. Therefore a network analysis would be handy to explore which characteristics differ between the states.

As for my previous analysis, I have calculated the Manhattan distance pairwise between the states; and I am displaying this in the figure using a NeighborNet network. States that have similar retirement characteristics are near each other in the network; and the further apart they are in the network then the more different are their characteristics.

In the network graph I have highlighted Bankrate's top 10 ranked states in green and their bottom 10 states in red. Note that they do not cluster neatly in the network, emphasizing the importance of considering the different characteristics, rather than just averaging them into a single ranking.

So, the network does not represent a single trend (from best to worst) — this would produce a long thin graph. Instead, the network scatters the states broadly, indicating that they have multiple relationships with each other — the eight retirement characteristics are not highly correlated. Indeed, the network is L-shaped, suggesting two main trends. The main part of the L has the north-eastern and west-coast states at one end and the mid-western and western states at the other, while the short part of the L separates out the south-eastern and south-western states. There are several obvious exceptions to these broad patterns (eg. Kentucky).

You can see that the north-eastern states tend to cluster together as being among the most desirable retirement locations (in Bankrate's ranking), and that the southern states tend to cluster together as being among the least desirable.

California is interesting because it ranks in the top two for Weather and Culture, but near the bottom for everything else. Hawaii ranks highly on Well-being and Culture but very poorly on Taxes, Crime rate, and Cost of living (where it is dead last). Florida, naturally, ranks first for Prevalence of seniors, but it is ranked mediocre to poor on everything else (including its hurricane-prone weather). New York is ranked first for Culture but mediocre to poor for everything else (and is ranked last for Taxes).

Alaska is ranked best for Taxes, Mississippi is best for Cost of living, Vermont is top for Crime rate (being low!), and Maine is best for Health care. Of these, only the latter state scores well for other characteristics, being second for Crime Rate and Prevalence of seniors. This puts it in the overall top three states, along with New Hampshire and Colorado.

New Hampshire gets the top spot by ranking well on everything except Cost of living and Weather — it is close to last for the latter characteristic!

So, the bottom line is that there is no state that particularly stands out as most suitable for retirees — in terms of desirable characteristics, what you win on the swings you lose on the roundabouts. Hardly surprising, really.

If you are interested in retiring to a particular city, then this recent web page may also be of relevance to you: Top 25 cities where you can live large on less than $70k.

Neither this nor the previous analysis (for countries) has addressed the issue of politics. Political voting is not randomly distributed, and some people prefer to live surrounded by voters similar to themselves. If this is you, then Wikipedia has a map indicating which states you might prefer.

Tuesday, October 3, 2017

Clades, cladograms, cladistics, and why networks are inevitable

During the work for another post, I stumbled on a kind of gap-in-knowledge that has nagged me for quite some time. This gap exists because researchers like to stay within chosen philosophical viewpoints, rather than reassessing their stance.

This gap involves the use of cladistic methodology in a manner that obscures information about evolutionary history, rather than revealing it. A clade, a subtree in a rooted tree that fulfills the parsimony criterion (or, indeed, any other criterion), may or may not reflect monophyly in a Hennigian sense, i.e. inclusive common origin. This is especially true for studies of extinct lineages.

I will explore this idea here in some detail.

Assumptions when studying fossils

Phylogenetic papers dealing with the evolution of extinct groups of organisms frequently use strict consensus trees (typically cladograms) of a sample of equally parsimonious trees (MPT) as the sole or main basis for their conclusions. They do this under two important implicit assumptions:
  • The morphological differentiation patterns encoded in a character matrix provide a generally treelike signal. In other words, the data patterns in the morphological matrix can be explained by a single, dichotomous, 1-dimensional graph. This assumption is also the basis for posterior filtering or down-weighting of characters that support splits (taxon bipartitions) conflicting with the branches in the inferred tree(s).
  • Morphological evolution is generally parsimonious. Although this may apply for characters that evolved only once or only evolve under very rare conditions, total evidence and DNA-constrained analysis demonstrate that this is not generally the case: the tree inferred by total-evidence or molecular constraints is typically longer than the tree(s) with the fewest character changes inferred on the morphological partition alone.
Another implicit assumption seems to be that all fossil specimens must represent extinct sister clades, and that no fossil specimen is ancestral to any other (or to an extant species) — hence, all taxa can be treated as terminals (not ancestors). Rooting typically relies on outgroups, under the assumption that ingroup-outgroup branching artefacts (such as long-branch attraction) play no role for parsimony inference when using morphological data sets.

In many of these morphology-phylogenetic papers (using parsimony or other methods) the authors state that they have conduct a “cladistic” study (I also made this error in my masters thesis; Grimm 1999). Cladistics is a classification system established by Hennig (1950) that relies on synapomorphies, exclusively shared, derived traits, that are linked with groups of inclusive common origin, the so-called monophyla.

Over 90 years earlier, Haeckel (1866) used the German word monophyletisch to refer to “natural” groups defined by a shared evolutionary history (a common origin). The latter could also include what Hennig identified as paraphyla: groups that have a common origin, but are not inclusive. To avoid confusion between Haeckelian and Hennigian monophyletic groups, Ashlock (1971) suggested the term holophyletic for the latter. This can be useful when a classification should recognise evolutionary relationships but needs to classify potentially or definitely paraphyletic groups for reasons of practicality (see e.g. Bomfleur, Grimm & McLoughlin 2017). Here, I will stick to Hennig’s terminology, as it is much more commonly used (although not necessarily correctly applied).
Hennig’s monophyla are from a theoretical (and computational) point of view a brilliant concept, as they can be inferred using a rooted tree. The test for monophyly is simple: Do A and B have a common ancestor? If yes, identify all taxa that are part of the same subtree as A and B. Unfortunately, we often find more than one possible tree, and roots can be misleading.

Strict consensus trees poorly represent the alternative topologies in a MPT sample

All consensus-tree approaches are limited to depicting the topological alternatives in a tree sample, but strict consensus trees are probably the worst (see e.g. Felsenstein 2004, chapter 30). They also have become obsolete with the development of consensus networks (Holland & Moulton 2003), and their subsequent implementation in freely accessible software packages such as SplitsTree (Huson 1998; Huson & Bryant 2006) and, more recently, the PHANGORN library for R (Schliep 2011; Schliep et al. 2017).

Figure 1 illustrates this difference for two extreme cases of binary matrices and their MPT collections. The two datasets in Fig. 1 reflect a substantially different data situation. The data in one matrix are perfectly tree-unlike (completely “confused about relationships”): any possible non-trivial bipartition of the 5-taxon set is supported by one (parsimony-informative) character. The data in the other matrix reflect two incongruent trees: each character is compatible with either one of the trees (parsimony-informative characters) or both trees (unique characters). The non-treelike matrix allows for many more MPTs than does the tree-like matrix, which results in two MPTs perfectly matching the two conflicting true trees. But both consensus analyses result in the same, unresolved (polytomous) strict consensus tree. In contrast, the two consensus networks highlight the difference in the quality between the data sets and the MPT sample.

Fig. 1 Non-treelike and treelike data, and the representation of their most-parsimonious tree collections as strict consensus trees and networks

Another example is shown in Figure 2, which shows four trees that differ only in the placement of one taxon (T8). This is a common phenomenom, particularly when dealing with extinct groups of organisms. The three main reasons for such topological ambiguity are:
  1. Indicisive data regarding the exact position of T8 with respect to the members of the red (T1–T4) and green clades (T5–T7).
  2. Conflicting data, T8 shows a combination of traits that are otherwise restricted to (parts of) the green or red clade.
  3. T8 is an ancestor or primitive member of the green or red clade, or both. 

Fig. 2 A single rogue taxon (T8) with ambiguous affinities collapses the strict consensus tree. In contrast, the conensus network can simultaenously show all alternatives, and identifies T8 as the source of topological ambiguity.

The strict consensus tree shows only three clades (three pairs of sister taxa) and a large polytomy, but the strict consensus network shows simultaneously the topology of all four trees and the position of T8 in these trees. From the consensus network, it is clear that the members of the red and green clades share a common origin. T8 can easily be identified as the rogue taxon (lineage).

Cladograms are incomplete representations of evolutionary trees

Figure 3 shows one of the first phylogenetic trees ever produced, and how it would look in the results section of a cladistic study. The tree was produced 150 years ago by Franz Martin Hilgendorf — more than 100 years before Hennig’s ideas were introduced to the Anglo-Saxon world and became mainstream. Hilgendorf was a palaeontology Ph.D. student at the same institute (in Tübingen, Germany) that also promoted me. Quenstedt, his supervisor, forced a quick promotion to get him and his heretic Darwinian ideas out of his university; there are thus no figures in Hilgendorf's thesis, and he published a phylogenetic tree only after he left Tübingen. It shows the evolution of derived forms (terminals) from putative ancestral forms (placed at the nodes) of fossils snails from the Steinheimer Becken, and clearly distinguishes ancestors and sisters. At some point, Hilgendorf even considered including the reticulation of lineages to better explain some forms, but later dropped this idea, feeling it would violate Darwin’s principle (Rasser 2006; see The dilemma of evolutionary networks and Darwinian trees).

Fig. 3 Hilgendorf's phylogenetic tree of fossil snails and its representation in form of a cladogram. The coloured fields and boxes refer to a series of nested clades, which here equal monophyletic groups.

Translating Hilgendorf’s tree into a cladogram comes with a loss of information about the evolution of the snails. Some ancestors are placed as sisters to their descendants (e.g. 18 vs. 18a and 19) and others are collected in a polytomy together with their descendants/descending lineages (e.g. 15, the ancestor of the siblings 16, 17, and the 18+). The loss of information regarding assumed ancestor-descendant relationships is dramatic. But this is no problem for cladistic classification: all clades in the cladogram in Fig. 3 (boxes) refer to Hennigian monophyletic groups seen in the original phylogenetic tree (coloured backgrounds). The polytomies in the cladogram are hard polytomies and do not reflect uncertainty or ambiguity. This contrasts with most cladograms depicted in the phylogenetic (“cladistic”) literature, where polytomies can also reflect lack of support or topological ambiguity.

Accepting the possibility that some fossils (fossil forms) may be ancestral to others (or their modern counterparts), or at least represent an ancestral, underived form, we actually should not infer plain parsimony trees but median networks (Bandelt et al. 1995). Median networks and related inferences (reduced median networks: Bandelt et al. 1995; median joining networks: Bandelt, Forster & Röhl 1999) work under the same optimality criterion (evolution is parsimonious) but allow taxa to be placed at the nodes (the “median”) of the graph. In doing so, they depict ancestor-descendant relationships. That they have not been used for morphological data so far, nor in palaeophylogenetic studies (as far as I know), may have to do with their vulnerability to homoplasy and missing data. High levels of homoplasy are common in morphological matrices, and missing data can be a problem when working with extinct organisms.

An ideal matrix, in which each divergence is followed by the accumulation of synapomorphies (or “autapomorphies”, unique traits, close to the tips), results in a median network perfectly depicting the evolutionary tree (Figure 4). As soon as convergent evolution steps in, a median network can easily become chaotic, although less so for a median-joining network. Note that half of the characters are homoplasious, and yet the median-joining network is still largely treelike (Fig. 4), with only one 2-dimensional box. The true tree is included in the network; but an E-G clade evolving from D is indicated as alternative to the correct (and monophyletic) FGH clade, with G and H evolving from F. Another deviation from the true tree is that A, the ancestor of B and C, is not placed at the node, but is closer to the all-common ancestor X.

Fig. 4 Two datasets, one without (left) and one with homoplasy (right), and their median(-joining) networks. Green branches refer to exact fits with the true tree, red indicate deviation or conflict with the true tree.

Paraphyletic clades...

Figures 5A and B show the corresponding MPT for the ideal matrix and the strict consensus tree vs. strict consensus network for the matrix affected by homoplasy. As our ideal matrix includes actual ancestors, the MPT rooted with the most primitive taxon X (the common ancestor of A–H) cannot resolve the exact relationships, in contrast to the median network. It thus represents the true tree only partly. But it also does not show any clade that is not monophyletic.

In the case of the partly homoplasious data, the median-joining network reconstructs a synapomorphy of the clade BC, because A is not placed on the node. This is because one character in our matrix is a methodologically undetectable parallelism — the same trait evolved in the sister taxa B and C, but only after both evolved from A. Clade BC is non-inclusive (paraphyletic), since A is the direct ancestor of both B and C and the clade BC lacks a real synapomorphy (if we go back to Hennig's concept). The reconstructed A would, however, be a stem taxon and clade BC would be inclusive (monophyletic) with one (inferred) synapomorphy. But this is a purely semantic problem of cladistics. In the real world, we will hardly have the data to discern whether A represents: the last common ancestor of B and C, a stem taxon of the ABC-lineage (a’), a very early precursor of B or C (b/c), or an ancient sister lineage of A, B, and/or C (a*). For practicality, one would eventually include all fossil forms with A-ish appearance in a paraphyletic taxon A (Fig. 5C), in (silent) violation of cladistic classification, to name only monophyletic groups.

Fig. 5A The median network compared to the single most-parsimonious tree inferred based on the ideal matrix

Fig. 5B The median-joining network compared to the strict consensus tree and networks of five most-parsimonious trees inferred based on the matrix with homoplasy. Red edges indicate deviations from or conflicts with the true tree.

Fig. 5C Potential monophyla that could be inferred from the median-joining network (Clades XY), when rooted with the most ancient taxon X. Groups that are monophyletic according to the true tree in blue, groups that are not in orange.

The strict consensus tree of the five MPTs that can be inferred from the homoplasious matrix shows only the paraphyletic (pseudo-monophyletic) clade BC and two monophyletic clades (ABC and D–H); and it contains no further information about the actual topology of the five MPTs. Its lack of resolution is due to the ancestors, which have typically less derived traits (no autapomorphies and fewer synapomorphies), in combination with the homoplasy-induced topological ambiguity. In contrast, the strict consensus networks reveal that all five MPTs place D, the ancestor of the D–H lineage, as (zero branch length) sister to a technically paraphyletic E–H clade, thereby identifying D as the most primitive form of the monophyletic D–H clade. Furthermore, all MPTs recognise a paraphyletic FH clade (F again a zero-length branch). They disagree in the placement of G, which is either sister to F+H (monophyletic FGH clade) or sister to E (a wrong EG clade).

... and monophyletic grades

Figure 6 shows a scenario in which paraphyletic groups are resolved as clades and monophyletic groups form grades, both because of outgroup-ingroup branching artefacts. The derived outgroup O is notably distinct from all ingroup taxa showing a character suite of convergently evolved traits that are randomly shared with parts of the ingroup. Within the ingroup, members of clade DEF are much more derived than are A and C.

Fig. 6 Ingroup-outgroup long-branch attraction can turn monophyla into grades and paraphyla into clades. The ingroup (A–F) consists of a sequence of nested monophyletic lineages (green shades) including two taxa (lowercase letters) that are ancestral to others. Each ingroup lineage evolved (convergent) traits also found in the outgroup O. The data allow inferring two MPTs that misplace O. The outgroup-misinformed root leads to a series of nested clades that a paraphyletic. Splits congruent with the actual monophyletic groups in green, those in conflict with the true tree in red.

Parsimony-tree inference finds two MPTs, which, rooted with the outgroup O, recognise a distinctly paraphyletic A–D+X clade. In both outgroup-rooted MPTs, the monophyletic DEF group is dissolved into a grade. By the way: using neighbour-joining (NJ) to find a tree fulfilling the least-squares (LS) criterion based on the corresponding pairwise mean distance matrix, the outgroup-inferred root is still misplaced with respect to the primitive taxa (X, A–C), but the DEF monophylum is correctly resolved as a clade. Call the Spanish Inquisition! A “phenetic” clustering algorithm finds a tree that is less wrong than the MPTs.

The most comprehensive display of the misleading signal in this matrix is nevertheless the neighbour-net (NNet; Figure 7), which includes both the parsimony and LS-solutions, and it can be used to map the competing support patterns surfacing in a bootstrap analysis of the data. In this network we can see that the signal is not compatible with a single tree, and that the signal from the distant outgroup O is too ambiguous for rooting the ingroup. Based on this graph, one can argue to delete the outgroup, thereby deleting all non-treelike signal — a NNet (or median network) excluding O matches exactly the true tree.

Fig. 7 Neighbour-net based on mean pairwise distances (same data in Fig. 6). The outgroup O provides a strongly ambiguous (non-treelike) signal, thus, triggering a series of splits (in red) conflicting the true tree (shown in grey). Edges compatible with the true tree shown in green. The numbers refer to non-parametric bootstrap support estimated under three optimality criteria: least-squares (LS; via neighbour-joinging), maximum likelihood (ML; using Lewis' 1-parameter Mk model), and maximum parsimony (MP) and 10,000 (pseudo)replicates each. Upper right: A splits-rose illustrating the competing support patterns for proximal splits involving O: green — split seen in the true tree, reddish — the competing splits seen in the two MPTs.

We need to accept that a clade, a subtree in a rooted tree (see e.g. Felsenstein 2004) fulfilling the parsimony criterion (or any other criterion), may or may not reflect monophyly in a Hennigian sense, i.e. inclusive common origin. Thus, it is imperative to distinguish between a classification concept that interprets trees (cladistics) and the method used to infer trees (typically parsimony, in the case of extinct lineages). This is especially so when one has to work with stand-alone data, such as morphological data of extinct groups of organisms.

Aside from the clades/grades ↔ monophyla / paraphyla / can't-say problem, the instability of clades in a parsimony or otherwise optimised rooted tree, or the alternative clades that can be inferred from the more data-comprehensive networks, make it difficult to enforce a strictly cladistic naming scheme. For the example shown in Fig. 2, we would be unable to name the red and green clades until the exact position of T8 is settled (see also Bomfleur, Grimm & McLoughlin 2017). In the end, the overall diversity patterns (studied using exploratory data analysis) may remain the most solid ground for classification.

It should also be obligatory in phylogenetic studies to use networks to display both competing topological alternatives and incompatible data patterns. There should also always be some information on edge-lengths. Consensus trees are insufficient, as they mask conflicting data patterns, and cladograms mask the amount of change.


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