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Home , Aequornithes, Afroaves, Australaves, Columbimorphae, Coraciimorphae, Gruae

Zoologica Scripta

Invited Review The phylogenomic forest of trees contains a hard polytomy at the root of

ALEXANDER SUH

Submitted: 11 April 2016 Suh, A. (2016). The phylogenomic forest of bird trees contains a hard polytomy at the root Accepted: 11 August 2016 of Neoaves. — Zoologica Scripta, 45,50–62. doi:10.1111/zsc.12213 have arguably been the most intensely studied group for their phylogenetic relationships. However, the recent advent of genome-scale phylogenomics has made the for- est of bird phylogenies even more complex and confusing. Here, in this perspective piece, I show that most parts of the avian Tree of Life are now firmly established as reproducible phylogenetic hypotheses. This is to the exception of the deepest relationships among Neoaves. Using phylogenetic networks and simulations, I argue that the very onset of the super-rapid neoavian radiation is irresolvable because of eight near-simultaneous speciation events. Such a hard polytomy of nine taxa translates into 2 027 025 possible rooted bifurcat- ing trees. Accordingly, recent genome-scale phylogenies show extremely complex conflicts in this (and only this) part of the avian Tree of Life. I predict that the upcoming years of avian phylogenomics will witness many more, highly conflicting tree topologies regarding the early neoavian polytomy. I further caution against bootstrapping in the era of genomics and suggest to instead use reproducibility (e.g. independent methods or data types) as sup- port for phylogenetic hypotheses. The early neoavian polytomy coincides with the Creta- ceous–Paleogene (K-Pg) mass extinction and is, to my knowledge, the first empirical example of a hard polytomy. Corresponding author: Alexander Suh, Department of Evolutionary Biology, Evolutionary Biology Centre (EBC), Uppsala University, SE - 752 36, Uppsala, Sweden. E-mail: alexander.suh@ ebc.uu.se Alexander Suh, Department of Evolutionary Biology, Evolutionary Biology Centre (EBC), Uppsala University, SE - 752 36, Uppsala, Sweden. E-mail: [email protected]

Introduction publicly available trees/data sets (Jarvis et al. 2014, 2015; The last few years have produced so many different phylo- Prum et al. 2015; Suh et al. 2015). This was complemented genetic trees of birds that it has become almost impossible with networks based on simulated nine-taxon gene trees to see the forest for the trees. It is thus about time to revisit from a random tree generator (Boc et al. 2012) and simu- recent contradictory claims from genome-scale studies and lated nine-taxon retroposon presence/absence patterns (i.e. to disentangle the degrees of conflict present in different all 512 possible strings of nine binary digits). parts of the avian Tree of Life. Nearly all of these conflicts involve the super-rapid radiation of Neoaves, the taxon The emerging consensus of a largely resolved comprising all extant birds but the lineages leading to chick- neoavian Tree of Life ens, , ostriches, cassowaries and . The There have been numerous attempts to solve the Neoaves Neoaves problem has been intensely debated for decades. In problem. Each of these has seen extensive disagreement the following, I show that genome-scale phylogenomics has between and among studies, irrespective of whether the resolved most of these disputes, and I argue that the data type was morphology (e.g. Livezey & Zusi 2007), presence of a hard polytomy explains all of the remaining DNA–DNA hybridization (e.g. Sibley & Ahlquist 1990), irresolvable branches of the neoavian Tree of Life. mitochondrial genes (e.g. Pratt et al. 2009; Pacheco et al. To test for a hard polytomy, I generated phylogenetic 2011) or single nuclear genes (e.g. Fain & Houde 2004; networks in Splitstree4 (Huson & Bryant 2006) using Poe & Chubb 2004). If one were to draw a strict consensus

50 ª 2016 The Authors. Zoologica Scripta published by John Wiley & Sons Ltd on behalf of Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. A. Suh  A hard polytomy at the root of Neoaves of all these analyses, it would be an entirely unresolved studies (McCormack et al. 2013; Jarvis et al. 2014; Prum neoavian ‘Bush of Life’. et al. 2015; Suh et al. 2015), for example Otidimorphae Over the last ten years, this picture has changed dramati- ( + + ), ( cally with analyses of more and more nuclear loci. While + + doves), the /Phaethontimor- exons have been shown to be problematic for resolving the phae and (core landbirds excluding Aus- neoavian radiation (Chojnowski et al. 2008; Jarvis et al. tralaves) (Fig. 1). 2014), other nuclear data types such as introns, ultracon- served elements (UCEs) and retroposon presence/absence Some neoavian relationships are more complex than patterns have become increasingly important. Analysing a bifurcating tree even just a few handfuls of unlinked nuclear loci has It is relieving that there are now robust and reproducible revealed some surprising relationships that since then have phylogenetic hypotheses for most aspects of the Neoaves been recovered relatively consistently, such as Psittaco- problem (Fig. 1). But genome-scale phylogenomics passerae ( + ), (flamingos notwithstanding, why do there continue to be controversial + ), (core landbirds) and Aequornithes branches at the base of core landbirds and unresolved (core waterbirds) (van Tuinen et al. 2001; Ericson et al. branches at the onset of the neoavian radiation? As so often 2006; Hackett et al. 2008; Suh et al. 2011, 2012; Wang in biology, things are a bit more complex than they seem. et al. 2012). Other, equally surprising relationships have First of all, the current mainstream approach of measur- only been discovered through analysis of hundreds or thou- ing and comparing ‘support’ in genome-scale phyloge- sands of unlinked nuclear loci, such as Phaethontimorphae nomics may be flawed, as nicely illustrated by the recent ( + ), the / clade and the example of the enigmatic . In their Nature paper, sandgrouse/ clade (McCormack et al. 2013; Jarvis Prum et al. (2015) stated that their ‘[...] concatenated Baye- et al. 2014; Prum et al. 2015; Suh et al. 2015). sian analyses resulted in a completely resolved, well supported Since the end of 2015, it has been safe to say that there phylogeny. [...] Almost all in the maximum likelihood is now a new and robust consensus for the neoavian Tree tree were maximally supported with bootstrap scores (BS) of of Life (Fig. 1). With the exception of the eight deepest 1.00’. They used this ‘support’ to group the hoatzin with speciation events of the neoavian radiation, there appear to core landbirds and name the ‘novel’ clade ‘Inopinaves’ be reproducible phylogenetic hypotheses for all other long- (Fig. S1B). However, the same argument was used in the standing aspects of the Neoaves problem. I emphasize that Science paper by Jarvis et al. (2014) to group the hoatzin this reproducibility lies not in high values for bootstrap or with cranes + shorebirds and name the ‘novel’ clade Bayesian posterior probabilities, the reasons for which will ‘Gruae’ (Fig. S1A). How can we trust bootstrap or Baye- be discussed below. Instead, the new neoavian Tree of Life sian posterior probabilities if different trees exhibit discor- is largely robust and reproducible because most relation- dant topologies that all have maximum ‘support’? Salichos ships have been recovered in at least two phylogenetic trees & Rokas (2013) foresaw this problem a few years ago based on independent methods or data types (Fig. 1). when they stated that ‘[...] relying on bootstrap to analyse These are four genome-scale phylogenies, namely the Jar- phylogenomic data sets is misleading, [...] because its application vis et al. (2014) TENT tree (48 taxa, 14 536 nuclear loci), will, even in the presence of notable conflict or systematic error, the McCormack et al. (2013) tree (32 taxa, 1541 UCE loci), almost always result in 100% values’. Genome-scale phyloge- the Prum et al. (2015) tree (198 taxa, 259 nuclear loci) and nomics thus urgently needs a paradigm shift, away from the Suh et al. (2015) tree (48 taxa, 2118 retroposon loci). overselling ‘novel’ clades and instead defining support as For comparison in Fig. 1, I also included the Jarvis et al. reproducibility of a phylogenetic hypothesis – that is, the (2014) UCE tree (48 taxa, 3769 UCE loci) and the histori- emergence of the same bifurcation in independent data cally important and widely known Hackett et al. (2008) tree sets analysed by independent research groups using inde- (169 taxa, 19 intron loci). pendent methods. Additionally, I hope that measures such What is the lesson to be learnt from all this? It is not as internode certainty (Salichos & Rokas 2013), now simply the addition of more data, but the replication of implemented in RAxML (Salichos et al. 2014), will soon such analyses via independent data types, independent replace bootstrap and conventional Bayesian posterior taxon samplings and different teams of researchers, that probabilities in genome-scale phylogenomics. A recent lends credibility to the new consensus of the avian Tree of study on complex speciation in tomatoes shows that this is Life. Not only does this apply to the aforementioned rela- feasible (Pease et al. 2016). Furthermore, Bayesian analyses tionships that have now been recovered consistently in using a reversible-jump Markov chain Monte Carlo phylogenomic analyses, but also to some clades that were approach yield more reliable Bayesian posterior probabili- newly found in at least two of the four genome-scale ties than conventional Bayesian analyses under uniform

ª 2016 Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 51 A hard polytomy at the root of Neoaves  A. Suh

Fig. 1 The new consensus of the avian Tree of Life. Topological support by six different trees is colour-coded. Branches recovered by less than two independent studies are collapsed and white boxes denote conflicting topologies at the respective node. Higher-level names follow the nomenclature in Jarvis et al. (2014), with the exception of those taxon names (asterisks) that were described in prior studies (Sangster 2005; Mayr 2011; Suh et al. 2011; Yuri et al. 2013).

52 ª 2016 Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 A. Suh  A hard polytomy at the root of Neoaves branch length priors (Lewis et al. 2005; Yang & Rannala The phylogenetic network approach can be extended to 2005; Yang 2007). compare gene trees or species trees from different studies. Genome-scale phylogenomics requires a second para- A consensus network (Fig. 2) based on three species trees digm shift. For reasons mentioned above, the addition of from the largest genome-scale analyses to date (Jarvis et al. more and more data yields increasingly ‘resolved’ species 2014; Prum et al. 2015; Suh et al. 2015) largely resembles trees (Salichos & Rokas 2013). At the same time, sampling the neoavian Tree of Life (cf. Fig. 1, cf. Fig. S2A). Reticu- more genomic loci increases the variance among the gene lations are restricted to the controversial branches at the trees that underlie the species tree, leading to more, not onset of the core landbird radiation and the unresolved less, phylogenetic discordance (Jeffroy et al. 2006; Philippe branches at the onset of the neoavian radiation. Below, I et al. 2011; Liu et al. 2015; Hahn & Nakhleh 2016). The address these two issues separately because they constitute most striking example for this is the hitherto largest gen- two different phylogenetic problems. ome-scale analysis of any animal group, the Jarvis et al. (2014) TENT tree in which not a single one of the 14 536 Why is the root of core landbirds controversial? individual gene trees is identical to the species tree. Fur- The controversial branches at the onset of the core land- thermore, gene trees from different data types differ from bird radiation result from changes in the respective posi- the species tree on most branches (Jarvis et al. 2014). This tions of (eagles + NW vultures), dilemma illustrates that ‘resolved’ species trees are a prob- Coliiformes () and Strigiformes () (Fig. 2, lematic metaphor for genome-scale data because they hide Fig. 3A–B). However, the majority of independent gen- ubiquitous discordance among gene trees (Hahn & Nakh- ome-scale species trees have grouped Accipitriformes and leh 2016). Thus, I hope that in the near future every analy- owls as illustrated in Fig. 1 (McCormack et al. 2013; Jarvis sis of bifurcating trees will be complemented by et al. 2014; Suh et al. 2015). The only remaining contro- phylogenetic networks, because these visualize alternative versy is the problem with two recurrent alterna- topologies as reticulations (Whitfield & Lockhart 2007; tive positions (Suh et al. 2015). In analyses that include Hallstrom€ & Janke 2010; Bapteste et al. 2013). One such exons and/or introns, mousebirds have grouped within analysis of retroposon presence/absence patterns revealed (Jarvis et al. 2014; Prum et al. 2015) as sis- that Neoaves in fact comprise three rapid radiations, ter to Cavitaves ( + bee-eaters + hornbills + namely an initial super-radiation, the core waterbird radia- roller; Yuri et al. 2013). However, independent data tion and the core landbird radiation (Fig. 4A of Suh et al. sets of UCEs or retroposon markers have consistently 2015). The neighbour network analysis also showed that all recovered mousebirds as sister to the remaining Afroaves the controversial and unresolved parts of the neoavian Tree (Jarvis et al. 2014; Suh et al. 2015). A possible explanation of Life (cf. Fig. 1) contain high degrees of phylogenetic for this discordance would be strong genomic signal for discordance (Suh et al. 2015). As carefully analysed retro- the two competing hypotheses, resulting from introgressive poson presence/absence patterns contain negligible hybridization (i.e. postspeciation gene flow) between the amounts of homoplasy (Shedlock et al. 2004; Ray et al. ancestor of mousebirds and the ancestor of Cavitaves. 2006; Han et al. 2011), these discordances have been attrib- However, tests of retroposon support suggested that uted to an extreme prevalence of incomplete lineage sort- hybridization did not play a measurable role in the mouse- ing (ILS) in some parts of the neoavian Tree of Life (Suh bird problem (Suh et al. 2015), and the only prevalent et al. 2015). ILS, the persistence of ancestral polymor- genomic signal is that for the grouping of mousebirds as phisms across multiple speciation events, is ubiquitous in sister to the remaining Afroaves (Fig. 3C). I suggest an nature (Maddison & Knowles 2006). Subsequent fixation of alternative explanation for the grouping of mousebirds with one of the two alleles of a polymorphism occurs indepen- Cavitaves in genome-scale analyses involving exons and dently in each descendant lineage, leading to discordances introns. Compared to the more slowly evolving raptorial that have been dubbed ‘hemiplasy’ because, unlike homo- lineages within Afroaves, mousebirds and Cavitaves have plasy, the alleles are identical by descent (Avise & Robin- longer branches (cf. Fig. 3 of Hackett et al. 2008 and son 2008). ILS is particularly prevalent when effective Fig. S7 of Jarvis et al. 2014), raising suspicion of long- population size (Ne) is large and time between consecutive branch attraction (LBA) in sequence analyses of exons and speciation events is short (Maddison 1997; Maddison & introns. This would explain the previous notion that Knowles 2006). It is thus interesting that there are very mousebirds are a ‘rogue’ taxon in such analyses (Wang high levels of ILS at the respective onsets of the initial et al. 2012). It remains to be tested whether minimizing neoavian super-radiation and the core landbird radiation, LBA in existing intron/exon data (e.g. via the LS3 method followed by a gradual slowdown of ILS towards the tips of of Rivera-Rivera & Montoya-Burgos 2016) might over- the neoavian Tree of Life (Suh et al. 2015). come the rogue effect of mousebirds and recover them as

ª 2016 Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 53 A hard polytomy at the root of Neoaves  A. Suh

Fig. 2 The avian Tree of Life contains local reticulations and a polytomy zone. Average consensus network based on the three largest, independent genome-scale phylogenies to date (Jarvis et al. 2014; Prum et al. 2015; Suh et al. 2015). The Prum et al. (2015) tree was pruned to match the taxon sampling of the Jarvis et al. (2014) and Suh et al. (2015) trees (see Fig. 1 for information about methods and data types employed in the three studies and Figure S2A for a majority-rule consensus tree). The polytomy zone is marked in yellow and the remaining colour coding corresponds to the three neoavian radiations defined by Suh et al. (2015), namely the initial superradiation (red), the core waterbird radiation (blue) and the core landbird radiation (green).

sister to the remaining Afroaves, consistent with UCEs and level[s] of up to 100% ILS [in retroposon markers] [...] [creat- retroposon markers. ing arrangements that] may neither resemble a fully bifurcating tree nor are they resolvable as such’. Although this notion was Why is the root of Neoaves unresolved? ignored in the subsequent phylogenomic study of Prum The unresolved relationships at the very onset of the neoa- et al. (2015), their ‘resolved’ tree in fact further strengthens vian radiation (Fig. 1) perfectly overlap with those branches the aforementioned suggestion that the root of Neoaves is that Suh et al. (2015) recently found to exhibit ‘[...] extreme not resolvable. Not only are the major tree hypotheses of

54 ª 2016 Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 A. Suh  A hard polytomy at the root of Neoaves

Fig. 3 The onset of the core landbird radiation (i.e. the mousebird problem) is not a hard polytomy. All avian phylogenetic networks were pruned to one representative for each of the five taxa (pictured). (A) Pruned version of fig. 5D of Suh et al. (2015) (B) Pruned version of Fig. 2 of this study. (C) Pruned version of Fig. 4A of Suh et al. (2015). Consensus networks (panels A–B) show consensus splits present in at least 5% of trees and the neighbour network (panel C) shows reticulations among 0/1-coded character distributions. The five taxa involved in the mousebird problem are reciprocally monophyletic (cf. Figs 1–2), namely Accipitriformes (Ac), (Au), Cavitaves (Cv), Coliiformes (Ci) and Strigiformes (St). Colour coding of phylogenetic networks corresponds to Fig. 2.

Jarvis et al. (2014) and Prum et al. (2015) entirely discor- present in at least 5% of the respective trees. The resultant dant with regard to the eight deepest neoavian relationships consensus networks generally exhibit tree-like reticulations, (Fig. S1), but these conflicts are additionally so complex with the exception of the root of Neoaves (Fig. S3, cf. that they collapse into an unresolved ‘tangle’ in attempts to Fig. 2). Illustrating the extent of the conflict between gen- reconcile them (Fig. 2, Fig. 4C, Fig. 2A). It is important to ome-scale species trees (Figs 1–2), a total of nine recipro- keep in mind that this star-like tangle is different from the cally monophyletic taxa originate in this unresolved tangle previously discussed mousebird problem, which exhibits at the root of Neoaves. Pruning networks to one represen- tree-like reticulations that are much less complex and tative for each of these nine taxa again reveals a star-like resolvable as a bifurcating species tree (cf. Fig. 4A–D vs. tangle (Fig. 4A–B). The same star-like signal was previ- Fig. 3). ously noted in a neighbour network analysis of low-homo- What is this early neoavian tangle? Previous studies in plasy retroposon markers (Fig. 4A of Suh et al. 2015) and the pregenomic era have repeatedly argued for (Poe & persists when pruning this network to one representative Chubb 2004) or against (Gibb et al. 2007; Chojnowski et al. for each of the nine critical taxa (Fig. 4D). Thus, neither 2008) a polytomy within Neoaves. In this context, it is adding more data nor reducing homoplasy decreases the important to distinguish between two types of polytomies. complexity at the root of Neoaves. On the contrary, irre- In contrast to ‘soft polytomies’ that result from insufficient spective of whether one looks at genome-scale species data, ‘hard polytomies’ reflect the biological limit of phylo- trees, individual gene trees or low-homoplasy retroposon genetic resolution because of near-simultaneous speciation presence/absence patterns (Fig. 4), the tangle persists. This (Hoelzer & Meinick 1994; Walsh et al. 1999; Whitfield & suggests a lack of underlying tree-like signal. I thus argue Lockhart 2007). Now that genome assemblies are available that the very onset of the neoavian radiation was character- for all major lineages of Neoaves (Jarvis et al. 2014; Zhang ized by eight near-simultaneous speciation events that led et al. 2014), there are finally sufficient data to rule out the to a nine-taxon hard polytomy. presence of soft polytomies via genome-scale phyloge- How can we test the hypothesis of a nine-taxon hard nomics. To do so, I conducted phylogenetic network analy- polytomy at the root of Neoaves? Slowinski (2001) ses of existing data sets derived from 48 bird genomes. The suggested that ‘[...] when an internode on a species tree is results can be considered minimum estimates for phyloge- zero-length, each possible gene tree corresponding to each possible netic conflict, because an increase in taxon sampling is resolution of the species polytomy is equiprobable’. It is possible likely to recover more discordances due to ILS. I analysed to determine the number of possible gene trees for nine 3679 UCE gene trees and 2022 binned intron gene trees taxa following the formula by Cavalli-Sforza & Edwards (Jarvis et al. 2014, 2015) for the presence of reticulations (1967). Accordingly, 2 027 025 rooted bifurcating trees are

ª 2016 Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 55 A hard polytomy at the root of Neoaves  A. Suh

Fig. 4 The onset of the neoavian radiation is a nine-taxon hard polytomy. All avian phylogenetic networks were pruned to one representative for each of the nine taxa (pictured). (A) Pruned network of 3,679 UCE gene trees from Jarvis et al. (2014) (cf. Fig. S3A), (B) pruned network of 2022 binned intron gene trees from Jarvis et al. (2014) (cf. Fig. S3B), (C) pruned version of Fig. 2 of this study. (D) Pruned version of Fig. 4A of Suh et al. (2015), (E) simulated nine-taxon polytomy based on a sample of 1000 random gene trees and (F) simulated nine-taxon polytomy based on a random sample of 95% of the 512 possible retroposon presence/absence patterns (cf. Fig. S2B). Consensus networks (panels A–C, panel E) show consensus splits present in at least 5% of trees whereas neighbour networks (panels D and G) show reticulations among 0/1-coded character distributions. The nine taxa originating in this polytomy zone are reciprocally monophyletic (cf. Figs 1–2, Fig. S1), namely Aequornithes/Phaethontimorphae (Ae/Ph), (Ca), (Ch), Columbimorphae (Co), (Gr), Opisthocomiformes (Op), Mirandornithes (Mi), Otidimorphae (Ot) and Telluraves (Te). Colour coding of phylogenetic networks corresponds to Fig. 2. possible (Felsenstein 1978) and these should be equiproba- Assuming that these ancestral polymorphisms are biallelic ble under the null hypothesis of a nine-taxon hard poly- markers (e.g. single-nucleotide polymorphisms or retro- tomy. I therefore simulated a nine-taxon hard polytomy by poson presence/absence polymorphisms), this translates generating a random set of 1000 gene trees for nine taxa. into 2n possible character distributions which should be The resultant consensus network (Fig. 4E) is strikingly equiprobable under the null hypothesis of a hard polytomy. similar to the aforementioned star-like tangle among Consequently, a nine-taxon polytomy leads to 512 possible empirical gene trees (Fig. 4A–B). I propose an additional character distributions. Following the formula of Suh et al. test of this null hypothesis by simulating the data underly- (2015), 96.5% of these show hemiplasy, which means that ing individual phylogenetic trees. Under the scenario of a this percentage of character distributions is discordant with hard polytomy giving rise to n taxa, all of the shared ances- each of the 2 027 025 possible bifurcating species tree tral polymorphisms will undergo ILS and subsequent topologies. I thus simulated retroposon presence/absence stochastic sorting in each of the n descendant lineages. patterns by picking a random subset of 95% of all 512

56 ª 2016 Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 A. Suh  A hard polytomy at the root of Neoaves possible strings of nine binary digits. Neighbour network hard polytomy, the polytomy zone and anomaly zone analysis again reveals star-like signal (Fig. 4F, cf. Fig. S2B) should thus be mutually exclusive. that is strikingly similar to the empirical retroposon data Is the polytomy zone visible in dated species trees of (Fig. 4D). Taken together, the analysed empirical and sim- Neoaves? If this were the case, its near-simultaneous speci- ulated data suggest that the root of Neoaves is a nine-taxon ation events should translate into extremely short intern- hard polytomy. odes. I tested this by mapping the early neoavian polytomy Is it possible to invoke hybridization as a full or partial zone onto the species tree of Jarvis et al. (2014) and mea- explanation of this hard polytomy-like pattern? According suring the time from the onset of the neoavian radiation to to the ‘hybrid swarm theory’, hybridization may spur the youngest divergence still within in the polytomy zone. rapid adaptive diversification (Seehausen 2004). In this This way of forcing the polytomy zone on a bifurcating context, it is important to distinguish between gene flow tree suggests that its temporal extent was ~4.618 million during speciation (natural hybridization) and postspecia- years and internode lengths were similar to those in the tion gene flow (introgressive hybridization) (Harrison & resolvable parts of the neoavian radiation. How is this pos- Larson 2014). Gene flow during speciation is virtually sible? All these branches exhibit extreme levels of up to indistinguishable from ILS and adds to the overall extent 100% ILS (Suh et al. 2015) and thus, as mentioned before, and duration of ILS (Suh et al. 2015). On the other either Ne must have been large or internode length extre- hand, postspeciation gene flow can be distinguished from mely short (Maddison 1997; Maddison & Knowles 2006). ILS, and there are now a number of methods to do so Now consider the following thought experiment. The aver- for single-nucleotide polymorphisms (e.g. Durand et al. age duration from mutation to fixation is 4Ne generations 2011; Martin et al. 2015; Pease & Hahn 2015) and retro- (Kimura & Ohta 1969). Assuming a generation time of two poson presence/absence patterns (Kuritzin et al. 2016). years (as present in Ficedula flycatchers, Nadachowska- However, these methods handle trees Brzyska et al. 2016), ILS across the putative 4.618 million with only four or five taxa and require a known species years of the neoavian polytomy zone yields an average Ne tree. New methods are therefore necessary for analysing of 1 732 352. Although there is evidence for similar Ne in complex phylogenetic scenarios which involve more than extant bird species (Nadachowska-Brzyska et al. 2015), it is five taxa and lack a ‘resolved’ species tree. Alternatively, hard to imagine that such a large ancestral neoavian popu- it remains to be tested whether existing phylogenetic net- lation persisted for such a long time and across eight speci- work methods (e.g. Yu et al. 2013, 2014; Solıs-Lemus & ation events. Instead, I suggest that the discrepancy Ane 2016) can handle the aforementioned complexity of between the neoavian polytomy zone and the relatively the Neoaves problem. At any rate, if the star-like tangle long internodes in the Jarvis et al. (2014) time tree results at the root of Neoaves was caused by introgressive from the fact that highly ILS-affected gene trees were hybridization rather than a hard polytomy, one would forced on a fully ‘resolved’, yet irresolvable species tree of have to assume near-equal frequencies of postspeciation Neoaves. Mendes & Hahn (2016) recently showed that an hybridization among nine species. This scenario seems ILS-related phenomenon, dubbed ‘substitutions produced much less plausible than assuming a hard polytomy due by incomplete lineage sorting’ (SPILS), erroneously to nine near-simultaneous speciation events at the onset increases or decreases internode lengths and thereby affects of the neoavian radiation. a wide range of downstream molecular analyses, including The explosive super-radiation at the onset of neoavian molecular dates. As mentioned before, the base of Neoaves diversification can thus be described as a polytomy zone. witnessed extreme levels of up to 100% ILS across at least How does the concept of a polytomy zone relate to eight speciation events and SPILS can be expected to another concept, the ‘anomaly zone’? Degnan & Rosen- strongly affect these internode lengths in any ‘resolved’ berg (2009) defined the anomaly zone as internodes where species tree. I thus agree that there is a current ‘[i]rrational there is a higher probability of observing an anomalous exuberance for resolved species trees’ (Hahn & Nakhleh 2016) gene tree than the actual species tree. This is caused by the and suggest that the internode lengths at the root of consecutive arrangement of very short internodes and leads Neoaves should be taken with a grain of salt. to symmetric anomalies in asymmetric species trees (Rosenberg 2013). Suh et al. (2015) suggested that some Outlook: what are the implications of hard neoavian relationships may lie within the anomaly zone. polytomies for the animal Tree of Life? However, a recent phylogenomic study on skinks empha- If the early neoavian polytomy is ‘real’, what catalysed the sized that an accurate species tree topology is essential to near-simultaneous emergence of nine bird lineages? Suh empirically detect the anomaly zone (Linkem et al. 2016). et al. (2015) noted an interesting coincidence between Considering that there is no bifurcating species tree in a those branches with extreme degrees of ILS (herein

ª 2016 Royal Swedish Academy of Sciences, 45, s1, October 2016, pp 50–62 57 A hard polytomy at the root of Neoaves  A. Suh collapsed within the polytomy zone; Fig. 2) and the overlap (Kutschera et al. 2014), Darwin’s finches (Lamichhaney of their divergence times with the K-Pg boundary in Jarvis et al. 2015), Ficedula flycatchers (Nater et al. 2015), gal- et al. (2014). In the meantime, the Jarvis et al. (2014)’s time lopheasants (Meiklejohn et al. 2016) and tomatoes (Pease tree was largely confirmed by recent independent analyses et al. 2016)]. Notably, none of these cases seem to be irre- (Claramunt & Cracraft 2015; Prum et al. 2015) whereas solvable or contain hard polytomies. However, I predict the much older dates estimated via mitochondrial or few that more examples of hard polytomies will be discovered nuclear loci (reviewed, e.g., by Brown & Van Tuinen 2011 once various types of densely sampled genome-scale data and van Tuinen 2009) seem somewhat outdated (but see are available for other super-rapid radiations. These might Acosta Hospitaleche & Gelfo 2015 and De Pietri et al. include, for example, gibbons (Veeramah et al. 2015), 2016). Although it was recently noted that current methods Hawaiian honeycreepers (Lerner et al. 2011), Lake Malawi are unable to confidently distinguish between events right cichlids (Takahashi et al. 2001), Lake Tanganyika cichlids before and right after the K-Pg boundary (Ksepka & Phil- (Meyer et al. 2015), Myotis bats (Platt et al. 2015), sylvioid lips 2015), these methods imply temporal proximity of the songbirds (Fregin et al. 2012), thrushes (Nylander et al. polytomy zone to the K-Pg boundary. If this assumption is 2008) and white eyes (Moyle et al. 2009). correct, then it seems plausible that in the wake of the K-Pg mass extinction of non-avian , archaic birds Conclusions and many other organisms, the super-rapid speciation of The Neoaves problem pictured in this perspective piece Neoaves began with an initial nine-taxon hard polytomy illustrates the necessity for three paradigm shifts avian phy- and subsequently slowed down into a bifurcating tree. logenomics as well as genome-scale phylogenomics in gen- There is another case of super-rapid speciation that eral: (i) Bootstrap and conventional Bayesian posterior appears to coincide with the K-Pg boundary, namely the probabilities should not be the only measures for topologi- emergence of the three major groups of placental mammals cal support. (ii) Every phylogenetic tree hypothesis should (O’Leary et al. 2013, but see Springer et al. 2013 and dos be accompanied by a phylogenetic network for visualization Reis et al. 2014). Various genome-scale studies have of conflicts. (iii) Hard polytomies exist in nature and should debated whether or not the relationships among Boreothe- be treated as the null hypothesis in the absence of repro- ria, Afrotheria and Xenarthra are resolvable as a bifurcating ducible tree topologies. tree (Murphy et al. 2007; Churakov et al. 2009; Nishihara In summary, the neoavian Tree of Life is now largely et al. 2009; Hallstrom€ & Janke 2010; Morgan et al. 2013; resolved, except for its deepest relationships which likely Romiguier et al. 2013; Tarver et al. 2016). The most recent constitute a nine-taxon hard polytomy. One may ask contribution to this controversy has argued that weak phy- whether the declaration of a hard polytomy should lead logenetic signal, not a hard polytomy, is causing the diffi- researchers to turn away from early neoavian relationships culty of resolving the root of placental mammals (Tarver and focus on other issues? On the contrary! Neoaves com- et al. 2016). Alternatively, only three bifurcating tree prise, to my knowledge, the first empirical example for a topologies are possible for a three-taxon polytomy of pla- hard polytomy in . The imminent genome sequenc- cental mammals (Felsenstein 1978) and I suggest that forc- ing of all ~10 500 bird species (Jarvis 2016) will provide an ing it on a bifurcating tree might stochastically yield one exciting system for studying the exact phylogenetic extent topology more often than the other two. But irrespective and genomic signatures of a hard polytomy. of whether or not there exists an early placental mammal polytomy, this situation is somewhat less complex than the Acknowledgements aforementioned 2 027 025 rooted bifurcating trees which I thank Lutz Bachmann, Per G. P. Ericson and Per Sund- are possible for a nine-taxon polytomy at the root of berg for the invitation to write this perspective piece. Fur- ̌ Neoaves. Thus, I hope that the future debate on the ther thanks go to Per Alstrom,€ David Cerny, Matthew W. Neoaves problem will be less about which species tree is Hahn, Erich D. Jarvis, John McCormack, Frank E. ‘correct’, and more about if there is such a thing as a fully Rheindt, Alexandre P. Selvatti and members of Hans Elle- bifurcating neoavian Tree of Life at all. In the meantime, gren’s lab and Jochen Wolf’s lab for numerous helpful dis- ̌ it is likely that we will see the publication of hundreds of cussions. I am grateful to Reto Burri, David Cerny, Verena highly discordant species trees that each will be hailed as E. Kutschera, Frank E. Rheindt and Alexandre P. Selvatti the ‘resolved’ one. for valuable comments on this manuscript. Further thanks In the recent era of phylogenomic research, the early go to the users of the online discussion platforms Twitter neoavian polytomy stands out for its complexity. There are and Reddit Science for inspiring conversations on the many other genome-scale studies that have resolved sub- topics of this manuscript. I am indebted to Jon Fjeldsa for stantial phylogenetic conflict in rapid radiations [e.g. bears the permission to use his bird paintings.

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