Accepted Manuscript

Short Communication

Outgroup effects on root position and tree topology in the AFLP phylogeny of a rapidly radiating lineage of

Paul C. Kirchberger, Kristina M. Sefc, Christian Sturmbauer, Stephan Koblmüller

PII: S1055-7903(13)00347-3 DOI: http://dx.doi.org/10.1016/j.ympev.2013.09.005 Reference: YMPEV 4706

To appear in: Molecular Phylogenetics and Evolution

Received Date: 5 December 2012 Revised Date: 4 September 2013 Accepted Date: 6 September 2013

Please cite this article as: Kirchberger, P.C., Sefc, K.M., Sturmbauer, C., Koblmüller, S., Outgroup effects on root position and tree topology in the AFLP phylogeny of a rapidly radiating lineage of cichlid fish, Molecular Phylogenetics and Evolution (2013), doi: http://dx.doi.org/10.1016/j.ympev.2013.09.005

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Outgroup effects on root position and tree topology in the AFLP phylogeny of a rapidly 2 radiating lineage of cichlid fish

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4 Paul C. Kirchbergera,b, Kristina M. Sefca, Christian Sturmbauera, Stephan Koblmüllera.*

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6 a Department of Zoology, Karl‐Franzens‐University Graz, Universitätsplatz 2, A‐8010 Graz, 7 Austria

8 b present address: Department of Biological Sciences, University of Alberta, Edmonton, AB, 9 Canada T6G 2E9

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11 * Corresponding author: Tel.:+43 316 380 3978; Fax: +43 316 380 9875; email: 12 stephan.koblmueller@uni‐graz.at (S. Koblmüller)

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14 Abstract

15 Phylogenetic analyses of rapid radiations are particularly challenging as short basal 16 branches and incomplete lineage sorting complicate phylogenetic inference. Multilocus 17 data of presence‐absence polymorphisms such as obtained by AFLP genotyping overcome 18 some of the difficulties, but also present their own intricacies. Here we analyze >1000 AFLP 19 markers to address the evolutionary history of the , a cichlid fish lineage 20 endemic to , and to test for potential effects of outgroup composition on 21 tree topology. The data support previous mitochondrial evidence on the tribe’s 22 by confirming the polyphyly of the and ‐ in contradiction to a recent 23 taxonomic revision ‐ nesting the genus within the Limnochromini. 24 Species relationships suggest that ecological segregation occurred during the rapid basal 25 radiation of the Limnochromini. The large phylogenetic distance between candidate 26 outgroup taxa and the Limnochromini radiation caused random outgroup effects. 27 Bootstrap support for ingroup nodes was lower in outgroup‐rooted than in midpoint‐ 28 rooted trees, and root positions and ingroup tree topologies varied in response to the

1 29 composition of the outgroup. These observations suggest that the predisposition for 30 homoplastic evolution makes AFLP‐based phylogenetic analyses particularly susceptible to 31 random biases introduced by too‐distant outgroup taxa.

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33 Keywords: ancient incomplete lineage sorting; Cichlidae; Limnochromini; outgroup rooting; 34 rapid radiation; Lake Tanganyika

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36 1. Introduction

37 Adaptive radiations such as the Darwin’s finches on the Galapagos archipelago, the Hawaiian 38 silver swords, the Caribbean anoles lizards, and the cichlid fish of the East African Great 39 Lakes provide opportunities for studying the processes underlying rapid speciation (Schluter, 40 2000; Brakefield, 2006; Salzburger, 2009). Yet, resolving the phylogeny of adaptive radiations 41 still remains a challenge. Due to the extreme rapidity of the radiations, lineage sorting often 42 lags behind cladogenesis, complicating phylogenetic inference based on single or a few 43 molecular markers (Pamilo and Nei, 1988; Maddison and Knowles, 2006). Furthermore, the 44 short basal branches typical for rapid radiations can make the phylogenetic reconstructions 45 sensitive to the choice of outgroup taxa. In particular if the phylogenetic distance between 46 outgroup and ingroup taxa is large, there is a high likelihood of homoplasy between ingroup 47 and outgroup taxa. Consequently, the outgroup may attach randomly to the ingroup and 48 bias the inferred ingroup tree topology (Wheeler, 1990; Huelsenbeck et al., 2002; Rota‐ 49 Stabelli & Telford, 2008; Rosenfeld et al., 2012). The outgroup effect on tree topology is well 50 established for sequence data, but has so far received no attention in connection with 51 multilocus binary presence‐absence data such as those obtained by AFLP genotyping. 52 However, the elevated potential for homoplasy in this type of data (Althoff et al., 2007; 53 García‐Pereira et al., 2010) implies that random outgroup effects on AFLP tree topologies are 54 at least as likely as in analyses of DNA sequences. Despite certain other AFLP‐specific 55 shortcomings, which are associated with low information content, dominance and phenetics 56 (e.g. García‐Pereira et al., 2010), AFLP data nonetheless harbor potential for improved 57 species tree approximation as the phylogenetic signal is integrated from numerous loci 58 distributed throughout the genome, thus reducing the confounding effects of incomplete

2 59 lineage sorting and hybridization on phylogenetic inference (Koopman, 2005). Consequently, 60 AFLP markers are frequently being applied to taxa originating from rapid radiations (Ogden 61 and Thorpe, 2002; Schliewen and Klee, 2004; Herder et al., 2006; Fink et al., 2010; Geiger et 62 al., 2010; Koblmüller et al. 2010; Sturmbauer et al., 2010; Joyce et al., 2011).

63 The present study addresses the phylogeny of the Limnochromini (Cichlidae, Perciformes), 64 an endemic cichlid fish tribe of Lake Tanganyika. The deep‐water habitat of the East African 65 rift lake formed 5‐6 million years ago (Tiercelin and Mondeguer, 1991) and provided a 66 prolific environment for intralacustrine speciation (Koblmüller et al., 2008a). The 67 Limnochromini originated in the course of a rapid radiation into several mouthbrooding 68 cichlid tribes (the C‐lineage, Clabaut et al., 2005), and subsequently diversified into 10 69 benthic, mainly deepwater‐dwelling (up to depths >100 m), species (Coulter, 1991; Konings, 70 1998; Duftner et al. 2005). Previous work on the Limnochromini entailed repeated 71 taxonomic revisions. Initially, the tribe included 13 species in 8 genera, Benthochromis, 72 Baileychromis, , Greenwoodochromis, Limnochromis, Reganochromis, 73 Tangachromis and Triglachromis (Poll 1986). Extending the morphological data, Takahashi 74 (2003) erected two new tribes, the Benthochromini and the Greenwoodochromini, for the 75 corresponding genera, and excluded from the Limnochromini. The 76 establishment of the Benthochromini and the exclusion of G. pfefferi were later 77 corroborated by mtDNA data, whereas the validity of Greenwoodochromini as a separate 78 tribe was rejected (Duftner et al. 2005; Fig. 1a). Since questions on the taxonomy and 79 evolutionary history of the Limnochromini have not been fully settled by the existing data, 80 additional information was sought by employing AFLP markers in the present study. The 81 phylogenetically unresolved radiation of the C‐lineage, from which the Limnochromini 82 originate (Clabaut et al. 2005), made it difficult to choose a suitable set of outgroup taxa 83 from among the C‐lineage tribes. Because of the large phylogenetic distance between the 84 Limnochromini and the candidate outgroup taxa (Duftner et al., 2005), particular attention 85 was paid to the effect of outgroup composition on ingroup topology.

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87 2. Material and Methods

88 2.1 Ingroup and outgroup taxa

3 89 The Limnochromini were represented by 31 specimens representing all described species 90 of the tribe, except for the small deepwater‐dwelling Tangachromis dhanisi, which is seldom 91 caught and rarely found in the aquarium trade. Outgroup effects were examined by 92 comparing a midpoint‐rooted tree with trees rooted with different sets of outgroup taxa, 93 which were assembled from a pool of 17 species in six tribes representing the entire C‐ 94 lineage (see “Phylogenetic analyses”).

95 Fish were either caught on site using gill nets, acquired from aquarium trade or bought 96 from local fishermen (Supplementary Table 1).

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98 2.2 DNA extraction and AFLP data collection

99 DNA extraction and AFLP genotyping (ten primer combinations for selective 100 amplification: EcoRI‐ACA/MseI‐CAA, EcoRI‐ACA/MseI‐CAG, EcoRI‐ACA/MseI‐CAC, EcoRI‐ 101 ACA/MseI‐CAT, EcoRI‐ACT/MseI‐CAA, EcoRI‐ACT/MseI‐CAG, EcoRI‐ACT/MseI‐CAC, EcoRI‐ 102 ACT/MseI‐CAT, EcoRI‐ACC/MseI‐CAA, EcoRI‐ACT/MseI‐CAC) were performed as described in 103 Egger et al. (2007). Initial scoring of electropherograms (size range of 50‐300 bps for each 104 primer combination; bin width was adjusted manually for each peak) was done using 105 GeneMapper 3.7 (Applied Biosystems). AFLPScore 1.4a (Whitlock et al., 2008) was then used 106 to convert the un‐normalized peak‐height data into a presence/absence matrix. 20 replicate 107 samples were used to estimate the mismatch error rate, and a rate of <2% was set as 108 threshold for the inclusion of data in the final matrix.

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110 2.3 Phylogenetic analyses

111 PAUP 4.05b (Swofford, 2000) was used to construct a neighbor joining (NJ) tree based on 112 Nei‐Li distances (Nei and Li, 1979) and estimate statistical support from 1000 bootstrap 113 replicates. To test for effects of outgroup composition, analyses were conducted with only 114 ingroup taxa (the “ingroup dataset”) and with eight different sets of outgroup taxa, i.e. the 115 “full dataset” using the complete outgroup (17 taxa from six tribes, encompassing the 116 diversity of the entire C‐lineage), and seven datasets with smaller outgroups (Ectodini; a 117 monophyletic clade of Perissodini + Benthochromini + Cyprichromini; Cyprichromini;

4 118 Benthochromini; Perissodini; Cyphotilapiini; ). For the ingroup dataset, a Neighbor‐ 119 Net analysis based on a Nei & Li (1979) distance matrix was conducted in SplitsTree v.4.10 120 (Huson and Bryant, 2006) as an explorative approach for identifying conflicting phylogenetic 121 signal in the data.

122 To test for consistency between mtDNA‐ and AFLP‐based tree topologies, we evaluated the 123 fit of the mtDNA sequence data (Duftner et al., 2005) to the unrooted NJ tree topology 124 derived from ingroup AFLP data, by constraining a maximum likelihood (ML) tree search with 125 the substitution model GTR+I+G (Duftner et al., 2005) to the AFLP tree. The Shimodaira ‐ 126 Hasegawa (SH) test (Shimodaira and Hasegawa, 1999; full optimization, 10,000 bootstrap 127 replicates; implemented in PAUP) was then used to test whether the likelihood of the 128 mtDNA data was significantly lower under the AFLP‐NJ topology than under an 129 unconstrained topology optimized from the mtDNA data.

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131 3. Results

132 3.2. Phylogenetic reconstruction of the ingroup dataset

133 Phylogenetic analyses were based on 1128 AFLP marker loci. Bootstrap support for the 134 monophyly of all species and for the reconstructed species clades was relatively good and 135 ranged from 71 to 100% (Fig. 1b). Polyphyly of the genus Limnochromis was confirmed, as L. 136 auritus was most closely related to T. otostigma, and L. abeelei and L. staneri clustered with 137 the genus Greenwoodochromis. Gnathochromis permaxillaris resulted as sister group to the 138 L. abeelei/L. staneri/Greenwoodochromis clade and B. centropomoides plus R. calliurus 139 constituted a well supported monophylum. Mid‐point rooting placed T. otostigma + L. 140 auritus as sister to the remaining limochromines. The SH‐test (p=0.059) revealed no 141 significant differences between the AFLP topology of the ingroup dataset and the 142 phylogenetic tree reconstructed from mitochondrial DNA sequence data by Duftner et al. 143 (2005).

144 The phylogenetic relationships inferred by the NeighborNet analysis (Fig. 1c; least 145 squares fit value = 95.633) were consistent with the tree‐based inferences. Most of the 146 internal splits in the network are represented by boxes, which is indicative of conflicting

5 147 signals in the data. Incomplete or differential lineage sorting of marker loci as well as 148 reticulation due to hybridization can underlie these ambiguities.

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150 3.1. Outgroup effects

151 The NJ analysis of the full dataset (Fig. 2) confirmed the monophyly of the tribe 152 Limnochromini (including the Greenwoodochromini). In contrast to the analysis of the 153 ingroup dataset, statistical support for most of the basal nodes within the Limnochromini 154 was low. The eight tested outgroup sets gave rise to five different tree topologies, with 155 variability in root position, ingroup topology and bootstrap support (Fig. 2).

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157 4. Discussion

158 4.1. Phylogenetic inference, rapid radiation and ecological diversification

159 Irrespective of the composition of the outgroup in the phylogenetic reconstruction, our AFLP 160 data corroborated the mitochondrial evidence (Duftner et al. 2005) for a monophyletic clade 161 consisting of the genera Greenwoodochromis, Limnochromis, Baileychromis, Reganochromis, 162 Triglachromis and Gnathochromis permaxillaris [the nominally congeneric G. pfefferi belongs 163 to the tribe Tropheini (Duftner et al. 2005), and a taxonomic revision is required]. A 164 monotypic genus, Tangachromis, was not included in the genetic studies, but clustered 165 within the Limnochromini according to morphological data (Takahashi 2003). The derived 166 monophyletic position of the genus Greenwoodochromis in both our nuclear and Duftner et 167 al.’s (2005) mitochondrial phylogeny strongly supports its retention within Limnochromini 168 and discards an earlier classification as Greenwoodochromini (Takahashi 2003). The 169 eponymous genus of the Limnochromini, Limnochromis, originally comprised multiple 170 species, most of which have meanwhile been assigned to alternative genera (Triglachromis, 171 Gnathochromis, Tangachromis, Greenwoodochromis; Poll, 1986; Takahashi, 2003). The 172 taxonomy of the remaining three Limnochromis species remains problematic, since neither 173 nuclear nor mitochondrial data support a monophyletic relationship, but separate L. auritus 174 from L. abeelei and L. staneri in all phylogenetic reconstructions (Figs. 1, 2).

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6 176 The root of the Limnochromini tree could not be determined in this study. Different sets of 177 outgroup taxa yielded different solutions, and midpoint rooting of the ingroup did not 178 resolve the basal branching order. Both mtDNA and AFLP data suggest an almost 179 simultaneous diversification of several ecologically distinct lineages at the onset of the 180 Limnochromini radiation. Some well supported clades were resolved differently in the AFLP 181 and mtDNA topologies. However, according to the SH test, these differences were not 182 significant. Despite some well known AFLP‐specific shortcomings, the genome‐wide 183 distribution of AFLP markers minimizes locus‐specific effects potentially problematic for

184 analyses of single or just a few loci (Koopman, 2005; Fink et al., 2010). In case of conflicting 185 outcomes, it is therefore usually the multi‐locus reconstruction that is considered a better 186 approximation of the true evolutionary history than the mitochondrial gene tree (e.g., 187 Kingston et al. 2009; Koblmüller et al. 2007a, 2010; Fink et al., 2010). According to the AFLP 188 ingroup dataset, which is probably the most reliable representation of ingroup relationships 189 given the obvious outgroup effect, one of the major lineages comprises Baileychromis and 190 Reganochromis, benthic predators with elongated bodies and large eyes, which prey upon 191 shrimp and small fish on deep muddy bottoms (Coulter, 1991). A second lineage consists of a 192 single species, G. permaxillaris, a deepwater zooplankton feeder with a strongly enlarged 193 upper lip and protractile mouth apparatus (Coulter, 1991; Konings, 1998). In the third 194 lineage, Greenwoodochromis christyi, G. bellcrossi, L. staneri and L. abeelei are all true 195 benthic deepwater fish. Both Limnochromis species are generalized predators on muddy 196 flats, feeding on small fish, molluscs and arthropods (Coulter, 1991). The two 197 Greenwoodochromis species occupy a slightly different habitat and feed on invertebrates 198 and small fish in the transition between sandy and rocky bottoms (Konings, 1998). Finally, a 199 somewhat less well supported clade comprises L. auritus and T. otostigma, whose habitat 200 extends from muddy bottoms at the shores far into the depths of the lake (Coulter, 1991). 201 is the only known detritus feeder among the Lake Tanganyika 202 (Coulter, 1991), and uses the pectoral fin ray tips as sensory organs to screen the 203 muddy ground, which is swallowed in large quantities when potential food items are 204 detected.

205 The ecological segregation during the radiation of the Limnochromini is consistent with 206 the proposition that in adaptive radiations, early bursts of cladogenesis in response to 207 ecological opportunity are followed by a decline in the rate of speciation due to the filling of

7 208 niches (Losos 2010). Indeed, cladogenesis among and within Lake Tanganyika’s cichlid tribes 209 appears to follow this pattern (Seehausen, 2006; Koblmüller et al. 2008a; Sturmbauer et al. 210 2011), and in particular in lineages unaffected by physical barriers to gene flow, ecological 211 segregation may be an important promoter of diversification (Koblmüller et al., 2005; Kidd et 212 al., 2006; Kirchberger et al., 2012).

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214 4.2. Outgroup effect on root position, tree topology and bootstrap support

215 Outgroup choice is a crucial step in phylogenetic analysis, in particular when potential 216 outgroup taxa are only distantly related to the ingroup. Regarding phylogenetic inference 217 from DNA and protein sequence data, numerous studies showed that highly divergent 218 outgroup taxa can underlie a loss of phylogenetic signal and can generate random outgroup 219 effects as well as systematic biases by attracting fast evolving and compositionally similar 220 ingroup species towards the base of the tree and thus heavily impact tree topology and root 221 position (Wheeler, 1990; Foster and Hickey, 1999; Huelsenbeck et al., 2002; Shavit et al., 222 2007; Rota‐Stabelli and Telford, 2008; Rosenfeld et al., 2012). Surprisingly, potential effects 223 of outgroup choice on phyogenetic inference have been largely ignored in AFLP‐based 224 studies. However, homoplastic non‐homologous bands in outgroup and ingroup taxa might 225 lead to the same effects as homoplasy in DNA and protein sequences. Indeed, analyses of 226 simulated AFLP data provided evidence for a dramatic decrease of phylogenetic accuracy, 227 both with respect to tree topology and relative branch length, with increasing divergence 228 within datasets, which was due to a rapid accumulation of non‐homologous AFLP bands 229 (e.g., Althoff et al., 2007; García‐Pereira et al., 2010). Likewise, examination of empirical data 230 shed doubt on the usefulness of AFLPs for the resolution of deep phylogenetic relationships 231 (Near and Keck, 2012).

232 The impact of outgroup composition on root position, bootstrap support and tree topology 233 in our AFLP dataset likely originates from the combination of the large genetic distance 234 between the available outgroup taxa and the ingroup in the one hand, and the short basal 235 branches within the ingroup on the other hand. Moreover, considerable differences in 236 branch lengths between ingroup and some outgroup taxa might promote random outgroup 237 effects (Wheeler, 1990; Huelsenbeck et al., 2002). With an age of 5‐6 MY (or more, 238 contingent on the calibration; Genner et al., 2007; Schwarzer et al., 2009; but see Koblmüller 8 239 et al., 2008ab), the split between the Limnochromini and the outgroup taxa is approximately 240 twice as old as the most recent common ancestor of the extant limnochromines, from which 241 the major limnochromine lineages radiated almost simultaneously (Duftner et al., 2005; 242 present data). This allowed different sets of outgroup taxa to attach to different branches of 243 the ingroup and pull certain ingroup taxa towards the base of the tree, which naturally 244 affected bootstrap support and topology of the ingroup clades.

245 Prompted by these observations, we examined whether previous AFLP‐based phylogenetic 246 reconstructions of various Lake Tanganyika cichlid lineages were robust to the removal of 247 the outgroup. No substantial outgroup effect on inferences of ingroup relationships was 248 detected in these datasets (Koblmüller et al., 2007b, 2010; Kirchberger et al., 2012), as 249 changes in branching order concerned only those clades, which had low statistical support 250 irrespective of whether or not the outgroup was included in the analyses (data not shown). 251 Notably, in none of these datasets was the basal ingroup radiation as rapid and the relative 252 divergence from the outgroup as large as it was in the Limnochromini dataset. However, 253 outgroup effects may underlie the different AFLP tree topologies of the Perissodini radiation 254 obtained by Koblmüller et al. (2007b) and Takahashi et al. (2007), as the latter study used 255 some highly divergent outgroup taxa.

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257 5. Conclusions

258 The effect of outgroup composition on our analyses prompt us to call for increased 259 awareness of potential outgroup problems in work with empirical data, and to suggest that 260 additional theoretical work on the causes and consequences of outgroup effects is 261 warranted, specifically with regard to AFLP data. Outgroup choice is often confounded by 262 the lack of sufficiently closely related taxa and the present study also demonstrates that 263 particular care has to be taken in such instances. With regard to implications for the 264 taxonomy of Limnochromini, AFLP data are in accordance with previous mtDNA sequence 265 data (Duftner et al. 2005) in that the genus Greenwoodochromis is nested in the 266 Limnochromini rather than constituting a separate tribe, as well as regarding the polyphyly 267 of the genus Limnochromis. Finally, ecological segregation appears to have occurred at the 268 early stage of the limnochromine radiation.

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270 6. Acknowledgements

271 We thank W. Salzburger, B. Egger, A. Indermaur and M. Muschick for providing DNA 272 samples. We further thank W. Gessl (www.pisces.at), W. Salzburger, B. Egger, A. Indermaur 273 and M. Muschick for the photographs of the fish used in this study (Table S1). This study was 274 supported by the Theodor Körner Foundation (to S.K.) and the Austrian Science Fund (Grant 275 P20883 to K.M.S.).

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13 449 Whitlock, R., Hipperson, Mannarelli, M., Butlin, R.K., Burke, T., 2008. An objective, rapid and 450 reproducible method for scoring AFLP peak‐height data that minimizes genotyping error. 451 Mol. Ecol. Res. 8, 725‐735. 452

14 453 Fig. 1: Phylogenetic relationships of the Limnochromini and tree‐based tests for ancient 454 hybridization. A) Mitochondrial phylogeny of the Limnochromini according to Duftner et al. 455 (2005). Solid lines represent the topology obtained by maximum likelihood, dotted lines 456 show the strict consensus of neighbor‐joining, maximum likelihood and maximum parsimony 457 trees. B) Nuclear (AFLP) phylogeny of the Limnochromini; NJ tree based on Nei & Li distances 458 (Nei and Li, 1979) with bootstrap values shown at the branches. TRO, Tropheini; CYPH, 459 Cyphotilapiini; PER, Perissodini; BEN, Benthochromini, CYPR, Cyprichromini, ECT, Ectodini; 460 GRE, Greenwoodochromini. Letters A, B, C, D, E, F in the NJ tree label nodes/clades for

461 homoplasy excess test (Fig. 1d). C) Neighbor‐Net graph based on Nei & Li distances.

462 Fig. 2: Random outgroup effect on root position, tree topology and bootstrap support. NJ 463 trees based on Nei & Li distances (Nei and Li, 1979) using different sets of outgroup taxa, 464 with bootstrap values shown at the branches.

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17 467 Highlights 468 469 ● AFLPs resolve the phylogeny of Lake Tanganyika’s endemic benthic deepwater cichlid 470 lineage 471 ● The recently proposed tribe Greenwoodochromini is nested within the Limnochromini 472 ● The Limnochromini underwent rapid initial radiation into eco‐morphologically distinct 473 lineages 474 ● Large phylogenetic distances between candidate outgroup taxa and the Limnochromini 475 radiation cause random outgroup effects. 476

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