Accepted Manuscript Short Communication Outgroup effects on root position and tree topology in the AFLP phylogeny of a rapidly radiating lineage of cichlid fish 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 3 4 Paul C. Kirchbergera,b, Kristina M. Sefca, Christian Sturmbauera, Stephan Koblmüllera.* 5 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 10 11 * Corresponding author: Tel.:+43 316 380 3978; Fax: +43 316 380 9875; email: 12 stephan.koblmueller@uni‐graz.at (S. Koblmüller) 13 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 Limnochromini, a cichlid fish lineage 20 endemic to Lake Tanganyika, and to test for potential effects of outgroup composition on 21 tree topology. The data support previous mitochondrial evidence on the tribe’s taxonomy 22 by confirming the polyphyly of the genus Limnochromis and ‐ in contradiction to a recent 23 taxonomic revision ‐ nesting the genus Greenwoodochromis 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. 32 33 Keywords: ancient incomplete lineage sorting; Cichlidae; Limnochromini; outgroup rooting; 34 rapid radiation; Lake Tanganyika 35 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, Gnathochromis, 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 Gnathochromis pfefferi 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. 86 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). 97 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. 109 110 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; Tropheini).