RESEARCH ARTICLE AMERICAN JOURNAL OF BOTANY Widespread paleopolyploidy, gene tree confl ict, and recalcitrant relationships among the carnivorous Caryophyllales 1 Joseph F. Walker 2,5 , Ya Yang 2,6 , Michael J. Moore 3 , Jessica Mikenas3 , Alfonso Timoneda 4 , Samuel F. Brockington 4 , and Stephen A. Smith 2,5 PREMISE OF STUDY: The carnivorous members of the large, hyperdiverse Caryophyllales (e.g., Venus fl ytrap, sundews, and Nepenthes pitcher plants) repre- sent perhaps the oldest and most diverse lineage of carnivorous plants. However, despite numerous studies seeking to elucidate their evolutionary rela- tionships, the early-diverging relationships remain unresolved. METHODS: To explore the utility of phylogenomic data sets for resolving relationships among the carnivorous Caryophyllales, we sequenced 10 transcrip- tomes, including all the carnivorous genera except those in the rare West African liana family Dioncophyllaceae. We used a variety of methods to infer the species tree, examine gene tree confl ict, and infer paleopolyploidy events. KEY RESULTS: Phylogenomic analyses supported the monophyly of the carnivorous Caryophyllales, with a crown age of 68–83 million years. In contrast to previous analyses, we recovered the remaining noncore Caryophyllales as nonmonophyletic, although the node supporting this relationship contained a signifi cant amount of gene tree discordance. We present evidence that the clade contains at least seven independent paleopolyploidy events, previously unresolved nodes from the literature have high levels of gene tree confl ict, and taxon sampling infl uences topology even in a phylogenomic data set, re- gardless of the use of coalescent or supermatrix methods. CONCLUSIONS: Our data demonstrate the importance of carefully considering gene tree confl ict and taxon sampling in phylogenomic analyses. Moreover, they provide a remarkable example of the propensity for paleopolyploidy in angiosperms, with at least seven such events in a clade of less than 2500 species. KEY WORDS Caryophyllales; gene tree confl ict; paleopolyploidy; phylogenomics; plant carnivory Carnivory in plants has long fascinated both the general public and soils that would otherwise be unsuitable for plant life by obtaining evolutionary biologists. Charles Darwin himself dedicated an entire nutrients unavailable in the soil through the digestion of animals. volume to carnivorous species in his Insectivorous Plants ( Darwin, Across angiosperms, carnivory is hypothesized to have indepen- 1875 ). Th e wide array of traps that are used to catch insects and dently evolved at least nine times ( Givnish, 2015). One of these events is other prey items make carnivorous plants some of the most mor- thought to have occurred relatively early on (~83 million years ago phologically diverse plants on Earth ( Ellison and Gotelli, 2001 ; [Ma]) in the noncore Caryophyllales ( Magallón et al., 2015 ), giving rise Heubl et al., 2006 ). Th ese plants are able to occupy nutrient poor to a “carnivorous clade” consisting of the fully carnivorous families Droseraceae, Drosophyllaceae, and Nepenthaceae, the small noncar- 1 Manuscript received 27 February 2017; revision accepted 12 May 2017. nivorous African family Ancistrocladaceae, and the rare west African 2 Department of Ecology & Evolutionary Biology, University of Michigan, 830 North family Dioncophyllaceae, which includes the unusual carnivorous University Avenue, Ann Arbor, Michigan 48109-1048 USA; 3 Department of Biology, Oberlin College, Science Center K111, 119 Woodland Street, liana Triphyophyllum peltatum and two other monotypic, noncarnivo- Oberlin, Ohio 44074-1097 USA; and rous genera (Dioncophyllum and Habropetalum ) ( Albert et al., 1992 ; 4 Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK Meimberg et al., 2000 ; Brockington et al., 2009 ; Soltis et al., 2011 ; 5 Authors for correspondence (e-mails: [email protected]; [email protected]) J.F.W. Hernández-Ledesma et al., 2015 ). Th e carnivorous clade of Caryo- ORCID id: 0000-0003-2928-8899 6 Present address: Department of Plant and Microbial Biology, University of Minnesota- phyllales comprises approximately 250 of the estimated 600 species of Twin Cities, 1445 Gortner Avenue, St. Paul, MN 55108 USA carnivorous angiosperms ( Heubl et al., 2006 ; Ellison and Gotelli, https://doi.org/10.3732/ajb.1700083 2009 ) and includes a diverse assemblage of trap plants and pitcher 858 • AMERICAN JOURNAL OF BOTANY 104 (6): 858 – 867 , 2017; http://www.amjbot.org/ © 2017 Walker et al. Published by the Botanical Society of America. This work is licensed under a Creative Commons Attribution License (CC-BY). JUNE 2017 , VOLUME 104 • WALKER ET AL. —PHYLOGENOMIC ANALYSIS OF CARNIVOROUS CARYOPHYLLALES • 859 plants that occupy a wide range of ecosystems, from the fully aquatic this article) (Bioproject: PRJNA350559). Assembled sequences, Aldrovanda vesiculosa to desert species of Drosera to the rainforest data fi les, programs, alignments, and trees are available from Dryad liana Triphyophyllum. Moreover, carnivory also appears to have been (data available from the Dryad Digital Repository: http://dx.doi. lost 1–3 times (Heubl et al., 2006 ) within the carnivorous clade, in- org/10.5061/dryad.vn730 ). cluding in the ancestor of the 16 species of Ancistrocladaceae ( Taylor et al., 2005 ) as well as in the ancestors of Dioncophyllum and Hab- Taxon sampling, tissue collection, sequencing, and data assem- ropetalum in Dioncophyllaceae ( Meimberg et al., 2000 ). bly— Tissue collection, RNA extraction, library preparation, and Despite broad appeal and interest, the evolutionary relationships quality control were carried out using a previously developed in the noncore Caryophyllales remain ambiguous, with studies seek- workflow ( Yang et al., 2017 ). Transcriptomes of eight noncore ing to resolve these relationships oft en resulting in individually well- Caryophyllales families representing nearly all of the major lineages supported but mutually confl icting topologies ( Meimberg et al., of noncore Caryophyllales were included in this study (Appendix S1). 2000 ; Cameron et al., 2002 ; Brockington et al., 2009 ; Hernández- Th e transcriptomes of Dionaea muscipula , Aldrovanda vesiculosa , Ledesma et al., 2015 ). Much of this confl ict involves the earliest Nepenthes ampullaria , and Reaumuria trigyna were downloaded branch in the noncore carnivorous clade, with studies fi nding Ne- from the NCBI Sequence Read Archive (accessions SRX1376794; penthaceae as sister to the remaining lineages ( Hernández-Ledesma SRR1979677; SRR2666506, SRR2866512, and SRR2866533 com- et al., 2015 ), others fi nding Droseraceae as sister to the rest of the bined; and SRX105466 and SRX099851 combined; respectively) group ( Meimberg et al., 2000 ) and yet others fi nding Droseraceae to (Dang et al., 2013 ; Brockington et al., 2015 ; Bemm et al., 2016; Wan be sister to the Nepenthaceae ( Brockington et al., 2009 ). Th e strong Zakaria et al., 2016 ). Th e assembly used for Frankenia laevis was the support for confl icting topologies from diff erent studies may be ex- same as that used by Yang et al. (2015) and can be found in Dryad plained by the reliance on one or a few genes leading to systematic ( http://dx.doi.org/10.5061/dryad.33m48 ). The genomes of Beta error ( Maddison, 1997 ; Rokas et al., 2003 ). Th is type of error can vulgaris (RefBeet-1.2) and Spinacia oleracea were downloaded arise from a variety of sources, including, but not limited to, incom- from Th e Beta vulgaris Resource ( http://bvseq.molgen.mpg.de/ plete lineage sorting, horizontal gene transfer, hybridization, and Genome/Download/index.shtml ; accessed July 10 2015) ( Dohm et al., hidden paralogy ( Galtier and Daubin, 2008 ). Untangling these pro- 2014 ). We generated 10 new transcriptomes for this study from cesses has proven to be a challenge and adds a strong level of com- fresh tissue collected from Drosera binata , Nepenthes alata , Ancis- plexity to phylogenomic analyses ( Smith et al., 2015 ). trocladus robertsoniorum , Plumbago auriculata , Ruprechtia salici- Transcriptomes have proven to be a powerful source of data for folia , and Drosophyllum lusitanicum . Th e D. binata and N. alata understanding this complexity and have helped provide insight data were also collected from trap tissue at three diff erent develop- into the evolutionary history of nonmodel species ( Dunn et al., mental stages (Appendix S1). Th e plant tissues were fl ash frozen in 2008 ; Cannon et al., 2015 ; Yang et al., 2015 ). Th e thousands of liquid nitrogen and stored at −80 °C. RNAs were extracted from the genes typically sequenced in a transcriptome provide a means of leaf tissue using the Ambion PureLink Plant RNA Reagent (Th er- identifying gene duplications and paleopolyploidy events ( Cannon moFisher Scientifi c, Waltham, Massachusetts, USA) following the et al., 2015 ; Yang et al., 2015 ; Barker et al., 2016 ), which may clarify manufacturer’s instructions and quantifi ed using the Agilent 2100 whether such events have been major drivers of evolutionary nov- Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). elty ( Ohno et al., 1968 ; Soltis et al., 2014 ). Moreover, analyses of Sequence libraries were prepared using either the TruSeq Stranded gene tree concordance and confl ict allow for a better understand- mRNA Library Prep Kit (Illumina, San Diego, California, USA) or ing of the formation of species relationships and the complexity the KAPA stranded mRNA library preparation