LETTERS TO THE EDITOR Pisciforma, Setisura, and Furcatergalia (Order: Ephemeroptera) Are Not Monophyletic Based on 18S rDNA Sequences: A Response to Sun et al. (2006) 1 2 3 T. HEATH OGDEN, MICHEL SARTORI, AND MICHAEL F. WHITING Sun et al. (2006) recently published an analysis of able on GenBank October 2003. However, they chose phylogenetic relationships of the major lineages of not to include 34 other mayßy 18S rDNA sequences mayßies (Ephemeroptera). Their study used partial that were available 18 mo before submission of their 18S rDNA sequences (Ϸ583 nucleotides), which were manuscript (sequences available October 2003; their analyzed via parsimony to obtain a molecular phylo- manuscript was submitted 1 March 2005). If the au- genetic hypothesis. Their study included 23 mayßy thors had included these additional taxa, they would species, representing 20 families. They aligned the have increased their generic and familial level sam- DNA sequences via default settings in Clustal and pling to include lineages such as Leptohyphidae, Pota- reconstructed a tree by using parsimony in PAUP*. manthidae, Behningiidae, Neoephemeridae, Epheme- However, this tree was not presented in the article, rellidae, and Euthyplociidae. Additionally, there were nor have they made the topology or alignment avail- 194 sequences available (as of 1 March 2005) for other able despite multiple requests. This molecular tree molecular markers, aside from 18S, that could have was compared with previous hypotheses based on been used to investigate higher level relationships. morphological data to “test” (but see below) which These include Cytochrome oxidase I, Elongation fac- morphology-based relationships were not signiÞ- tor I-␣, Histone 3, 28S rDNA, and 16S mitochondrial cantly different from the molecular topology. Al- rDNA. though molecular data can help shed light on many of The purpose of this short reply is to reexamine the the fundamental questions in insect phylogenetics, it Sun et al. (2006) analysis to investigate whether the is important to perform analyses correctly and to ac- data they present do in fact support 1) the monophyly curately report results in a way that allows subsequent of the suborders Setisura, Pisciforma, and Furcater- validation. galia; 2) the placement of Baetiscidae as the sister Sun et al. (2006) provided an adequate review of the lineage to all other represented clades; and 3) the major traditional hypotheses concerning higher level elevation of Pseudiron and Arthroplea as separate classiÞcation and relationships among mayßies. Some monophyletic families. We will then present an anal- of these hypotheses are based on cladistic analysis of ysis based on all the 18S mayßy data that were avail- morphology, but others are intuitive phylogenies that able to the authors to determine whether inclusion of were not derived from any formal repeatable and these additional data result in conclusions contrary to objective analysis. These authors failed to report pre- those reported. We further examine their analytical viously published molecular hypotheses that shed methodology and focus on their use of the Shimo- light on mayßy phylogeny, many of which disagree dairaÐHasegawa test to conclude that the groups de- with their results. For example, there have been pre- Þned via morphology are not signiÞcantly different viously published molecular studies investigating from the molecular topology. All of these results are higher level relationships among mayßies and relatives then discussed in light of a more comprehensive phy- (Hovmo¨ller et al. 2002, Ogden and Whiting 2003, Ball logenetic analysis of mayßies as presented in Ogden et al. 2005, Danforth et al. 2005) and investigating and Whiting (2005). relationships below the family level (Tojo and Mat- sukawa 2003, Monaghan et al. 2005, Rebora et al. 2005). All of these analyses include additional data and taxa Materials and Methods that could have been included in the Sun et al. (2006) Sun et al. (2006) have did not provide us their analysis to make a more robust estimate of mayßy original sequence alignment, molecular topologies, or phylogeny. details related to parameters used in the reconstruc- It is clear that the authors knew other ephemerop- tion of molecular topologies. Consequently, we down- teran sequences were deposited on GenBank, because loaded their sequences from GenBank (Table 1) in an they downloaded and included sequence data for the attempt to reproduce their analysis. Because the au- genus Rallidens (Rallidentidae), which became avail- thors were brief in their description of some analytical details, certain analytical parameters had to be as- The authors of Sun et al. (2006) were notiÞed of this critique of their sumed. For example, we treated gaps as missing data paper, but did not submit a response. and all characters were equally weighted in our par- 1 Corresponding author: Department of Biological Sciences, Idaho simony analysis. We constructed a second data set that State University, Pocatello, ID 83209 (e-mail: [email protected]). consisted of these authorÕs data and all mayßy 18S 2 Museum of Zoology, P.O. Box, CH-1014, Lausanne, Switzerland. 3 Department of Integrative Biology, Brigham Young University, rDNA sequences that had been deposited on 401 WIDB, Provo, UT 84602. GenBank as of 1 October 2003, to gauge whether the 0013-8746/08/0001Ð0006$04.00/0 ᭧ 2008 Entomological Society of America 2ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 1 Table 1. Taxonomic sampling and GenBank accession addseq ϭ random nreps ϭ 5 nchuck ϭ 10 chuck- numbers score ϭ 1) in PAUP* version 4.0b10 (Swofford 2002). The Templeton (Templeton 1983, Larson 1994) and Family Genus species GenBank winning-sites tests (Prager and Wilson 1988) were Lepismatidae Lepisma sp. AF005458 used to compare Sun et al. (2006) accepted intuitive Acanthametropodidae Analetris eximia AY338697 Analetris eximia DQ648716 topologies to the most parsimonious tree (or consen- Ameletidae Ameletus sp. AY338712 sus tree) computed in our reanalysis of their se- Ameletus celer DQ648730 quences. Ameletopsidae Chaquihua sp. AY338715 Sun et al. (2006) chose to use the ShimodairaÐ Chiloporter sp. DQ648721 Ametropodidae Ametropus neavei AY338700 Hasegawa (S/H) test (Shimodaira and Hasegawa Ametropus neavei DQ648734 1999) to investigate whether their intuitive hypothe- Arthropleidae Arthroplea bipunctata DQ648727 ses of mayßy relationships were signiÞcantly different Baetidae Baetis bicaudatus DQ648719 from the tree they reconstructed from the molecular Baetis buceratus AF461248 Baetis sp. AY338695 data. Sun et al. (2006) compared their 18S molecular Callibaetis ferrugineus DQ648714 topology to each of their intuitive topologies one at a Callibaeits ferrugineus AF370791 time. When the test showed that their intuitive phy- Centroptilum luteolum AF461251 logeny was not signiÞcantly different from the 18S Cloeon dipterum AF461249 Baetiscidae Baetisca sp. AY338707 topology, they accepted the intuitive topologyÕs rela- Baetisca lacustris DQ648715 tionships as a credible estimate of mayßy phylogeny. Behningiidae Behningia sp. AY338703 However, the authors misused this test, because the Caenidae Caenis sp. AY338710 S/H test is only appropriate for likelihood analyses Caenis luctuosa AF461250 Caenis youngi DQ648717 when all a priori topologies are simultaneously com- Coloburiscidae Coloburiscoides giganteus DQ648723 pared (Goldman et al. 2000, Shimodaira 2002). More Coloburiscus humeralis AY338706 appropriate tests would have been the Templeton Ephemerellidae Drunella coloradensis AY338694 (Templeton 1983, Larson 1994) and winning-sites Ephemerella sp. U65107 Ephemeridae Ephemera sp. X89489 tests (Prager and Wilson 1988) for comparisons of Ephemera simulans DQ648733 their intuitive topologies to molecular topologies de- Hexagenia sp. AY121136 rived from parsimony analysis. We performed these Hexagenia rigida AF461253 latter tests on the three intuitive topologies deemed Euthyplociidae Polyplocia sp. AY338705 Heptageniidae Cinygmula sp. AY121137 credible by Sun et al. (2006) by comparing these Epeorus grandis DQ648729 topologies to trees generated from the 18S data. Heptagenia sp. AY338709 Heptagenia diabasia DQ648731 Leucrocuta aphrodite AF461254 Results Stenonema sp. AF461252 Isonychiidae Isonychia sp. AY338708 Reanalysis of Sun et al. (2006) Sequences (Fig. 1). Isonychia rufa DQ648728 The reanalysis of the Sun et al. (2006) sequences Leptohyphidae Leptohyphes apache AY338714 resulted in an alignment consisting of 616 aligned Leptophlebiidae Paraleptophlebia memorialis DQ648722 Choroterpes sp. AY555525 characters: 422 characters were constant, 113 variable Metretopodidae Metretopus borealis AY338698 characters were parsimony-uninformative, and 81 Metretopus borealis DQ648718 characters were parsimony-informative. The heuristic Neoephemeridae Neoephemera youngi AY338702 search (6,846,620 rearrangements tried) resulted in Nesameletidae Ameletoides lacusalbinae DQ648732 Oligoneuriidae Lachlania saskatchewanensis AY338701 117 most parsimonious trees with a length of 323 steps. Lachlania talea DQ648725 A 50% majority rule consensus topology is presented Oniscigastridae Tasmanophlebia sp. DQ648720 in Fig. 1. This topology does not support the mono- Polymitarcyidae Tortopus primus DQ648724 phyly of the suborders Furcatergalia, Pisciforma, or Potamanthidae Anthopotamus sp. AY338711 Anthopotamus sp. AF461255 Setisura as suggested by Sun et al. (2006), nor does it Pseudironidae Pseudiron centralis AY338699 support the position