Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior Michael A. Thomas, Kathleen A. Walsh, Melisande R. Wolf, Bruce A. McPheron, and James H. Marden* 208 Mueller Laboratory, Department of Biology, Pennsylvania State University, University Park, PA 16802 Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved September 15, 2000 (received for review June 27, 2000) Insects in the order Plecoptera (stoneflies) use a form of two- evolutionary reduction in wing structural complexity. Surface dimensional aerodynamic locomotion called surface skimming to skimming and reduced wing complexity may have evolved as move across water surfaces. Because their weight is supported by correlated traits during an evolutionary reduction in flight water, skimmers can achieve effective aerodynamic locomotion proficiency in certain lineages of modern stoneflies. even with small wings and weak flight muscles. These mechanical To determine the evolutionary history of stonefly skimming features stimulated the hypothesis that surface skimming may and wing structural complexity, it is necessary to examine how have been an intermediate stage in the evolution of insect flight, these traits are distributed across the plecopteran phylogeny. which has perhaps been retained in certain modern stoneflies. This type of analysis has already been attempted for surface Here we present a phylogeny of Plecoptera based on nucleotide skimming (15), which resulted in the conclusion that skimming sequence data from the small subunit rRNA (18S) gene. By mapping behavior is most likely a derived, apomorphic condition, i.e., a locomotor behavior and wing structural data onto the phylogeny, relatively recent loss of flight. However, that analysis had two we distinguish between the competing hypotheses that skimming serious shortcomings. First, the morphological characters used is a retained ancestral trait or, alternatively, a relatively recent loss to construct that phylogeny were not compared with homologous of flight. Our results show that basal stoneflies are surface skim- traits of outgroup taxa to determine their polarity (ancestral vs. mers, and that various forms of surface skimming are distributed derived; ref. 16). Polarities were assigned based on resemblance widely across the plecopteran phylogeny. Stonefly wings show to assumed ancestral conditions. This is problematic, and the evolutionary trends in the number of cross veins and the thickness resulting tree has limited utility for assessing evolutionary his- of the cuticle of the longitudinal veins that are consistent with tory. Second, the analysis assumed that skimming was restricted elaboration and diversification of flight-related traits. These data to the single species in which the behavior had been originally support the hypothesis that the first stoneflies were surface described (3), despite the fact that presence or absence of skimmers, and that wing structures important for aerial flight have skimming in other stonefly taxa had not been determined. become elaborated and more diverse during the radiation of Lacking even a rudimentary knowledge of the taxonomic dis- modern stoneflies. tribution of the trait in question, the analysis and conclusions are questionable. nsect flight is an example of a complex trait whose origin is Here we reexamine this question by using a rooted phyloge- Idifficult to explain by using a model that depends on gradual netic analysis based on DNA sequence data from stoneflies and progression through intermediate stages (1, 2). How can tiny a number of outgroup taxa. Mapping wing structural data and wings, simple wing hinges, and weak muscles provide a func- skimming behavior onto this phylogeny allows us to test hypoth- tional advantage over no wings at all? A novel solution to this eses about the evolutionary direction of locomotor behavior and riddle was recently provided by the discovery of surface skim- wing structural complexity. ming, a nonflying form of aerodynamic locomotion used by Methods certain stoneflies (Plecoptera) and mayflies (Ephemeroptera) to move in two dimensions across water surfaces (3–7). By flapping Phylogenetic Analysis. The small subunit rRNA (18S) gene was their wings or by using them as nonflapping sails while their sequenced for 34 stonefly species representing all families of weight is supported by water, skimmers can achieve effective Plecoptera (GenBank accession nos. AF311439–AF311472; a aerodynamic locomotion even with small wings and weak flight complete list of taxa and collection information can be found in muscles (3, 4). Table 1, which is published as supplemental data on the PNAS Surface skimming is now widely accepted as a plausible web site, www.pnas.org). An additional stonefly sequence (Me- mechanical model for flight evolution (8–11), but there is soperlina pecirai; Perlodidae) was obtained from GenBank (ac- cession no. U68400). DNA was extracted by using standard considerably less support for the suggestion (3, 4) that skimming ͞ in modern stoneflies is a retained ancestral trait. Many pterygote phenol chloroform protocols for alcohol-preserved material (17). Amplification of the 18S gene by the PCR used two insects have lost the ability to fly, including numerous stonefly Ј species that are wingless or possess greatly reduced wings. There oligonucleotide primers, rev18G (5 -AGGGCAAGTCTGGT- also appears to have been an evolutionary reduction in the number of cross veins in the wings of stoneflies in the super- This paper was submitted directly (Track II) to the PNAS office. family Nemouroidea (12), the clade in which skimming was first Abbreviations: NJ, neighbor joining; ME, minimum evolution; ML, maximum likelihood; described. Cross veins are structural elements that link the main MP, maximum parsimony. longitudinal veins; in some locations they stiffen the wing, Data deposition: The sequences reported in this paper have been deposited in the GenBank whereas in others they contribute to active and passive defor- database (accession nos. AF311439–AF311472). mations of the wing planiform that enhance aerodynamic per- *To whom reprint requests should be addressed. E-mail: [email protected]. formance (13, 14). The stonefly taxa that are traditionally The publication costs of this article were defrayed in part by page charge payment. This thought to be the basal group have wings with abundant cross article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. veins, as do other basal pterygotes (mayflies, dragonflies, and §1734 solely to indicate this fact. various extinct fossil lineages), thereby suggesting that particular Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.230296997. lineages of more recently evolved stoneflies have undergone an Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.230296997 13178–13183 ͉ PNAS ͉ November 21, 2000 ͉ vol. 97 ͉ no. 24 Downloaded by guest on September 27, 2021 GCCA) and 18L (5Ј-CACCTACGGAAACCTTGTTAC- GACTT), generating an approximately 1,300-nt fragment. Sequencing primers included the two PCR primers and two internal primers 18H (5Ј-TCAATTCCTTTAAGTTTGAGC) and rev18H (5Ј-GCTGAAACTTAAAGGAATTGA), which generated sequence fragments of approximately 700 nucleotides in length. Cycle-sequencing reactions were performed by using 3Ј BigDye-labeled dideoxynucleotide triphosphates and run on an Applied Biosystems Prism 377 DNA Sequencer. Raw data were analyzed by using the DNA STAR SEQMAN II sequence analysis program (DNAstar, Madison, WI). Outgroup taxa used to root the phylogeny were selected by using a relative apparent synapomorphy analysis (RASA; ref. 18), which identifies outgroups that maximize the ratio of informative phylogenetic signal to uninformative noise, thereby increasing the probability that the correct phylogeny is recov- ered. Of the outgroup combinations we tested (a list can be found in Supplemental Table 2 published on the PNAS web site, www.pnas.org), a diverse set of Hemiptera, Orthoptera, Der- maptera, Phasmatodea, Embioptera, Grylloblattodea, and Blat- todea (GenBank accession nos. U06478, U06480, U09207, U06477, Z97573, Z97574, Z97594, Z97561, Z97575, Z97593, ϭ Z97569, and Z97592) yielded the highest tRASA statistic (tRASA Ͻ Fig. 1. (A) Regions of stonefly wings from which cross veins were counted 31.814; P 0.001). and location of the chordwise cross section used for measurements of vein Sequences were aligned by using the CLUSTAL W alignment morphology. (B and C) Examples of cross sections of longitudinal wing veins model (19). The alignment consisted of 1,696 sites, including and the measurements taken for cuticle thickness. B shows the medial vein of gaps. A total of 578 sites, primarily in one large hypervariable a relatively basal species, Taeniopteryx burksi; C shows the medial vein of the region, were unalignable and therefore were excluded from our relatively derived species, Pteronarcella badia. analyses (sites 122–510, 591–600, 662–670, 774–776, 1182–1240, 1353–1362, 1564–1608, and 1644–1696). The analyzed data set consisted of 331 variable (133 nonparsimony informative and 198 subcostal or radial vein (hereafter referred to as costal cross parsimony informative) and 787 constant sites. Average nucle- veins) and those between the anterior cubital vein and both the otide frequencies estimated by PAUP were 24:23:28:25 medial vein and the posterior cubital vein (hereafter referred to (A:C:G:T). There was no significant