SYSTEMATICS Tests of Current Hypotheses of Mayfly (Ephemeroptera) Phylogeny Using Molecular (18s rDNA) Data LU SUN, ANIKO SABO,1 M. D. MEYER,2 R. P. RANDOLPH,3 LUKE M. JACOBUS, W. P. MCCAFFERTY, AND VIRGINIA R. FERRIS Department of Entomology, Purdue University, West Lafayette, IN 47907 Ann. Entomol. Soc. Am. 99(2): 241Ð252 (2006) ABSTRACT Partial 18s rDNA sequences from 22 exemplar mayßy species (Ephemeroptera) rep- resenting 20 families were analyzed to obtain a best phylogenetic tree for comparison to previous phylogenetic hypotheses. With respect to relationships among the three major groupings, our mo- lecular data support the hypothesis that Pisciforma and Setisura comprise a monophyletic sister group to the Furcatergalia, rather than the hypothesis that Setisura and Furcatergalia comprise a mono- phyletic group stemming from the Pisciforma. Within Pisciforma, acceptable trees show that Baetidae separates at the base of the Pisciforma clade. The data suggest that Pisciforma is paraphyletic and do not support the grouping of all Southern hemisphere families as a monophyletic group. An evolu- tionary sequence favored by the data suggests a grouping of Siphlonuridae, Rallidentidae, Nesame- letidae, and Ameletidae and a grouping of Oniscigastridae, Ameletopsidae, and Acanthametropodidae. The data support the monophyly of Setisura (Heptageniidae, Arthropleidae, Pseudironidae, Oligo- neuriidae, Isonychiidae, and Coloburiscidae). Within Setisura, a bootstrap/jackknife test places the families Heptageniidae, Arthropleidae, and Pseudironidae in one clade at 100% frequency. Also supported are hypotheses that Pseudironidae is a sister group to a HeptageniidaeÐArthropleidae group and that a sister relationship exists between the latter two families. Hypotheses that Pseudironidae separated from other Setisura families at an earlier stage and comprises a sister group to a Heptage- niidaeÐOligonuriidae lineage or that Pseudironidae should be moved out of Setisura are not supported. KEY WORDS Ephemeroptera, 18s rDNA, phylogeny EPHEMEROPTERA IS ONE OF the most archaic of extant four recent suborders, Furcatergalia, which was some- winged insect orders (Kukalova´-Peck 1985). The times previously referred to as Rectracheata (McCaf- higher classiÞcation of Ephemeroptera has evolved ferty 1991b), includes Leptophlebiidae, Behningiidae, considerably over the past 200 yr to include progres- all pannote families (Pannota), and all tusked bur- sively more higher taxa. However, the use of strict rower families (Scapphodonta); Setisura includes the phylogenetic classiÞcation is relatively recent (Mc- ßat-headed mayßies and their hypothesized minnow- Cafferty 1991b), and cladistic data have been mostly like mayßy ancestral groups, including all forms that morphological in nature. In this study, we used mo- passively Þlter food with their forelegs; Pisciforma lecular data to test the various hypotheses outlined includes all other minnowlike mayßies; and Carapacea below regarding certain aspects of the phylogeny of includes Baetiscidae and Prosopistomatidae. Ephemeroptera. Relationship between Carapacea and Other May- Suborder Level Classification of Ephemeroptera. flies. As a distinct monophyletic group supported by At present, four major groupings are generally recog- series of synapomorphies (e.g., the presence of a larval nized in Ephemeroptera under various names. We carapace), the BaetiscidaeÐProsopistomatidae rela- follow a classiÞcation system (Table 1) taken from tionship was suggested by various researchers (Ed- McCafferty (1991b) and its modiÞcations by Wang munds and Traver 1954). Kluge (1998) and McCaf- and McCafferty (1995), McCafferty (1997, 2004), and ferty and Wang (2000) showed the group to be basally McCafferty and Wang (2000). In this classiÞcation of derived with its sister group represented by all other extant mayßies, which share synapomorphic fore- wings with the tornus located between the ends of 1 Human Genome Sequencing Center, Baylor College of Medicine, veins CuA and CuP (CuP and A1 terminating in the Houston, TX 77030. anal margin, rather than the outer margin, of the 2 Department of Biology, Christopher Newport University, wings). McCafferty (1997) Þrst referred to the Baetis- Newport News, VA 23606. 3 Section of Evolution and Ecology, University of California, Davis, cidaeÐProsopistomatidae grouping as the suborder CA 95616. Carapacea. In our study using molecular data, all trees 0013-8746/06/0241Ð0252$04.00/0 ᭧ 2006 Entomological Society of America 242 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 99, no. 2 Table 1. McCafferty classification system of extant taxa followed in this study, with families arranged alphabetically within their groupings Suborder Infraorder Superfamily Family Carapacea Baetiscidae Prosopistomatidae Furcatergalia Lanceolata Leptophlebiidae Palpotarsa Behningiidae Scapphodonta Potamanthoidea Potamanthidae Euthyplocioidea Euthyplociidae Ephemeroidea Ephemeridae Ichthybotidae Palingeniidae Polymitarcyidae Pannota Caenoidea Caenidae Neoephemeridae Ephemerelloidea Coryphoridae Dicercomyzidae Ephemerellidae Ephemerythidae Leptohyphidae Machadorythidae Melanemerellidae Teloganellidae Teloganodidae Tricorythidae Vietnamellidae Pisciforma Acanthametropodidae Ameletidae Ameletopsidae Ametropodidae Baetidae Dipteromimidae Metretopodidae Nesameletidae Oniscigastridae Rallidentidae Siphlaenigmatidae Siphlonuridae Siphluriscidae Setisura Arthropleidae Coloburiscidae Isonychiidae Heptageniidae Oligoneuriidae Pseudironidae that we modiÞed and tested recognize the hypothesis by a series of synapomorphies (McCafferty 1991a, that Baetiscidae, as a representative of Carapacea, is Wang and McCafferty 1995, Kluge 1998). An alterna- the most basal clade. tive hypothesis regarding the relationships among the Phylogenetic Hypotheses Regarding Furcatergalia, three major groupings was given by Kluge et al. (1995) Setisura, and Pisciforma. Among the suborders Fur- and Kluge (1998, 2004), in which the Pisciforma catergalia, Setisura, and Pisciforma, which accommo- group (called Tridentiseta) and a Setisura-Furcater- date the majority of mayßy families, Pisciforma and galia group (called Bidentiseta) were considered sis- Setisura were considered to be derived together, ter clades, although Kluge admitted that Pisciforma constituting a sister clade to the Furcatergalia (Mc- was probably paraphyletic. KlugeÕs grouping of Seti- Cafferty and Edmunds 1979, Landa and Soldan 1985, ´ sura and Furcatergalia together was supported by a McCafferty 1991b, Tomka and Elpers 1991). The presumed initial presence of two dentisetae on larval monophyly of the Furcatergalia and the PisciformaÐ Setisura groups were supported by anatomic synapo- maxillae. Within this grouping, Setisura (or Branchit- morphies, including the presence of the ventral anas- ergaliae) and Furcatergalia (or Furcatergaliae) also tomosis of tracheae in segments other than 8 and 9 of were considered monophyletic groups. Solda´n and the abdomen, and the absence of branches of a dorsal Putz (2000) proposed another hypothesis based on trunk of tracheae formed in the head, respectively karyotype evidence that generally resembled KlugeÕs (Tomka and Elpers 1991). Within the PisciformaÐ hypothesis. It differed from the latter in the arrange- Setisura clade, no clear evidence has supported the ment of lineages within the hypothesized Furcater- monophyly of Pisciforma, and McCafferty and Ed- galiaÐSetisura group by treating Scapphodonta, Lep- munds (1979) indicated it was extremely paraphyletic. tophlebiidae, and Setisura as a monophyletic sister Monophyly of Setisura, however, is clearly supported group to Pannota (Fig. 3). March 2006 SUN ET AL.: MAYFLY PHYLOGENY 243 Phylogeny among Major Groups within Furcater- eages, which were deduced from a systematic study galia. The suborder Furcatergalia is generally thought of mayßy anatomy. Based on internal anatomical data to be monophyletic, with major clades Leptophlebi- and other morphological evidence of Landa and Sol- idae, Scapphodonta, and Pannota containing Ͼ15 fam- da´n (1985), Tomka and Elpers (1991) suggested an ilies. It has been generally recognized that within the evolutionary tree for mayßy families in which most of Furcatergalia, Leptophlebiidae is a basal lineage the clades were supported by presumed synapo- (Kluge 1998), and a sister group to a relatively derived morphies. Within the PisciformaÐSetisura grouping, clade that includes a pair of sister groups, Scapph- Ametropodidae was thought to branch Þrst, with odonta and Pannota (McCafferty and Wang 2000) in other families sharing the nerve ganglion of abdominal addition to a more basal lineage represented by the segment one merging with that of the metathorax. Behningiidae (McCafferty 2004). Scapphodonta and Siphlonuridae (Siphlonuridae and Dipteromimidae Pannota are both monophyletic and are supported by in Table 1) and Rallidentidae (Rallidentidae, Nesa- a series of morphological synapomorphies (McCaf- meletidae, and Ameletidae in Table 1) were thought ferty and Wang 2000, McCafferty 2004). In our study, to be sister groups that branched next, with other we followed this arrangement of branches within Fur- families lacking a nerve ganglion in abdominal seg- catergalia in our construction of hypothesized phylo- ment 8. Acanthametropodidae and Ameletopsidae genetic trees. were thought to branch next from the remainder that Relationships Regarding the Pisciforma–Setisura included Baetidae, Metretopodidae, and Setisura, Grouping. When Ulmer (1920) Þrst introduced