Molecular Phylogenetics and Evolution 45 (2007) 289–310

Phylogeny of the higher (Anisoptera: ): An exploration of the most speciose superfamily of dragonflies

Jessica Ware a,*, Michael May a, Karl Kjer b

a Department of Entomology, Rutgers University, 93 Lipman Drive, New Brunswick, NJ 08901, USA b Department of Ecology, Evolution and Natural Resources, Rutgers University, 14 College Farm Road, New Brunswick, NJ 08901, USA

Received 8 December 2006; revised 8 May 2007; accepted 21 May 2007 Available online 4 July 2007


Although libelluloid dragonflies are diverse, numerous, and commonly observed and studied, their phylogenetic history is uncertain. Over 150 years of taxonomic study of Libelluloidea Rambur, 1842, beginning with Hagen (1840), [Rambur, M.P., 1842. Neuropteres. Histoire naturelle des Insectes, Paris, pp. 534; Hagen, H., 1840. Synonymia Libellularum Europaearum. Dissertation inaugularis quam consensu et auctoritate gratiosi medicorum ordinis in academia albertina ad summos in medicina et chirurgia honores.] and Selys (1850), [de Selys Longchamps, E., 1850. Revue des Odonates ou Libellules d’Europe [avec la collaboration de H.A. Hagen]. Muquardt, Brux- elles; Leipzig, 1–408.], has failed to produce a consensus about family and subfamily relationships. The present study provides a well- substantiated phylogeny of the Libelluloidea generated from gene fragments of two independent genes, the 16S and 28S ribosomal RNA (rRNA), and using models that take into account non-independence of correlated rRNA sites. Ninety-three ingroup taxa and six outgroup taxa were amplified for the 28S fragment; 78 ingroup taxa and five outgroup taxa were amplified for the 16S fragment. Bayesian, likelihood and parsimony analyses of the combined data produce well-resolved phylogenetic hypotheses and several previously suggested monophyletic groups were supported by each analysis. Macromiinae, s. s., and are each monophy- letic. The corduliid (s.l.) subfamilies Synthemistinae, Gomphomacromiinae, and Idionychinae form a monophyletic group, separate from the Corduliinae. Libellulidae comprises three previously accepted subfamilies (Urothemistinae, a very restricted Tetrathemistinae, and a modified Libellulinae) and five additional consistently recovered groups. None of the other previously proposed subfamilies are sup- ported. Bayesian analyses run with an additional 71 sequences obtained from GenBank did not alter our conclusions. The evolution of adult and larval morphological characters is discussed here to suggest areas for future focus. This study shows the inherent problems in using poorly defined and sometimes inaccurately scored characters, basing groups on symplesiomorphies, and failure to recognize the widespread effects of character correlation and convergence, especially in aspects of wing venation. Published by Elsevier Inc.

Keywords: Odonata; Libellulidae; Dragonflies; PHASE; RNA7A; rRNA alignment; Phylogeny

1. Introduction able progenitors date to the (360-290 million years ago) and are probably the most widely known Dragonflies are among the most recognizable of , extinct insects. Anisoptera (in their present form) arose even having become subjects of extensive folklore (Sarot, later, with earliest known fossils from the (250– 1958) and, moreover, have been used in a wide array of 200 million years ago; Grimaldi and Engel, 2005). While studies dealing with functional morphology, behavior, clearly identifiable libelluloids are not well known from ecology, and evolution (Corbet, 1999). Odonata are consid- the (206–142 million years ago), Jarzembowski ered to be among the earliest flying insects. Their recogniz- and Nel (1996) suggest that libelluloids were already well established in the Early (142–65 million years ago). Extant libelluloids include, among others, the wide- * Corresponding author. Fax: +1 732 932 7229. spread Macromiinae and Corduliinae and the most abun- E-mail address: [email protected] (J. Ware). dant and familiar dragonflies, Libellulidae. Libellulidae

1055-7903/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.ympev.2007.05.027 290 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 are readily recognizable, often with colored or patterned and Tobin, 1985; Carle and Louton, 1994; Carle, 1995; wings and a boot shaped series of veins (the anal loop) in Bechly, 1996; Lohmann, 1996a,b; Trueman, 1996; Jarzem- the hindwing. They are commonly seen in territorial flight bowski and Nel, 1996; Carle and Kjer, 2002; Rehn, 2003). around lakes and ponds, or perched along the bank. Despite progress in understanding homologies in Odonata Among libelluloids, adult reproductive and feeding venation (e.g., Carle, 1982b; Riek and Kukalova´-Peck, behavior, larval behavior and ecology (Corbet, 1999), 1984), many wing vein characters may support convergent and (Carle, 1995) vary widely and have been relationships when used to the exclusion of other charac- investigated intensively. While it is clear that a well-sup- ters (Hennig, 1969; Carle, 1982b). The 11 subfamilies of ported phylogenetic hypothesis is needed in order to reach Libellulidae recognized in Davies and Tobin (1985) and an understanding of the evolution of these traits, phyloge- Bridges (1994) were largely based on wing vein morphol- netic relationships among libelluloid families remain highly ogy. Bechly’s (1996) morphological study of the Odonata contentious, with numerous hypotheses proposed (Fig. 1 also relied heavily on venational characters to break up and Table 1). For descriptive purposes, we use the taxon- the higher Libelluloidea into numerous families: Gompho- omy of Davies and Tobin (1985) unless otherwise indi- macromiinae and Synthemistinae were split into eight fam- cated, since it is widely used today (Table 2). ilies and the remaining Corduliidae and the Libellulidae Morphological studies of libelluloid phylogeny have were each divided into two families. relied heavily, although not exclusively, on wing vein char- Some studies have focused on egg, genitalic, flight mus- acters (Kirby, 1890; Needham, 1903, 1908; Martin, 1907; culature, color, and larval characteristics (St. Quentin, Ris, 1909–1919; Tillyard, 1910; Needham and Broughton, 1939; Gloyd, 1959; Lieftinck, 1971; Pfau, 1971; Theischin- 1927; Fraser, 1957; Gloyd, 1959; Geijskes, 1970; Lieftinck, ger and Watson, 1984; Pfau, 1991, 2005; May, 1995a; 1971; Theischinger and Watson, 1978; Carle, 1982a; Davies Bechly, 1996; Lohmann, 1996a; Carle and Kjer, 2002). Libellulinae Corduliinae Sister Taxa Cordulegastridae Libellulidae Macromiinae Sister Taxa Corduliinae Libellulidae Synthemistidae Sister Taxa Corduliidae Sister Taxa Cordulegastridae Cordulegastridae Libellulinae Macromiidae Corduliinae

Libellulidae Libellulidae Corduliidae

(a) Rambur, 1842* (b) Kirby, 1890* (c) Gloyd, 1959 (d) * Libellulidae Urothemistidae Hemicorduliidae Corduliidae Oxygastridae Petaluroidea Libellulidae Corduliidae Synthemistidae Idomacromiidae Idionychidae Libellulidae Corduliidae Macromiidae Macromiidae Synthemistidae Libellulidae Macromiidae Sister Taxa Corduliidae Cordulegastridae Macrodiplactidae Pseudocorduliidae Synthemistidae Synthemistidae Sister Taxa Gomphomacromiidae Sister Taxa Cordulegastridae Cordulegastridae

(e) Fraser, 1957 (f) Carle and (g) Bechly, 1996; (h) Pfau, 1971, 1991, 2005 Louton, 1994* Lohmann, 1996

Fig. 1. Hypotheses, also listed in Table 1, of relationships within Libelluloidea. Topologies are (a) one higher Libelluloid family (Libellulidae) comprises two subfamilies: Corduliinae and Libellulinae. *Some who use this include a third subfamily, the Macromiinae; (b) Two higher Libelluloid families (Corduliidae and Libellulidae). The Corduliidae comprises two subfamilies: Corduliinae, Macromiinae. *This is loosely based on Kirby, 1890, but he used the term ‘Corduliinae’; (c) three higher Libelluloid families (Macromiidae, Synthemistidae and Libellulidae). The Libellulidae comprises Corduliinae and Libellulinae; (d) four higher Libelluloid families (Synthemistidae, Corduliidae, Macromiidae, Libellulidae). *This is loosely based on Fraser (1957) but excludes Macrodiplactidae; (e) four higher Libelluloid families Synthemistidae, Corduliidae, Libellulidae, Macrodiplactidae). Fraser used the name ‘‘Synthemidae’’ for Synthemistidae; (f) five higher Libelluloid families (Synthemistidae, Gomphomacromiidae, Corduliidae, Macromiidae and Libellulidae). *This is loosely based on Carle and Louton, 1994; (g) twelve higher Libelluloid families (Synthemistidae, Gomphomacromiidae, Idomacromiidae, Austrocorduliidae, Oxygastridae, Idionychidae, Cordulephyidae, Hemicorduliidae, Macromiidae, Corduliidae, Urothemistidae, Libellulidae). This scheme is loosely based on Bechly (1996) and Lohmann (1996a,b) although the nomenclature differed slightly; (h) three higher Libelluloid families (Synthemistidae, Corduliidae, and Libellulidae). Petaluroidea includes Cordulegastridae. Placement of Macromiidae not discussed. In 2005, Pfau also includes the families Cordulephyidae and Gomphomacromiidae, placed basal to Synthemistidae + Libellulidae. J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 291

Table 1 A summary of previous work in libelluloid systematics; with reference to the hypotheses of internal libelluloid relationships that are shown in Fig. 1 Author and year Taxa studied Extant Libelluloid families MCL Dataset Analysis studied hypothesis supported Carle and Kjer 9 Non-Libelluloid Anisopteran Neopetaliidae, A Morphology Computer-assisted (2002) families; 4 Libelluloid families Cordulegastridae, parsimony Corduliidae, Libellulidae Carle (1982a) Odonata Cordulegastridae, A Morphology Manual parsimony Corduliidae, Libellulidae St. Quentin (1939) 3 Libelluloid families Cordulegastridae, A Morphology: Intuition Corduliidae, Libellulidae genetalia Martin (1914) 1 Libelluloid family Corduliidae A Morphology Intuition Tillyard (1917) Odonata Neopetaliidae, A Morphology Intuition Cordulegastridae, Corduliidae, Libellulidae Needham (1908) 2 Libelluloid families Corduliidae, Libellulidae A Morphology Intuition Martin (1907) 1 Libelluloid family Corduliidae A Morphology Intuition Needham (1903) Anisoptera Neopetaliidae, A Morphology Intuition Cordulegastridae, Corduliidae, Libellulidae Selys (1892) Anisoptera Cordulegastridae, A Morphology Intuition Corduliidae Kirby (1890) Odonata Cordulegastridae, A Morphology Intuition Corduliidae, Libellulidae Hagen (1861) Odonata Corduliidae, Libellulidae, A Morphology Intuition Rambur (1842) Odonata Neopetaliidae, A Morphology Intuition Cordulegastridae, Corduliidae, Libellulidae Misof et al. (2001) 4 non-Libelluloid Anisopteran families; Neopetaliidae, B Molecular: Computer-assisted 4 Libelluloid families Cordulegastridae, 16s and 12s parsimony and Corduliidae, Libellulidae likelihood Bridges (1994) Odonata Neopetaliidae, B—— Cordulegastridae, Corduliidae, Libellulidae Davies and Tobin Anisoptera Neopetaliidae, B Morphology Manual parsimony (1985) Cordulegastridae, Corduliidae, Libellulidae Steinmann (1997) Odonata Neopetaliidae, B—— Cordulegastridae, Corduliidae, Libellulidae Lieftinck (1977) 1 Libelluloid families Corduliidae B Morphology Manual parsimony Lieftinck (1971) 1 Libelluloid families Corduliidae B Morphology Intuition Tillyard (1928) 2 Libelluloid families Corduliidae, Libellulidae B Morphology Intuition Gloyd (1959) 1 Libelluloid family Corduliidae C Morphology Intuition Jarzembowski and Libelluloid fossils 4 Libelluloid families Neopetaliidae, D Morphology Computer-assisted Nel (1996) Cordulegastridae, parsimony Corduliidae, Libellulidae Nel and Paicheler 2 Libelluloid families Cordulegastridae, D*No Morphology Manual parsimony (1994) Corduliidae, Libellulidae studied Theischinger and 1 Libelluloid families Corduliidae E Morphology Manual parsimony Watson (1978) Fraser (1957) Zygoptera + Anisozygoptera 3 Non- Neopetaliidae, E Morphology Intuition Libelluloid families 2 Libelluloid Cordulegastridae, families Corduliidae, Libellulidae Carle (1995) Libelluloid fossil: Nothomacromia 3 Neopetaliidae, F Morphology Manual parsimony Libelluloid Anisoptera Cordulegastridae, Corduliidae, Libellulidae Carle and Louton 4 Non-Libelluloid Anisoptera families 4 Neopetaliidae, F* No Morphology Manual parsimony (1994) Libelluloid families Cordulegastridae, Corduliidae Libellulidae studied Lohmann (1996a,b) 4 Non-Libelluloid Anisopteran Neopetaliidae, G Morphology Manual parsimony families, 4 Libelluloid families Cordulegastridae, Corduliidae, Libellulidae (continued on next page) 292 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310

Table 1 (continued) Author and year Taxa studied Extant Libelluloid families MCL Dataset Analysis studied hypothesis supported Bechly (1996) Zygoptera, 4 Non-Libelluloid Anisopteran Neopetaliidae, G Morphology Manual families, 4 Libelluloid families Cordulegastridae, Corduliidae, parsimony Libellulidae Pfau (2005) 3 Non-libelluloid families 4 libelluloid Neopetaliidae, H Morphology: Manual families Cordulegastridae, Cordullidae, genitalia parsimony Libellulidae Pfau (1991) Zygoptera + Anisozygoptera 2 Non- Cordulegastridae, Cordullidae, H Morphology: Manual libelluloid families 3 libelluloid families Libellulidae secondary parsimony genitalia Pfau (1971) Zygoptera + Anisozygoptera 2 Non- Cordulegastridae, Cordullidae, H Morphology: Manual libelluloid families 3 libelluloid families Libellulidae secondary parsimony genitalia

Pfau’s (2005) study of sperm transfer mechanisms lead him 2. Materials and methods to an alternate phylogenetic hypothesis that placed Cord- ulegastridae, , and Neopetaliidae within 2.1. Taxon sampling Petaluroidea rather than Libelluloidea. Much of the cur- rent confusion over libelluloid taxonomy and phylogeny The superfamily Libelluloidea includes the Libellulidae, may be the result of uncertain character homology and with 143 genera and 969 , the most species-rich and independence (reviewed in Carle, 1982b). An independent commonly observed family of dragonflies worldwide, as molecular dataset may help resolve conflicting phylogenetic well as the Corduliidae Kirby, 1890 (43 genera, 406 species) hypotheses. (Davies and Tobin, 1985; Steinmann, 1997; number of spe- Several recent molecular studies (Kambhampati and cies from Schorr et al., 2006). These are the ‘‘higher’’ Charlton, 1999; Artiss et al., 2001; Saux et al., 2003; Hovm- Libelluloidea, which are the main focus of this study. We oller and Johansson, 2004) have included many libelluline also include the basal libelluloids (sensu Carle, 1995) Cord- taxa, and some (Misof et al., 2001; Misof and Fleck, ulegastridae Calvert, 1893 (4 genera, 49 species), Chloro- 2003; Hovmoller and Johansson, 2004; Hasegawa and Kas- Needham, 1903 (3 genera, 46 species), and the uya, 2006) included a broader sampling across the super- monotypic Neopetaliidae Tillyard and Fraser, 1940. family. Most of these studies were based on a single gene Taxa sequenced are listed in Table 2. Cordulegastridae, (Kambhampati and Charlton, 1999; Misof et al., 2001; Chlorogomphidae, and Neopetaliidae served as outgroups, Artiss et al., 2001; Hovmoller et al., 2002; Misof and Fleck, with the tree rooted using Neopetalia punctata (2 species 2003; Saux et al., 2003). Because their question focused on from 2 cordulegastrid genera, 2 species from 2 chloro- subordinal relationships, Saux et al. (2003) used Locusta gomphid genera and 1 species from the monotypic Neope- migratoria as an outgroup, which may have been too dis- taliidae). We sampled as broadly as we could across each of tantly related to answer questions about the internal order the libelluloid families, extracting from individuals of every of the families (Farris, 1982; Lyons-Weiler et al., 1998; subfamily in every libelluloid family (3 species from 3 macr- Graham et al., 2002). The most recent study (Fleck et al., omiine genera, 14 species from 11 corduliine corduliid gen- in press), includes a large taxon sample, primarily consist- era, 18 species from 17 other non-corduliine corduliid ing of tetrathemistine and libelluline Libellulidae, and com- genera, and 70 species from 56 libelullid genera) (Table 2). bines molecular data with larval morphology. Our purpose here is to present a phylogenetic hypothesis 2.2. Gene selection, DNA extraction, and PCR amplification of the higher Libelluloidea (i.e., Corduliidae and Libelluli- dae of Davies and Tobin, 1985), generated from two inde- Freshly collected dragonflies were used when possible; pendent gene fragments, (mitochondrial and nuclear large other taxa were obtained from personal and museum col- ribosomal RNA subunits; 16S and 28S), structurally lections (Table 2). We amplified the second, third, and sev- aligned, using basal libelluloid outgroups, and the follow- enth hypervariable (divergent) regions (D2, D3, and D7) of ing methods that model the correlated rRNA sites as the nuclear large subunit rDNA (28S) and the third domain non-independent. Extensive taxon sampling has allowed of the mitochondrial large subunit rDNA (16S). us to assess several regions of contention in the higher Muscle tissue was extracted using a Qiagen Dneasy tis- Libelluloidea and to propose historical relationships within sue kit overnight at 55 °C with 180 ll of ATL Buffer and this group. The phylogenetic reassessment provides a basis 20 ll Proteinase-K. Older specimens (collected prior to for improving the taxonomy in the historically difficult 1980) were extracted with 40 ll (twice the suggested Libelluloidea. amount) of Proteinase-K buffer for several days (a sugges- J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 293

Table 2 Table 2 (continued) Taxon list for the present study. Taxonomy based on Bridges (1994) and Taxon Locality Genbank Davies and Tobin (1985) Number Taxon Locality Genbank membranulata (F.L. Carle) D7: EF631211 Number D3: EF631308 Cordulegastridae D2: EF631415 Taeniogaster obliqua USA: NJ (M. L. May) D7: EF631216 16S: EF631528 D3: EF631312 xanthosoma USA: AK (M. L. May) D7: EF631242 D2: EF631420 D3: N/A 16S: EF631533 D2: EF631447 Pangaeagaster maculata USA: NJ (M. L. May) N/A 16S: N/A Kalyptogaster erronea USA: NJ (M. L. May) D7: EF631245 grayi New Zealand (R. Rowe) D7: EF631199 D3: N/A D3: N/A D2: EF631450 D2: EF631399 16S: EF631561 16S: EF631513 Zoraena bilineata USA: MD (M. L. May) N/A Procordulia smithi New Zealand (R. Rowe) D7: EF631200 D3: EF631295 Chlorogomphidae D2: EF631400 Chloropetalia soarer Sequences from F.L. Carle D7: EF631248 16S: EF631514 D3: EF631339 Rialla villosa Chile (Heppner) D7: EF631273 D2: EF631453 D3: EF631364 16S: N/A D2: EF631480 Sinorogomphus sp Sequences from F.L. Carle D7: EF631249 16S: EF631590 D3: EF631340 tenebrosa USA: NJ (M. L. May) D7: EF631215 D2: EF631454 D3: EF631311 16S: EF631564 D2: EF631419 Neopetaliidae 16S: EF631532 Tetragoneuria cynosura Neopetalia punctata Sequences from F.L. Carle D7: EF631247 USA: NJ (M. L. May) D7: N/A D3: EF631338 D3: EF631379 D2: EF631452 D2: N/A 16S: EF631563 16S: N/A Tetragoneuria cynosura USA: NJ (M. L. May) D7: EF631231 Corduliidae: Cordulephyinae D3: EF631325 pygmea Australia (Theischinger) D7: EF631255 D2: N/A D3: EF631346 16S: N/A D2: EF631460 Williamsonia fletcheri USA: MA (M. L. May) N/A 16S: EF631570 Corduliidae: Gomphomacromiinae Corduliidae: Corduliinae Apocordulia macrops Australia N/A Aeschnosoma forcipula French Guiana (J. Huff) D7: EF631223 (G. Theischinger) D3: EF631319 magnifica Australia (K. M. Kjer) D7: N/A D2: EF631427 D3: EF631356 16S: EF631540 D2: EF631470 shurtleffii Canada: Ontario D7: EF631232 16S: EF631580 (J. L. Ware, J.Huff) D3: EF631326 refracta Australia D7: EF631243 D2: EF631435 (G. Theischinger) D3: EF631336 16S: N/A D2: EF631448 Cordulia aenea Sweden (K.M. Kjer) D7: EF631286 16S: EF631559 D3: EF631383 Austrophya mystica Australia (F. L. Carle) D7: EF631236 D2: EF631500 D3: EF631332 16S: EF631603 D2: EF631441 lepida USA: NJ (M. L. May) N/A 16S: N/A princeps USA: NJ (J. L. Ware) D7: EF631205 Gomphomacromia chilensis Chile (F. L. Carle) D7: EF631206 D3: EF631302 & paradoxa D3: EF631303 D2: EF631407 D2: EF631408 16S: EF631521 16S: EF631522 uhleri USA: NJ (M. L. May) D7: EF631227 Hespercordulia berthoudi Australia (F. L. Carle) D7: EF631244 D3: N/A D3: EF631337 D2: EF631431 D2: EF631449 16S: EF631544 16S: EF631560 tau Australia (J. L. Ware, D7: EF631233 Lathrocordulia metallica Australia (F. L. Carle) D7: EF631239 K. M. Kjer, F. L. Carle) D3: EF631328 D3: EF631334 D2: EF631437 D2: EF631444 16S: EF631550 16S: EF631556 USA: FL (M. L. May D7: EF631196 atrifrons Australia (M. L. May D7: EF631240 and K. Tennessen) D3: N/A and F. L. Carle) D3: N/A D2: EF631395 D2: EF631445 16S: EF631509 16S: EF631557 elongata N/A Neocordulia batesi longipollex Panama (M. L. May) N/A (Tobin & Davies) (continued on next page) 294 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310

Table 2 (continued) Table 2 (continued) Taxon Locality Genbank Taxon Locality Genbank Number Number Neocordulia campana Panama (M. L. May) N/A Libellulidae: Tetrathemistinae Oxygastra curtisii Spain (P. Corbet) D7: N/A Calophlebia interposita CAS ( project) D7: N/A D3: N/A D3: EF631381 D2: EF631413 D2: N/A 16S: EF631526 16S: N/A Pseudocordulia Australia (G. Theischinger) D7: EF631251 risi Australia (M. L. May, D7: EF631254 circularis D3: EF631342 K. M. Kjer and F. L. Carle) D3: EF631345 D2: EF631456 D2: EF631459 16S: EF631566 16S: EF631569 Syncordulia gracilis South African (P. Grant) D7: N/A pauliani CAS (Madagascar Project) D7: EF631279 D3: N/A D3: EF631368 D2: EF631439 D2: EF631486 16S: N/A 16S: N/A polleni 1 (M. L. May) D7: EF631222 Corduliidae: Idionychinae D3: EF631318 selysi Hong Kong (K. Wilson) D7: EF631193 D2: EF631426 D3: EF631290 16S: EF631600 D2: EF631391 Tetrathemis polleni 2 South Africa (M. L. May) D7: N/A 16S: N/A D3: EF631376 rapida 1 Hong Kong (K. Wilson) D7: EF631209 D2: EF631495 D3: EF631306 16S: EF631539 D2: EF631411 16S: N/A Libellulidae: Brachydiplacinae Macromidia rapida 2 Hong Kong (K. Wilson) D7: EF631271 Anatya guttata Trinidad (S. Dunkle) N/A D3: EF631362 Australia (K. M. Kjer D7: EF631246 D2: EF631478 denticauda and F. L. Carle) D3: N/A 16S: EF631588 D2: EF631451 Corduliidae: Idomacromiinae 16S: EF631562 Idomacromia proavita Cameroon (CAS) N/A Brachydiplax Thailand (J. Michalski) N/A Corduliidae: Macromiinae c. chalybea transversa USA: NJ (M. L. May) D7: N/A flavifrons -Bissau (J. Huff) D7: EF631260 D3: EF631327 D3: EF631351 D2: EF631436 D2: EF631465 16S: EF631549 16S: EF631575 illionoiensis USA: IL (M. L. May) D7: EF631208 Elga leptostyla Trinidad (M. L. May) D7: EF631274 D3: EF631305 D3: N/A D2: EF631410 D2: EF631481 16S: EF631524 16S: N/A (T. W. Donnelly) D7: EF631197 aequalis Panama (M. L. May) D7: EF631195 contumax D3: EF631293 D3: EF631291 D2: EF631397 D2: EF631393 16S: EF631511 16S: EF631508 Belize (J. L. Ware, J. Huff) D7: EF631250 Corduliidae: Synthemistinae D3: EF631341 Choristhemis Australia (M. L. May, K. M. D7: EF631237 D2: EF631455 flavoterminata Kjer and F. L. Carle) D3: EF631333 16S: EF631565 D2: EF631442 albipuncta (J. Huff) D7: EF631256 16S: EF631554 D3: EF631347 Eusynthemis brevistyla Australia (J. L. Ware, K. M. D7: EF631230 D2: EF631461 Kjer, F. L. Carle) D3: EF631323 16S: EF631571 D2: EF631434 dalei Australia (J. L. Ware, K. M. D7: EF631241 16S: EF631547 Kjer and F. L. Carle) D3: EF631335 Synthemiopsis Australia (M. L. May, D7: EF631213 D2: EF631446 gomphomacromioides K. M. Kjer and F. L. Carle); D3: N/A 16S: EF631558 D2 and D7 sequences D2: EF631417 bella USA: FL (M. L. May) D7: EF631210 from F. L. Carle 16S: EF631530 D3: EF631307 Synthemis eustalacta Australia (J. L. Ware, D7: N/A D2: EF631412 K. M. Kjer, F. L. Carle) D3: EF631296 16S: EF631525 D2: EF631401 Nephepeltia phyryne Trinidad (M. L. May) N/A 16S: EF631515 Thermochoria Cameroon (Mbida Mbida) N/A Synthemis leachii Australia (D. Pryce) D7: EF631201 equivocata D3: EF631297 Oligoclada walkeri Trinidad (M. L. May) N/A D2: EF631402 imbuta Panama (M. L. May) D7: EF631228 16S: EF631516 D3: N/A Corduliidae: Neophyinae D2: EF631432 Neophya rutherfordi (J. Lempert) N/A 16S: EF631545 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 295

Table 2 (continued) Table 2 (continued) Taxon Locality Genbank Taxon Locality Genbank Number Number Libellulidae: Leucorrhiniinae sp 3 South Africa (K. M. Kjer) N/A herbida Venezuela (R.West) N/A Orthetrum sp 4 Senegal (J. Huff) N/A elisa USA: NJ (M. L. May) D7: EF631224 CAS (Madagascar) D7: EF631275 D3: EF631320 D3: N/A D2: EF631428 D2: EF631482 16S: EF631541 16S: EF631591 glacialis USA: NY (M. L. May) D7: EF631207 (X. Zhou) D7: EF631263 D3: EF631304 D3: EF631354 D2: EF631409 D2: EF631468 16S: EF631523 16S: EF631578 Libellulidae: Libellulinae 1 South Africa (K. M. Kjer) D7: EF631285 Australia (M. L. May, D7: EF631235 D3: EF631380 longitudinalis K. M. Kjer and D3: EF631331 D2: EF631498 F. L. Carle) D2: EF631440 16S: EF631601 16S: EF631553 Orthetrum julia 2 South Africa (K. M. Kjer) D7: N/A vibex Panama (M. L. May) N/A D3: EF631382 Dasythemis esmeralda Trinidad (J. Michalski) D7: N/A D2: EF631499 D3: N/A 16S: EF631602 D2: EF631386 CAS (Madagascar Project) D7: EF631267 16S: N/A neglectum D3: N/A defecta Uganda (T. W. Donnelly) D7: EF631277 D2: EF631473 D3: EF631366 16S: EF631583 D2: EF631484 Plathemis lydia USA: NJ (F. L. Carle) D7: EF631234 16S: EF631592 D3: EF631330 pulchella USA: NJ (J. L. Ware) D7: N/A D2: EF631438 D3: EF631329 16S: EF631552 D2: N/A 16S: EF631551 Libellulidae: Sympetrinae Libellula luctuosa USA: NJ (J. L. Ware) D7: EF631194 panorpoides Guinea-Bissau (J. Huff) D7: EF631229 D3: N/A D3: EF631322 D2: EF631392 D2: EF631433 16S: EF631507 16S: EF631546 Libellula USA: NJ (M. L. May) D7: EF631272 strachani Guinea-Bissau (J. Huff) D7: EF631257 quadrimaculata 1 D3: EF631363 D3: EF631348 D2: EF631479 D2: EF631462 16S: N/A 16S: EF631572 Libellula Sweden (K. M. Kjer) D7: N/A Guinea-Bissau (J. Huff) D7: EF631258 quadrimaculata 2 D3: N/A leucosticta D3: EF631349 D2: EF631497 D2: EF631463 16S: EF631589 16S: EF631573 Ladona julia USA: WI (M. L. May) D7: EF631219 erythraea South Africa (M. L. May) D7: EF631225 D3: EF631315 D3: EF631321 D2: EF631423 D2: EF631429 16S: EF631536 16S: EF631542 Japan (M. L. May) D7: EF631276 Crocothemis servilla USA: FL (M. L. May) D7: EF631192 pachygastra D3: EF631365 D3: EF631289 D2: EF631483 D2: EF631390 16S: N/A 16S: EF631506 Misagria parana French Guiana (J. Huff) D7: EF631268 Crocothemis sp Senegal (J. Huff) N/A D3: EF631359 Deielia Japan (M. L. May) D7: EF631226 D2: EF631475 D3: EF631543 16S: EF631585 D2: EF631430 ferruginea 1 Dominican Republic D7: EF631265 16S: N/A (J. Huff) D3: EF631357 haematodes Australia (J. L. Ware, D7: EF631238 D2: EF631471 K. M. Kjer, F. L. Carle) D3: N/A 16S: EF631581 D2: EF631443 Orthemis ferruginea 2 Dominican Republic D7: EF631266 16S: EF631555 (J. Huff) D3: N/A simplicicollis USA: TX (M. L. May) D7: EF631191 D2: EF631472 D3: EF631288 16S: EF631582 D2: EF631389 Orthetrum sp 1 Guinea-Bissau (J. Huff) D7: N/A 16S: EF631505 D3: N/A minuscula USA: FL (J. L. Ware D7: EF631190 D2: EF631396 and J. Huff) D3: EF631287 16S: EF631510 D2: EF631388 Orthetrum sp 2 South Africa (K. M. Kjer) D7: EF631261 16S: EF631504 D3: EF631352 (continued on next page) D2: EF631466 16S: EF631576 296 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310

Table 2 (continued) Table 2 (continued) Taxon Locality Genbank Taxon Locality Genbank Number Number Pachydiplax longipennis USA: NJ (F. L. Carle) D7: EF631198 Onychothemis Thailand (T. W. Donnelly) D7: EF631284 D3: EF631294 testacea 2 D3: EF631375 D2: EF631398 D2: EF631494 16S: EF631512 16S: N/A hollandi Trinidad (M. L. May) N/A Libellulidae: Palpopleurinae janeae USA: NJ (F. L. Carle) D7: EF631214 jucunda South Africa (M. L. May) D7: N/A D3: EF631310 D3: N/A D2: EF631418 D2: EF631414 16S: EF631531 16S: EF631527 USA: DE (M. L. May) D7: N/A Senegal (J. Huff) D7: EF631262 D3: EF631324 D3: EF631353 D2: N/A D2: EF631467 16S: EF631548 16S: EF631577 Libellulidae: Trithemistinae tenera New Jersey (J. L. Ware) D7: EF631212 mendax USA: TX (M. L. May) D7: EF631572 D3: EF631309 D3: N/A D2: EF631416 D2: EF631385 16S: EF631529 16S: EF631502 fasciata 1 French Guiana (J. Huff) D7: EF631283 fugax USA: TX (M. L. May) D7: N/A D3: EF631371 D3: N/A D2: EF631490 D2: EF631387 16S: EF631595 16S: EF631503 Zenithoptera fasciata 2 French Guiana (J. Huff) D7: N/A Dythemis multipunctata Panama (M. L. May) D7: EF631259 D3: EF631372 D3: EF631350 D2: EF631491 D2: EF631464 16S: EF631596 16S: EF631574 oreophila (J. Michalski) D7: EF631270 Libellulidae: Trameinae D3: EF631361 onusta USA: NJ (M. L. May) D7: EF631281 D2: EF631477 D3: N/A 16S: EF631587 D2: EF631488 celeno Puerto Rico (M. L. May) D7: EF631282 16S: EF631593 D3: EF631370 Tramea lacerata USA: NJ (J. L. Ware) D7: EF631221 D2: EF631489 D3: EF631317 16S: EF631594 D2: EF631425 Macrothemis hemichlora Panama (M. L. May) D7: N/A 16S: EF631538 D3: EF631292 South Africa (M. L. May) D7: EF631204 D2: EF631394 semihyalina 1 D3: EF631301 16S: N/A D2: EF631406 Macrothemis pulmila Trinidad (M. L. May) N/A 16S: EF631520 lineatipes USA: CA (M. L. May) D7: N/A Rhyothemis South Africa (C. Chaboo) D7: N/A D3: EF631373 semihyalina 2 D3: EF631358 D2: EF631492 D2: EF631474 16S: EF631597 16S: EF631584 Scapanea archboldi Dominican Republic (J. Huff) D7: EF631269 marcella USA: FL (M. L. May) N/A D3: EF631360 flavescens 1 South Africa (M. L. May) D7: EF631220 D2: EF631476 D3: EF631316 16S: EF631586 D2: EF631424 basileri South Africa (K. M. Kjer) N/A 16S: EF631537 Trithemis dorsalis South Africa (M. L. May) D7: N/A Pantala flavescens 2 Senegal (J. Huff) D7: EF631280 D3: EF631299 D3: EF631369 D2: EF631404 D2: EF631487 16S: EF631518 16S: N/A Trithemis monardi Guinea-Bissau (J. Huff) D7: EF631264 Australia (M. L. May, K. M. D7: EF631252 D3: EF631355 brevistylus Kjer and F. L. Carle) D3: EF631343 D2: EF631469 D2: EF631457 16S: EF631579 16S: EF631567 amazonica Bolivia (Mauffray) D7: N/A Libellulidae: Onychothemistinae D3: EF631377 Onychothemis Thailand (T. W. Donnelly) D7: N/A D2: N/A culminicola D3: EF631374 16S: N/A D2: EF631493 tillarga Guinea-Bissau (J. Huff) D7: EF631202 16S: EF631598 D3: EF631298 1 Thailand (T. W. Donnelly) D7: EF631278 D2: EF631403 D3: EF631367 16S: EF631517 D2: EF631485 australis Panama (M. L. May) N/A 16S: EF631599 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 297

Table 2 (continued) ous purines), Y (for ambiguous pyrimidines), or N (for all Taxon Locality Genbank other ambiguities). Number Libellulidae: Urothemistinae rezia Guinea-Bissau (J. Huff) D7: EF631188 2.3. Alignment and phylogenetic reconstruction D3: N/A D2: EF631384 Initial sequence alignments were made using Clustal, 16S: EF631501 and the resulting files were then aligned manually in Micro- balteata USA: FL (M. L. May) N/A assignata Senegal (J. Huff) D7: EF631217 soft Word using the structural methods described in Kjer D3: EF631313 et al. (1994), Kjer (1995), Kjer et al., 2007 and secondary D2: EF631421 structure models based on Guttell et al. (1993). Ambigu- 16S: EF631534 ously aligned regions were defined as single stranded elgneri Australia (K. M. Kjer, D7: EF631253 F. L. Carle) D3: EF631344 regions with multiple insertions and deletions (indels) of D2: EF631458 variable length (and thus unclear nucleotide homology), 16S: EF631568 bounded by hydrogen bonded base pairs. These regions Bali (A. Rowat) D7: N/A D3: EF631378 were excluded from the dataset. For parsimony analyses, D2: EF631496 these characters were recoded as single multistate charac- 16S: N/A ters with the program INAASE (Lutzoni et al., 2000), with Libellulidae: Zygonychinae the stepmatrices applied. Alignments are available on the torridus South Africa (M. L. May) D7: EF631218 Kjer lab website, D3: EF631314 Although we regard manual alignment based on secondary D2: EF631422 16S: EF631535 structure to be quite strongly supported (e.g., Kjer, 1995, South Africa (M. L. May) D7: EF631203 2004; Titus and Frost, 1996; Schnare et al., 1996; Hickson D3: EF631300 et al., 2000; Lutzoni et al., 2000; Mugridge et al., 2000; Ellis D2: EF631405 and Morrison, 1995; Morrison and Ellis, 1997; Gillespie 16S: EF631519 et al., 2005) as the most accurate method for rDNA, we See Appendix A for taxa omitted from analyses. recognize that alignment methodology is a contentious issue. For those who prefer a different alignment proce- tion made by R. Caesar, pers. comm.). All other steps fol- dure, however, the original sequences are, of course, depos- lowed the manufacturer’s protocol. PCR primers, and their ited in GenBank and thus will be available for any sources, are given in Table 3. Programs used for amplifica- reanalysis that is desired. tions were (a) 96 °C, 3 min; 94 °C, 30 s; 50 °C, 30 s; 72 °C, The data were analyzed using parsimony, maximum 45 s for 35–40 cycles; 72 °C, 10 min and (b) 96 °C, 3 min; likelihood, and Bayesian criteria. For the parsimony recon- 94 °C, 30 s; 46 °C, 30 s, 72 °C, 45 s for 10 cycles; 94 °C, struction, a tree bisection-reconnection (TBR) branch 30 s; 48 °C, 40 s; 72 °C, 45 s for 30 cycles; 72 °C, 10 min. swapping heuristic search was run using PAUP 4.0b10 A Qiagen PCR purification kit was used to purify amplified (Swofford, 2001) with 10,000 random additions. Gaps of product, which was then sequenced on an ABI 3100 capil- uniform length were each treated as presence/absence char- lary sequencer. Sequences from both strands were com- acters; other gaps were treated as missing data, except pared and edited in Sequence Navigator (Applied those encoded with INAASE as described above. To esti- Biosystems). Lowercase letters were used to indicate nucle- mate branch support, 500 bootstrap pseudoreplicates (Fel- otides that were readable but difficult to interpret with cer- senstein, 1985) were performed using 10 random addition tainty in either strand (i.e., there was competing searches per pseudoreplicate. background peaks). These lowercase letters were changed Prior to maximum likelihood and Bayesian analyses, we to uppercase letters only if there was agreement in the used MODELTEST 3.06 Akaike weights (Posada and two complementary strands. When there was conflict about Crandall, 1998; Posada and Buckley, 2004) and DT-Mod- a single base call between reads from complementary Sel (Minin et al., 2003) to select an appropriate model of strands, this nucleotide was coded with an R (for ambigu- evolution for each of the two independent gene fragments.

Table 3 Primers used in the present study D2 region of the 28S D3 region of the 28S D7 region of the 28S 16S primers by E. Pilgrim 16s primers from Misof et al., (2001) Forward primer 50TGCTTGAGAG 50ACCCGTCTTGA 50CGSGCGACGA 50GTAAGAGTTTAAA LR-J-12887 sequence TGCAGCCCAA30 AACACGGAC30 GTAGGAGGG3’ SGTCGAACAGA30 50GGAGCTCCGGTTTG AACTCAGATC30 Reverse primer 50CCTTGGTCCGT 50ATAGTTCACCATCT 50CTTCAGAGCC 50AGGATTAGATACCCT LR-N-13398 sequence GTTTCAAGAC30 TTCGGGTCC30 AATCCTTAT30 TTTATTTTAAATG30 50CGGCCGCCTGTTAT CAAAAACAT30 298 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310

Both programs suggested a GTR + I + G model for the 3.2. Phylogenetic relationships 28S and 16S (Yang, 1994; Yang et al., 1994; Gu et al., 1995). GARLI (Zwickl, 2006; available at http:// Bayesian, likelihood and parsimony analyses of the was combined data produced well-resolved phylogenetic used to run a rapid maximum likelihood analysis. The hypotheses (GARLI and PHASE results: Fig. 2; parsi- GARLI bootstrap analysis was run using 100 replicates mony: Fig. 3). Minor differences occur in areas for which of a 500,000-generation search; the heuristic search ran there is low branch support (<50%). Several previously 500,000 generations. Datasets were unable to be parti- suggested monophyletic groups were supported by all three tioned in GARLI, although future versions of GARLI will methods: (1) Libellulidae (100% support from all analyses) be able to do so (Derrick Zwickl, pers. comm.). Because it and Macromiinae (100% support from all analyses). These considers the non-independence of hydrogen-bonded , plus Corduliinae, together form a monophyletic rRNA sites, we also used the RNA7A seven state model group, hereafter the MCL group in the PHASE and available in the PHASE program (Jow et al., 2002), to GARLI analyses (similar but not identical to the MCL run Markov-chain Monte Carlo (MCMC) analyses of par- group of Carle, 1995). (2) Corduliidae s.l is polyphyletic. titioned RNA data (10 million generations each) and a Corduliinae is monophyletic (89% PHASE posterior prob- REV model for the loop regions. Unlike the current ver- ability; 60% GARLI bootstrap; 86% parsimony bootstrap). sion of Mr. Bayes, which uses a 16 by 16 rate matrix, the (3) Surprisingly, Gomphomacromiinae + Synthemisti- RNA7A seven state model (Higgs, 2000) uses a 7 by 7 rate nae + Cordulephyinae + Idionychinae (hereafter, the GSI matrix (7 frequencies, 21 rate parameters).This biologically group) together form a monophyletic group (100% realistic model is useful for studies of rRNA. The REV PHASE; 79% GARLI; 71% parsimony bootstrap) that, model is the most general loop model with the time revers- however, is not clearly divided into the traditional ible constraint (four frequencies, five rate parameters). (sub)families and does not nest within Corduliidae. Using our PHASE trees, we tested the classifications of The results do not support most of the 17 families pro- Davies and Tobin (1985) and Bechly (1996) using the con- posed by Bechly (1996) and Lohmann (1996a,b), or the straint function in PAUP. Constraints were written for subfamilies of Corduliidae suggested by Davies and Tobin each of Davies and Tobin’s subfamilies, and for each of (1985) and Bridges (1994). Furthermore, the puzzling Aus- Bechly’s families. We then filtered our PHASE tree file tralian species Cordulephya pygmea, variously placed as (1) using these constraints and recorded the number of trees a monogeneric subfamily (Fraser, 1957; Davies and Tobin, that contained these clades. 1985), (2) with Hetronaias and Libellulosoma (Bridges, To recheck our data for possible contaminants, and to 1994) or (3) with Neophya (Bechly, 1996), is well nested incorporate additional available taxa, we ran a parsimony within the GSI as sister to the nominal gomphomacr- analysis, as described above, with 71 libelluloid sequences omiine Pseudocordulia circularis (Hetronaias, Libelluloso- downloaded from GenBank and aligned them with our ma, and Neophya were not sequenced). The two genera data (Appendix A). Most of the libelluloid sequences in of Idionychinae, Macromidia and Idionyx, form a mono- GenBank are mitochondrial. Some of the data available phyletic but poorly supported (35% PHASE) subclade in GenBank included a longer fragment of mitochondrial within the GSI clade. Corduliinae and Macromiinae are rRNA that included a fragment of the 12S. These charac- separate monophyletic clades in all analyses. In terms of ters were included in the analysis and coded as missing the MCL interfamilial relationships, the PHASE analysis for our taxa (for a discussion of missing data see Weins, placed the Corduliinae as sister to the Libellulidae, 2005). although with such low support that we would prefer to consider the relationship unresolved. 3. Results Within the Libellulidae, three subfamilies were consis- tently recovered: Leucorrhiniinae Tillyard 1917 (99% 3.1. Molecular data collection PHASE; 94% GARLI, 80% parsimony), Urothemistinae Lieftinck, 1954 (96% PHASE; 79% GARLI; 85% parsi- Ninety-three ingroup taxa and six outgroup taxa were mony), and Libellulinae Rambur 1842 (76% PHASE; lar- amplified for the 28S fragment; 78 ingroup taxa and five gely, though not entirely recovered). Otherwise, members outgroup taxa were amplified for the 16S (Appendix A) of the subfamilies listed in Davies and Tobin (1985) and are included in the analysis. After 192 ambiguously aligned Bridges (1994) are scattered throughout the Libellulidae. characters were excluded and coded in INAASE, 1418 nts Conspecifics and congenerics (with the exceptions of Libell- remained from the nuclear gene fragment and 430 nts from ula, Cordulia, Zyxomma,andOrthetrum) form monophy- the mitochondrial fragment. Five hundred and forty-three letic groups. characters were parsimony informative (nuclear = 346, To assess congruence among independent data sets, we mitochondrial = 189, INAASE = 8); 370 were parsimony also ran separate PHASE analyses of the mitochondrial uninformative and 943 characters were constant. All and nuclear genes. The nuclear PHASE data lends strong sequences are deposited in GenBank under Accession support to monophyly of the GSI (97%) but the mitochon- No. EF631188–EF631603. drial data place them instead as a polytomy at the base of J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 299

Neopetalia punctata 100* Kalyptogaster erronea Taeniogaster obliqua 100* Sinorogomphus sp Chloropetalia soarer Syncordulia gracilis 30 Idionyx selysi 36 99 Macromidia rapida 100* Macromidia rapida Oxygastra curtisii 43 Gomphomacromia sp 37 73 Synthemis leachii 95 Synthemis eustalacta 99 Cordulephya pygmea 55 40 Pseudocordulia circularis Lathrocordulia metallica 53 85* Hesperocordulia berthoudi 'GSI' 44 Micromidia atrifrons 72* Austrophya mystica Eusynthemis brevistyla 83 27 Synthemiopsis gomphomacromioides 100* 86 75* Choristhemis flavoterminata Archaeophya magnifica 100* Pentathemis membranulata Aeschnosoma forcipula 'Higher' Libelluloids 89* 100* 70 Tetragoneuria cynosura Tetragoneuria cynosura 100* 100* Neurocordulia obsoleta Hemicordulia tau 65 85 100* Procordulia smithii 56 Procordulia grayi 99* Cordulia shurtleffi 94 Rialla villosa Corduliinae 60 87 62 Cordulia aenea 'MCL' 100* Phyllomacromia contumax 100* Calophlebia interposita A 100* 38 94* Tetrathemis polleni Tetrathemis polleni 96* B 100* 100* Zygonyx natalensis C79 98* Nannophlebia risi 87 Huonia oreophila Onychothemis culminicola 100* Macromiidae 60 100* Onychothemis testacea Onychothemis testacea 100* Rhyothemis semihyalina 23 D 100* Sympetrum ambiguum Urothemistinae 100* Sympetrum janeae Libellulidae 99* 36 Hydrobasileus brevistylus Tramea onusta 100* E18 75* Tramea lacerata Dasythemis esmeralda Leucorrhiniinae 53 100* Chalcostephia flavifrons Brachydiplax denticauda 95 100* Deielia phaon 97 Zyxomma petiolatum 44 14 96 92 Idiataphe 68 Zyxomma elgneri amazonica Pachydiplax longipennis 33 Elga leptostyla 53 F 93* Micrathyria aequalis 56 Micrathyria aequalis Zenithoptera fasciata 100* Zenithoptera fasciata 88 100* Dythemis multipunctata 97 100* Macrothemis celeno Macrothemis hemichlora 13 99 71 Scapanea frontalis 45 Brechmorhoga mendax 40 Bradinopyga strachani 98 Hemistigma albipuncta G 100* 97* 60 Palpopleura lucia Nannophya dalei 22 Uracis fastigiata 46 41 35 Nannothemis bella 50 55 99 100 Crocothemis erythraea Crocothemis servilia Perithemis tenera 49 Trithemis monardi 100* Trithemis dorsalis H 70 35 100* Pantala flavescens Plathemis lydia Neodythemis pauliani 76 52 Misagria species 42 Hadrothemis defecta 47 Agrionoptera longitudinalis 0.1 40 Orthemis ferruginea 100* Orthemis ferruginea 68 100* Libellula pulchella 54 Libellula luctuosa Lyriothemis pachygastra Libellulinae 94 Libellula quadrimaculata 98* Libellula quadrimaculata 21 Ladona julia 69 Orthetrum sp 96* Orthetrum pruinosum 38 Orthetrum abbotti 100* Orthetrum sp 73 Orthetrum chrysis 100* Orthetrum julia Orthetrum julia

Fig. 2. Phylogenetic reconstruction from a 10 million generation PHASE mcmc analysis. The numbers above the branch indicate posterior probabilities. The ‘‘*’’ indicates GARLI support greater than 50%. the tree (not shown). Both datasets support the monophyly port for this group was low (50%), probably due to the of Macromiinae and of Libellulidae. The 16S data support unstable position of the genera Pentathemis + Aeschnoso- the monophyly of the Corduliinae (80% 16S), but 28S sup- ma. Within the Libellulidae, only the Libellulinae are lar- 300 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310


94 Chloropetalia soarer Sinorogomphus sp Neopetalia punctata 76 100 Taeniogaster obliqua Kalyptogaster erronea Macromiidae 100 Phyllomacromia contumax 99 Macromia illinoiensis Didymops transversa 100 Pentathemis membranulata Aeschnosoma forcipula Corduliinae 88 Hemicordulia tau 98 Procordulia grayi 99 30 Procordulia smithii 83 Epitheca princeps 61 Tetragoneuria cynosura 55 Tetragoneuria cynosura Somatochlora tenebrosa Helocordulia uhleri 19 Cordulia aenea 90 Neurocordulia obsoleta Neurocordulia xanthosoma 53 Cordulia shurtleffi Rialla villosa 'GSI' Syncordulia gracilis 76 91 Macromidia rapida Macromidia rapida 30 Idionyx selysi 28 Oxygastra curtisii Gomphomacromia sp 21 Synthemis eustalacta 24 Synthemis leachii 100 Synthemiopsis gomphomacromioides 22 40 Austrocordulia refracta 44 Choristhemis flavoterminata Archeophya magnifica 'Higher' 81 Eusynthemis brevistyla Austrophya mystica Libelluloids 42 Pseudocordulia circularis 7 Cordulephya pygmea 32 Lathrocordulia metallica 57 Micromidia atrifrons Hespercordulia berthoudi A 98 Calophlebia interposita 95 Tetrathemis polleni * Tetrathemis polleni B 9 Hydrobasileus brevistylus 76 Aethriamanta rezia Urothemistinae Urothemis assignata D* Perithemis tenera 97 Rhyothemis semihyalina Rhyothemis semihyalina 99 81 Leucorrhinia glacialis D* 52 Celithemis elisa 93 Sympetrum janeae Leucorrhiniinae Sympetrum ambiguum 5 Uracis fastigiata 15 Acisoma panorpoides 48 Erythemis simplicicollis G 18 Pachydiplax longipennis Nannothemis bella 27 3 Nannophya dalei Bradinopyga strachani Libellulidae 17 100 Hemistigma albipuncta 78 Palpopleura jucunda Palpopleura lucia Erythrodiplax minuscula 19 Diplacodes haematodes 100 Crocothemis servilia Crocothemis erythraea Elga leptostyla 96 Zenithoptera fasciata Zenithoptera fasciata 60 Macrothemis hemichlora 32 Macrothemis celeno 3 29 Scapanea frontalis 34 Paltothemis lineatipes Brechmorhoga mendax E&F 100 Dythemis fugax Dythemis multipunctata 84 Micrathyria aequalis Micrathyria aequalis 100 Tramea onusta 66 Dasythemis esmeralda Tramea lacerata 50 Brachydiplax denticudata Chalcostephia flavifrons 5 68 Deielia phaon 16 Brachythemis leucosticta 37 Tholymis tillarga Zyxomma petiolatum Zyxomma elgneri Idiataphe amazonica 94 Trithemis dorsalis Trithemis monardi 100 Pantala flavescens 5 Pantala flavescens 100 Zygonyx natalensis Zygonyx torridus 76 Nannophlebia risi 58 Huonia oreophila 3 100 Onychothemis culminicola C&H 97 Onychothemis testacea Onychothemis testacea 28 Misagria species Hadrothemis defecta Neodythemis pauliani 18 16 Agrionoptera longitudinalis 97 Orthemis ferruginea Orthemis ferruginea 19 Ladona julia Plathemis lydia Libellula luctuosa 18 Libellula pulchella 20 Lyriothemis pachygastra 83 Libellula quadrimaculata Libellulinae Libellula quadrimaculata Orthetrum species Orthetrum chrysis 81 Orthetrum species 70 Orthetrum pruinosum Orthetrum abbotti 97 Orthetrum julia Orthetrum julia

Fig. 3. Phylogenetic reconstruction from 10,000 replicate parsimony heuristic search. The ‘‘*’’ indicates that this clade differs in composition in the PHASE analysis. Bootstrap support is written above the branches. gely supported by both genes. Subtle differences occur in the 28S analysis while there is little support for the sub- between the 16S and 28S in the composition and/or posi- family Urothemistinae in the 16S analysis; Clade F tion of Clades B and F (Clade B includes Hydrobasileus includes Rhyothemis in the 28S analysis, while Clade F is J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 301 not supported by the 16S analysis). Other differences mistinae as sister to the Libellulidae. In the remainder of between the 16S and 28S were found only in areas with less this section, we compare in more detail our hypothesis with than 50% support. previous systematic treatment of Libelluloidea. Our hypothesis testing revealed that neither Gompho- macromiinae nor Synthemistinae were present in any of 4.1.1. The GSI clade the 69,009 trees (after burnin discarded) created by our Our analysis places Synthemistinae relatively basally PHASE analysis. Idionychinae was recovered, in 15,736 among higher libelluloids, as have most earlier studies trees (23%). Bechly’s (1996) Synthemistidae, Gompho- (Trueman’s (1991) study of egg morphology and early lar- macromiidae, Austrocorduliidae, and Oxygastridae were val characteristics; wing venation by Tillyard (1917), Fraser never present. In addition, the monogeneric Pseudocordu- (1957), Davies and Tobin (1985), Carle (1995) Lohmann liidae and Cordulephyidae are nested well within the GSI (1995) and Bechly (1996)). Since Tillyard and Fraser clade and thus do not form the pectinate arrangement of (1940) and Fraser (1957), all authors have regarded Gom- Bechly (Fig. 1g) that would justify family status. Bechly’s phomacromiinae and Idionychinae as distinct from Synthe- Hemicorduliidae (Hemicordulia and Procordulia), is recov- mistinae, although with various internal subdivisions ered in 84% of the trees (Fig. 2), but its nested position does (Carle, 1995; Bechly, 1996, and see Table 1). Our analysis, not suggest that family status is warranted. The libellulid however, fails to support this distinction, with the three subfamilies of Davies and Tobin (1985), with the exception groups mingled within the GSI group. This is a surprising of Leucorrhiniinae and Libellulinae were never recovered. result, since Theischinger and Watson (1984) identified sev- We constrained Pantala and Tramea only but found that eral convincing larval morphological characters favoring they were never recovered together. Dasythemis never such a division. Subsequent authors (e.g., Carle and Lou- occurred within Libellulinae. ton (1994), Carle (1995), Lohmann (1996a,b) and Bechly Parsimony and PHASE analyses of the dataset that (1996)), using additional characters, also found support included GenBank sequences were largely consistent with for the separation of these Synthemistinae and Gompho- our other analyses (Fig. 7). The taxon sample in GenBank macromiinae. In our phylogeny, support is low for many consists mostly of libellulids. In all cases, except for Eryth- relationships within the GSI clade, but, Synthemistinae emis, Pachydiplax, Plathemis, Ladona, Lyriothemis, and and Gomphomacromiinae were never found in any of the Libellula congenerics group together. Because the support near optimal trees from the Bayesian treefile. Consider- values are so low within the Libellulinae, we consider rela- ation of additional morphological or molecular data may tionships among Plathemis, Ladona, Lyriothemis, and suggest a more traditional structure within the clade. It Libellula to be unresolved. Erythemis and Pachydiplax would be very difficult, however, to reconcile our conclu- GenBank sequences are available for the 12S fragment sions with the hypothesis that the Gomphomacromii- only, a fragment we did not sequence, and so they may nae + Idionychinae are paraphyletic with respect to not have enough information to place them with our con- Corduliinae s. s. (e.g., May, 1995b; Bechly, 1996; Loh- generic taxa. Similarly, data may be insufficient to correctly mann, 1996a,b). place Idiataphe, for which we have only the D3 fragment sequence: in the larger dataset, it is placed in Clade A, with 4.1.2. Corduliidae and Macromiinae low support. The Urothemistinae, Leucorrhiniinae, and Corduliidae s. s. and Macromiinae have long been con- Libellulinae remain as monophyletic groups, and because sidered closely related and have been regarded as confamil- the support along the backbone of Libellulidae is low, ial by many workers (Martin, 1906, 1909; Tillyard, 1917; the other groupings (Clades A, C, D, E, F, and G) are ran- Fraser, 1957; Lieftinck, 1971; Davies and Tobin, 1985; domly arranged (and present as a polytomy in the boot- Steinmann, 1997). They share a number of characters, most strap analysis). The composition of the clades does not of which, however, are either plesiomorphies or are also differ greatly. Not surprisingly, the differences between shared with Libellulidae (see below). Gloyd (1959) pro- the phylogenetic reconstructions were found in areas with posed that Macromiinae be raised to family status, but less then 50% bootstrap support. her arguments were based almost entirely on autapomor- phies of Macromiinae. Nevertheless, Gloyd’s suggestion 4. Discussion is not inconsistent with our results. Our data support the monophyly of the Macromiinae; whether this monophy- 4.1. Taxonomic implications letic group deserves family status is a matter of taxonomic preference. The relationship of these two families to Libell- Our results agree with those of most previous dragonfly ulidae is not strongly supported by our data, although systematists in placing Synthemistinae as basal and Libell- most morphological features suggest a corduliid–libellulid ulidae as terminal libelluloids (e.g., Tillyard, 1917; Fraser, sister group relationship. 1957; Carle, 1995; Bechly, 1996). Pfau’s (1991, 2005) inno- vative morphological study of the odonate vesica spermalis 4.1.3. Libellulidae (penis), however, differs radically in placing Cordulegastri- Three putative libellulid subfamilies (Fraser, 1957; dae and related taxa in the Petaluroidea and the Synthe- Davies and Tobin, 1985) are supported, with some modifi- 302 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 cation, as monophyletic by all our analyses (Urothemisti- caused Pantala and Tramea to be placed together previ- nae, a modified Libellulinae and a restricted Tetrathemist- ously (Fig. 4). inae). Urothemistines are recovered as monophyletic near Finally, Diastatopidinae is distributed among Clades F the base of the libellulids (Clade B): this group was consid- (Zenithoptera), G (Palpopleura), and H (Perithemis, ered a family, Macrodiplactidae, by Fraser (1957) and Bec- although with considerable uncertainty). Several authors hly (1996), but their nested position within the Libellulidae (e.g., Fraser, 1957) have noted that members of these gen- in some analyses suggests caution should be used in elevat- era have unusually patterned wings and may mimic Hyme- ing the urothemistines to family status (e.g., Davies and noptera. Possibly this convergence on wasp-like color and Tobin, 1985). The Libellulinae also is apparently mono- behavior has resulted in similarities of morphology that phyletic (Clade H) except for the exclusion of Dasythemis misled previous workers. The larvae of these genera differ esmeralda, which falls into Clade E in all analyses. A markedly in lateral and dorsal abdominal spine develop- monophyletic Libellulinae agrees with the results presented ment, abdomen shape, and epiproct length (Zenithoptera, by Fleck et al. (in press), including their placement of Costa et al., 2004; Palpopleura, Fraser, 1955; Perithemis, Agrionoptera, Misagria, and most notably, Neodythemis Needham et al., 2000). Palpopleura + Hemistigma, placed (they did not include Dasythemis in their analysis). In addi- together in our phylogeny with high support, share several tion, the Leucorrhiniinae (Leucorrhinia + Celithemis, larval characteristics. They both lack dorsal spines, have placed in Clade D) are sister to Sympetrum, which is con- prominent and share a striking pale dorsal stripe sistent with Pilgrim (2006) and Fleck et al. (in press). Sub- (Whiteley et al., 1999). Adults of these two genera also family status for Leucorrhiniinae is probably unwarranted share markedly bicolored pterostigma. None of these char- because they are a small distinct group within a much lar- acters is definitive, but the combination tends to support ger clade (apophyletic sensu Carle (1995), i.e., its distinctive separation of the diastatopidines and the grouping of Pal- autapomorphies have resulted in its assignment to an exag- popleura and Hemistigma. gerated ). Most subclades of Libellulidae recovered in our phylog- Three well-established taxa are clearly polyphyletic. Spe- eny comprise a mixture of taxa from various previously cies usually attributed to Tetrathemistinae, commonly recognized subfamilies. Members of Sympetrinae, Trithe- regarded as the most plesiotypic of libellulid subfamilies, mistinae, and Brachydiplacinae are scattered throughout are scattered throughout Libellulidae, with Tetrathemis Libellulidae and none of these subfamilies were present and Calophlebia in Clade A, Nannophlebia in Clade C, among our PHASE trees. and Neodythemis in Clade H. Thus, a very restricted Tetr- athemistinae might remain as the most basal Libellulidae 4.2. Character evolution (Clade A), but clearly its composition and delimiting char- acters are very different than previously defined. As noted 4.2.1. Adult characters already by Dijkstra and Vick (2006) and Fleck et al. (in As in many taxa, traditional systematic treatment of press), the venational traits used heretofore to define Tetr- Libelluloidea has typically emphasized an essentially linear athemistinae are correlated with narrowing of the wing transformation of characters, especially of wing veins, lead- base and thus are probably subjected to convergence. ing from an ‘‘archaic’’ to a ‘‘modern’’ state. As Fraser Second, the Trameinae is also polyphyletic, although (1957) expressed it, the superfamily ‘‘...exhibits an almost most species cluster loosely in clade E. This clade also unbroken chain of evolution....’’ Such a progression would includes some genera generally thought to be closely only be expected if all phylogenies were perfectly pectinate related to trameines but placed by Fraser (1957) and oth- and without homoplasy. The situation is almost certain to ers in the Zyxommatinae (Tholymis, and Zyxomma). The be less clear-cut in the real world, and such appears to be placement of Idiataphe in Clade E is ambiguous. Morpho- the case based on our phylogeny. logical evidence is inconclusive about its position. For example, the elongation of the anal loop and the Although Davies and Tobin (1985) place it in Trameinae, development of a midrib (Fig. 4) can be considered to pro- its position is unstable in our analyses, quite possibly gress from its ‘‘absence’’ in most non-libelluloids to its because only the D3 fragment was sequenced for this spe- extreme (a boot-shaped structure, with a ‘‘toe’’ and a mid- cies. In addition, its branch is suspiciously, long compared rib) in Libellulidae, although with notable generic excep- to other nearby taxa. Rhyothemis, also previously thought tions (Needham, 1903; Tillyard, 1910; Tetrathemis, to be closely related to trameines, is placed by PHASE Nannothemis, Misagria, and Fylgia for example, are libellu- (with low support) in clade D. The most surprising result lids with reduced anal loops). Our reconstruction, however, with respect to the polyphyly of the trameines is that suggests that either elongation of the anal loop and devel- Pantala is very distantly related to the other trameines, opment of a midrib occurred in parallel, perhaps multiple falling to the base of Clade H in the smaller dataset, times, in the GSI and the MCL clades or this condition and nested within Clade C in the larger dataset. Again, was replaced by a short broad loop independently in Syn- convergent modification of the hindwing base, in this case themistinae and Macromiinae. Moreover, the loop proba- expansion as an adaptation to extended periods of glid- bly has been secondarily lost several times independently ing, could explain the morphological similarity that has in Libellulidae and possibly in members of GSI. A charac- J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 303

Cordulegaster Gomphomacromia* Synthemis*

Cordulia Macromia Palpopleura

Tetrathemis Pantala Libellula

Fig. 4. Forewing and hindwing fragments of several libelluloids. Yellow, anal loop; blue, supra-triangle; red, triangle. Wings are not to scale. The ‘‘*’’ indicates figure modified from Bridges (1994). ter often correlated with the loss of the anal loop is the and camber of the distal portion of the wings. Understand- presence of a ‘‘broken’’ costal side of the triangle, making ing of the details of wing kinematics and aerodynamics the triangle, in fact, quadrangular; this, too, seems to have during flapping flight, however, is as yet too rudimentary evolved multiple times. Several other characters show a to make confident predictions of these effects (Wootton considerable degree of homoplasy between the GSI and and Kukalova-Peck, 2000; Combes and Daniel, 2003a,b, MCL clades. The supposedly increasing alignment of the 2005). antenodal crossveins (these are crossveins at the leading Reduction in the ovipositor, convergently shared with edge of the wings, anterior to the nodus), for example, the Gomphidae (Carle, 1995), is a prominent feature of and the proximity of the HW triangle to the arculus (a flex- Libelluloidea (and considered a synapomorphy of Gom- ion point), both emphasized by Fraser (1957), and to some phidae + Libelluloidea by Bechly, 1996 and Lohmann, extent, by Bechly (1996), vary across the GSI and MCL 1996a,b; Fig. 5). The ovipositor of and Petalu- (although they are most strongly expressed in the Libellu- ridae (and Zygoptera) comprises three pairs of ventral pro- lidae and Corduliidae, respectively). It appears possible cesses. The first and second pairs (anterior and posterior that all of these changes are partially correlated. They gonapophyses) are enclosed by the third (gonoplacs). In may have the effect of reducing, or at least reconfiguring, libelluloids, including Cordulegastridae, the ovipositor is chordwise flexibility of the wing base, especially in the modified for exophytic oviposition (Tillyard, 1917; Carle, hindwing, and perhaps consequently increasing twisting 1995). In Cordulegastridae, the third processes (gonoplacs) 304 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310

Fig. 5. Terminal abdominal segments in lateral view of (a) female Zoraena (Cordulegastridae) and (b) (Libellulidae). Arrows indicate the well-developed first gonapophyses typical of Cord- ulegastridae and some Synthemistinae and Gomphomacromiinae (a) and the nearly obsolete ovipositor, often reduced to a short extension of the sternum of S8 and/or a pair of small scales at the base of S9 in Libellulidae and most Corduliidae (b). Figures from Needham et al. (2000). are vestigial. In the GSI clade, the third processes are absent and at least the second processes are reduced, although in some taxa the first pair is present and nearly as long as in Cordulegastridae. Our results do not allow Fig. 6. Lateral view of male secondary genitalia of (a) Zoraena diastatops us to conclude whether the latter GSI condition is plesio- (Cordulegastridae); (b) Didymops transversa (Corduliidae, Macromiinae); (c) Libellula quadrimaculata (Libellulidae, Libellulinae). In each case the morphic or secondarily (re)developed. In the MCL, the ventral side is upward and the anterior direction to the right, and the first processes are reduced to small flaps and the other abdominal terga are removed to reveal the genitalic structures. Accessory structures are apparently absent except for the probable secondary genitalia are the anterior lamina (AL), anterior hamules (AH; vestige of the styli emerging directly from the ninth sternite absent in L. quadrimaculata and other Libellulidae), anterior frame (AF), (Tillyard, 1917). In a few instances, the eighth (e.g., some posterior hamule (PH), posterior frame (PF), and genital ligula (GL); these structures engage the female ovipositor during copulation and assist Somatochlora) or eighth and ninth sternites (Uracis) are movement of the vesica spermalis. Segments of the vesica spermalis, which secondarily produced to form an ovipositor in MCL functions both for temporary sperm storage before copulation and for species. intromission, are indicated by Roman numerals I, II, III, and IV from Pfau’s (2005) detailed morphological study of the vesica base to apex; these medial structures are most easily visible in A. spermalis (v. s.; Fig. 6), and especially of the distal ‘‘sperm Terminology follows Pfau (1971). pump’’ implies a very different phylogeny than that found here or by most other workers. Most radically, Pfau sug- Carle and Kjer, unpublished data), we feel some confidence gests that cordulegastrids are members of a larger group that Cordulegastridae + Neopetaliidae + Chlorogomphi- that includes petalurids, and gomphids (Fig. 1). He reasons dae are the closest relatives to other Libelluloidea, and that because it appears impossible to derive the complex appropriate outgroups. sperm pump apparatus of libelluloids directly from the cor- Even if our tree were to be re-rooted, however, Pfau’s dulegastrid sperm pump, the multiple similarities of Cord- hypothesis clearly cannot be reconciled with ours for ulegastridae and libelluloids are the result of convergence. higher libelluloids. No previous molecular dataset applies Moreover, he maintains that Corduliinae s.s. are the most broadly to the higher Libelluloidea, but based on our plesiomorphic libelluloid group, with synthemistids as the results we must conclude that the v. s. morphology of Gom- sister taxon to Libellulidae at the apex of the libelluloids. phomacromia + synthemistids is convergent to that of Despite Pfau’s (1971, 1991, 2005) conclusion, our analysis libellulids. It is unclear whether the synthemistid-type or is rooted with Cordulegastridae. Given other, independent the corduliid-type v. s. is plesiomorphic for higher libellu- support, both morphological (Bechly, 1996; Gorb, 1999; loids. Although the GSI is strongly supported, there is such Carle and Kjer, 2002), and molecular (Misof et al., 2001; low internal support that it is hard to make any conclu- J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 305

Neopetalia punctata 100 Taeniogaster obliqua 65 sieboldii O 71 Cordulegaster picta O 48 Cordulegaster boltonii O Kalyptogaster erronea 100 Chloropetalia soarer 100 brunneus O Sinorogomphus sp 28 Idionyx selysi 35 Syncordulia gracilis 100 Macromidia rapida 100 Macromidia rapida 100 Oxygastra curtisii Oxygastra curtisii O 78 Gomphomacromia sp 58 94 Synthemis leachii 99 Synthemis eustalacta 81 100 Cordulephya pygmea GSI 41 Pseudocordulia circularis 64 Lathrocordulia metallica 93 Hespercordulia berthoudi 69 Micromidia atrifrons 62 Austrophya mystica 33 Eusynthemis brevistyla 94 Choristhemis flavoterminata 99 Archeophya magnifica 88 Austrocordulia refracta Synthemiopsis gomphomacromiodes 100 Macromiidae 100 Phyllomacromia contumax 100 Didymops transversa 60 42 Macromia illinoiensis Corduliinae 100 Pentathemis membranulata Aeschnosoma forcipula 86 100 Epitheca princeps Higher libelluloids 72 Tetragoneuria cynosura Tetragoneuria cynosura 100 100 Neurocordulia obsoleta Neurocordulia xanthosoma 66 85 Hemicordulia tau 100 Procordulia smithi 69 Procordulia grayi 100 Cordulia shurtleffi 65 Rialla villosa 93 Helocordulia uhleri 55 74 Cordulia aenea Urothemistinae Somatochlora tenebrosa B 23 Macrodiplax O 86 Urotnemis assignata Aethriamanta rezia robertsi O MCL A 50 58 Idiataphe amazonica 51 Tetrathemis polleni O 98 Calophlebia interposita 81 Tetrathemis polleni Tetrathemis polleni 100 imperatrixO D 100 Rhyothemis semihyalina 100 25 Rhyothemis semihyalina 92 Leucorrhinia glacialis 100 Celithemis elisa O Libellulidae 99 Celithemis elisa 62 Sympetrum corruptum 100 Sympetrum janeae 93 Sympetrum vulgatum O Sympetrum ambiguum 6 E* Hydrobasileus brevistylus 22 100 Tramea onusta Leucorrhiniinae 84 Tramea lacerata 7 66 Dasythemis esmeralda 100 Chalcostephia flavifrons 98 Brachydiplax denticauda 88 Brachythemis leucosticta 100 Deielia phaon 15 96 Zyxomma petiolatum 91 Zyxomma elgneri 98 Tholymis tillarga O 91 Micrathyria aequalis 50 Micrathyria aequalis F 33 Elga leptostyla O 35 50 Erythemis simplicicollis O 54 Pachydiplax longipennis O 43 Zenithoptera fasciata 36 Zenithoptera fasciata 100 Dythemis multipunctata 96 Dythemis fugax 95 Scapanea frontalis 32 Brechmorhoga mendax 5 39 Paltothemis lineatipes 99 Macrothemis celeno Macrothemis hemichlora Uracis fastigiata 30 70 Nannothemis bella 74 Nannophya pygmaea O G 55 Nannophya dalei 55 Acisoma panorpoides 46 63 Pacnydiplax longipennis Erythemis simplicicollis Diplacodes haematodes 40 96 Erythrodiplax minuscula 100 Crocothemis servilia 45 47 Crocothemis erythraea Crocothemis erythraea O 94 Bradinopyga strachani 100 Hemistigma albipuncta 96 Palpopleura jucunda Palpopleura lucia C 92 Perithemis lais O 18 Perithemis tenera 41 100 Pantala flavescens Pantala flavescens 68 76 aequatoris O 97 Trithemis monardi Trithemis dorsalis 47 100 Onychothemis culminicola 86 Onychothemis testacea 100 Onychothemis testacea O 62 Onychothemis testsacea 99 Zygonyx natalensis 58 Zygonyx torridus 100 Nannophlebia risi 41 99 Bironides sp O 89 Huonia epinephela O Huonia oreophila Plathemis depressa O 14 Oxythemis phoenicosceles O 95 Ladona deplaneta O 37 O 17 96 Ladona juliaO Ladona julia 45 Lyriothemis elegantissima O H Ladona fulva O 4 16 Hadrothemis infesta O 12 99 Plathemis lydia 49 34 Plathemis lydia O Platnenin subornata O 100 O Orthetrum cancellatum O 100 31 Orthetrum coerulescens O 58 Orthetrum sp 94 Orthetrum pruinosum 31 Lyriothemis pachygastra Libellulinae 51 Orthetrum sp Orthetrum abbotti 48 47 Orthetrum chrysis 43 97 Orthetrum julia Orthetrum julia 22 100 O 100 Orthetrum brunneum O 21 Orthetrum albistylum O 21Orthetrum brunneum O Orthetrum brunneum O Libellula croceipennis O 12.5 O O O 13 4 61 Libellula quadrimaculata O 38 Libellula quadrimaculata O 10 45 Libellula quadrimaculata Libellula quadrimaculata O 12 O 15 Libellula vibrans O 46 30 O 49 Libellula luctuosa Libellula pulchella 45 20 O 2 30 O 64 Libellula luctuosa O 19 O 11 O 25 Neodythemis pauliani 92 Neodythemis africana O 100 Allorhizucha preussi O Allorhizucha klingi O 4 O 1 18 Agrionoptera longitudinalis 36 O Thermorthemis madagascariensis O 94 Orthemis cultrifornis O 2 94 O 47 Orthemis ferruginea 100 Orthemis ferruginea O 6 Orthemis ferruginea metallica O 0.1 48 28 Libellula pulchella O 10 O 6 Libellula forensis O 15 camerunica O 41 Hadrothemis defecta 100 Misagria parana Misagria parana O

Fig. 7. Phylogenetic reconstruction from a ten thousand replicate parsimony heuristic search. The ‘‘*’’ indicates a clade that differs in composition from the smaller PHASE dataset. The ‘‘’’ indicates that the taxon sequence was downloaded from GenBank. Posterior probabilities are written above the branch except on very short branches where it is written below. sions about the evolution of characters within this group. loids, a more extensive taxon sampling in the GSI and To begin to understand the evolution of the v. s. in libellu- MCL must be examined to uncover any intra- and interfa- 306 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 milial differences that may shed light on v. s. development. although placement of several other genera not studied Studies of the ontogeny of the sperm pump within libellu- here certainly should be examined in the future (e.g., Ido- loids might help clarify its pattern of evolution. Pfau (1971) macromia, Neophya, Heteronias, Libellulosoma, Metaphya described its last larval instar form, but only in Cordulegas- and Williamsonia; the South American Navicordulia, Sant- ter and Libellula. osia, Schizocordulia, andRialla; and the geographically dis- Several authors have noted the tendency toward reduc- junct Antipodochlora of New Zealand). tion of the anterior hamule (a male secondary genetalic Libellulidae probably comprises three previously structure; Fig. 6) in higher libelluloids. Pfau (1971) showed accepted subfamilies, (i.e., Urothemistinae, a very a loss of hamular muscle 9a in Cordulia; once 9a is lost, a restricted Tetrathemistinae, similar to that suggested by correlated loss of 9b occurs in Libellulidae. Here too, how- Dijkstra and Vick (2006), and, with some modification, ever, homoplastic loss of 9a occurs in Synthemis eustalacta; Libellulinae) as well as five additional groups that are con- other synthemistids, as well as gomphomacromiines and sistently recovered (one of these including an apophyletic macromiids that have been investigated retain 9a, and loss Leucorrhiniinae). Closer examination of the morphological of 9b is a unique synapomorphy of Libellulidae, as far as basis of these groupings is needed before a definitive taxon- we know (May, unpublished). omy of the family can be proposed. It was beyond the scope of this study to sequence every in Libellulidae, 4.2.2. Larval characters but clearly the placement of nearly all the remaining genera The value of larval characters in understanding odonate remains uncertain. phylogeny has long been recognized (e.g., Fraser, 1957) Biogeographical studies may help to determine a pattern and often reveals relationships that are unclear from adult of origin with the libellulid groups and clarify the relation- morphology (Carle and Louton, 1994; Novelo-Gutierrez, ships among major taxa within GSI and MCL. In our 1995; Fleck et al., in press). Several larval apomorphies taxon sample several broad geographical patterns sug- unite the Libelluloidea including the scoop-like prementum gested. The GSI, for example, are virtually confined to (lower mouthparts) with numerous setae, and an asymmet- the southern Hemisphere, especially Australia (except for rical proventriculus (foregut) with large tooth-like lobes the Indomalayan Idionyx and Macromidia and the Palae- (Tillyard, 1917 [Libellulidae + Cordulegastridae]; Carle, arctic Oxygastra). Corduliinae and Macromiinae are pre- 1995; Bechly, 1996 [Cavilabiata]). Dentition of the distal dominantly Holarctic and Indomalayan, with substantial border of the labial palps differs among the libelluloids, radiations and/or expansions into South America by the however, again forming what has been interpreted as a lin- former and Africa by the latter. Libellulidae, as a whole, ear transformation from plesiomorphic to apomorphic: is cosmopolitan. from large, irregular teeth that lack setae in Cordulegastri- Future morphological work should be cautious about dae to more or less straight distal borders and shallow cren- creating families or subfamilies based on characters prone ulations with setal tufts or single setae in each concavity in to convergence. While some of the details of our tree are Libellulinae, with intermediates in Macromiidae and Cor- weakly supported and may change with further data and duliinae s. s.(Tillyard, 1917). Theischinger and Watson analysis, most of the framework appears sound. On a much (1984) noted that synthemistid larvae, plus those of Gom- broader scale, this reinforces suggestions that previous phomacromia, Archeophya and Pseudocordulia (GSI) have phylogenetic hypotheses have suffered from: (1) use of more cordulegastrid-like dentition. Cordulegastrid and poorly defined and sometimes inaccurately scored charac- synthemistid larvae also share wing sheaths that extend ters (O’Grady and May, 2003); (2) groups based on sympl- apart from one-another, while in other higher libelluloids esiomorphies; and (3) failure to recognize the widespread the wing sheaths lie parallel (Tillyard, 1910, 1917). Our effects of character correlation and convergence, especially analyses show no evidence of this divide. Furthermore, in aspects of venation (Carle, 1982b; Dijkstra and Vick, the molecular evidence uniting the GSI clade, which has 2006; Fleck et al., in press). An increase in the use of larval strong support (Fig. 2), argues that these characters in and genitalic character information is a promising step for- the gomphomacromiines (other than the three genera listed ward. Future work should use this phylogeny as a tool to above) are convergent with those of the Corduliidae s.s. focus in on phylogenetically problematic areas within the Libelluloidea. Although Libelluloidea is highly speciose, 5. Conclusions often collected, and familiar, our understanding of its phy- logenetic history is only just beginning. Based on our phylogeny, we suggest that higher Libellu- loidea comprises four families: the Gomphomacromiidae Acknowledgments (including Gomphomacromiinae, Idionychinae, Cordule- phyinae, and Synthemistinae of Fraser, 1957, and Davies We thank Jeremy Huff, Kenneth S. Macdonald III, and and Tobin, 1985), the Macromiidae, the Corduliidae, and Dana Price for careful review of the manuscript. Thanks the Libellulidae. The ‘‘Corduliidae’’ s.l. are polyphyletic. also to Frank Carle for many discussions of phylogeny We include in Corduliidae only the taxa defined as Cordu- and character evolution. Thanks to Dennis Paulson for liinae in Fraser (1957), and Davies and Tobin (1985), thoughtful comments on our phylogenetic reconstructions. J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 307

We are greatly indebted to those who collected or loaned us Appendix A (continued) specimens for DNA: F.L. Carle, C. Chaboo, T.W. Donnel- Taxon name Accession No. Author ly, S. Dunkle, P. Grant, J. Huff, B. Mauffray, M. Mbida, J. Michalski, D. Paulson, D.L. Price, A. Rowat, R. Rowe, K. Chlorogomphus AF266088 Misof et al. (2001) brunneus Tennessen, G. Theischinger, R. West, the American Mu- seum of Natural History and the California Academy of Corduliidae: Gomphomacromiinae Sciences. Many thanks to Dave Britton and Gu¨nter Theis- Oxygastra curtisii AF266103 Misof et al. (2001) chinger for assistance with Australian collecting permits Corduliidae: Macromiinae (permit numbers WT2004-10767, and WITK02489604; Macromia splendens AF266048 Misof et al. (2001) loan number 1914). We are also very thankful to George Baskinger, Mary McLaughlin, and Wlodek Lapkiewicz Libellulidae: Tetrathemistinae for their assistance in the lab. This work was supported Allorhizucha klingi DQ021437 Fleck et al. (in press) Allorhizucha DQ021434 Fleck et al. (in press) by NSF DEB-0423834. preussi Bironides sp DQ021432 Fleck et al. (in press) Appendix A Malgassophlebia DQ021433 Fleck et al. (in press) aequatoris Although we had samples for the following taxa we were Micromacromia DQ021436 Fleck et al. (in press) unable to amplify any of the gene fragments: Pangaeagas- camerunica ter maculata (Cordulegastridae); Apocordulia macrops, Neodythemis DQ021435 Fleck et al. (in press) , Idomacromia proavita, Metaphya africana elongata, Neocordulia campana, Neocordulia batesi longi- Notiothemis DQ021431 Fleck et al. (in press) pollex, Neophya rutherfordi, Williamsonia fletcheri (Cordu- robertsi liidae); Anatya guttata, Brachydiplax c. chalybea, Tetrathemis polleni DQ021430 Fleck et al. (in press) Thermorthemis DQ021438 Fleck et al. (in press) Cannaphila vibex, Crocothemis sp, , madagascariensis , Orthemis sp 1&2,Tauriphila australis, Thermochoria equivocata, and Trithemis basileri Libellulidae: Brachydiplacinae (Libellulidae). Micrathyria DQ021421 Fleck et al. (in press) In addition, we were unable to amplify the D2 portion didyma of the 28S for Zoraena bilineata (Cordulegastridae); Brac- Nannophya DQ021420 Fleck et al. (in press) pygmaea hymesia herbida, Oligoclada walkeri, and Rhodopygia hol- landi (Libellulidae). Since the D2 region provided many Libellulidae:Leucorrhiniinae of the parsimony-informative characters in our dataset Celithemis elisa DQ021425 Fleck et al. (in press) we chose to exclude these from the analysis. Some taxa suc- Libellulidae: Libellulinae cessfully sequenced for the 28S gene fragment failed to Agrionopuera DQ021439 Fleck et al. (in press) amplify for the 16S gene fragment but were included in insignis the analysis: Chloropetalia soarer (Chlorogomphidae); DQ021441 Fleck et al. (in press) Austrophya mystica, Cordulia shurtleffi, Idionyx selysii, Hadrothemis DQ021440 Fleck et al. (in press) Macromidia rapida, Syncordulia gracilis, andTetragoneuria infesta cynosura (Corduliidae); Dasythemis esmerelda, Elga lepto- Ladona deplaneta AF037187 Kambhampati and styla, Idiataphe amazonica, Libellula quadrimaculata 2, Lyr- Charlton (1999) iothemis pachygastra, Macrothemis celeno, Macrothemis Ladona exusta AF037188 Kambhampati and Charlton (1999) hemichlora, Neodythemis pauliani, Onychothemis testacea Ladona fulva AF266098 Misof et al. (2001) 1, Pantala flavescens 2, Rhodopygia hollandi, Tramea onu- Ladona julia AF037186 Kambhampati and sta, and Zenithoptera fasciata 1, Zenithoptera fasciata 2 Charlton (1999) (Libellulidae). Libellula angelina AF195726 Artiss et al. (2001) We included, in a separate analysis (Fig. 5), 12S and 16S Libellula auripennis AF037176 Kambhampati and sequences for several libelluloids from GenBank (below). Charlton (1999) Libellula axilena AF037175 Kambhampati and Taxon name Accession No. Author Charlton (1999) Cordulegasteridae and Chlorogomphidae Libellula comanche AF037182 Kambhampati and Cordulegaster AF266056 Misof et al. (2001) Charlton (1999) boltoni Libellula composita AF195727 Artiss et al. (2001) Cordulegaster AF266086 Misof et al. (2001) Libellula AF037183 Kambhampati and picta croceipennis Charlton (1999) Anotogaster AB127061 Hasegawa and Libellula cyanea AF037177 Kambhampati and sieboldi Kasuya (2006) Charlton (1999) (continued on next page) 308 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310

Appendix A (continued) Appendix A (continued) Taxon name Accession No. Author Taxon name Accession No. Author Libellula flavida AF195728 Artiss et al. (2001) Sympetrum AF037192 Kambhampati and Libellula forensis AF195729 Artiss et al. (2001) corruptum Charlton (1999) Libellula incesta AF037179 Kambhampati and Sympetrum DQ021426 Fleck et al. (in press) Charlton (1999) vulgatum Libellula jesseana AF037174 Kambhampati and Libellulidae: Trithemistinae Charlton (1999) Huonia epinephela DQ021429 Fleck et al. (in press) Libellula luctuosa 12S: AY282563 Saux et al. (2003) 16S: AF037178 Kambhampati and Libellulidae: Onychothemistinae Charlton (1999) Onychothemis DQ021427 Fleck et al. (in press) Libellula needhami AF195730 Artiss et al. (2001) testacea Libellula nodistica AF195731 Artiss et al. (2001) Libellula pulchella AF037180 Kambhampati and Libellulidae: Palpopleurinae Charlton (1999) Perithemis lais DQ021422 Fleck et al. (in press) Libellula DQ021418 Fleck et al. (in press) Libellulidae: Trameinae quadrimaculata Tholymis citrina DQ021423 Fleck et al. (in press) Libellula AF037171 Kambhampati and Rhyothemis variegata DQ021428 Fleck et al. (in press) semifasciata Charlton (1999) imperatrix Libellula vibrans AF037172 Kambhampati and Charlton (1999) Libellulidae: Urothemistinae Lyriothemis DQ021442 Fleck et al. (in press) DQ021424 Fleck et al. (in press) elegantissima Misagria parana DQ021419 Fleck et al. (in press) Orthemis DQ021444 Fleck et al. (in press) cultriformis References Orthemis discolor DQ021417 Fleck et al. (in press) Orthemis AF195732 Artiss et al. (2001) Artiss, T., Schultz, R.T., Polhemus, D.A., Simon, C., 2001. Molecular ferruginea phylogenetic analysis of the dragonfly genera Libellula, Ladona, and Orthetrum DQ021412 Fleck et al. (in press) Plathemis (Odonata: Libellulidae) based on mitochondrial cytochrome oxidase I and 16S rRNA sequence data. Molecular Phylogenetics and albistylum Evolution 18 (3), 348–361. Orthetrum DQ021413 Fleck et al. (in press) Bechly, G., 1996. Morphologische Untersuchungen am Flu¨gelgea¨der der albistylum rezenten Libellen und deren Stammgruppenvertreter (Insecta; Ptery- Orthetrum DQ021411 Fleck et al. (in press) gota; Odonata) unter besonderer Beru¨cksichtigung der Phylogenetis- brunneum chen Systematik und des Grundplanes der Odonata. Petalura special Orthetrum DQ021414 Fleck et al. (in press) vol. 2, 402. brunneum Bridges, C.A., 1994. Catalogue of the family-group, genus-group and Orthetrum DQ021415 Fleck et al. (in press) species-group names of the Odonata of the world, Third ed. C.A. brunneum Bridges, Urbana, IL. Orthetrum DQ021416 Fleck et al. (in press) Carle, F.L., 1982a. A contribution to the knowledge of the Odonata. Unpublished Ph.D. thesis, Virginia Polytechnic Institute and State brunneum University, Blacksburg, Virginia. 1095 pp. Orthetrum AF266097 Misof et al. (2001) Carle, F.L., 1982b. The wing vein homologies and phylogeny of the cancellatum Odonata: a continuing debate. Societas Internationalis Odonatologica Orthetrum DQ021445 Fleck et al. (in press) Rapid Communications, Rapid Communication 4, x+66 pp. coerulescens Carle, F.L., 1995. Evolution, taxonomy, and biogeography of ancient Oxythemis DQ021443 Fleck et al. (in press) Gondwanian libelluloides, with comments on anisopteroid evolution phoenicosceles and phylogenetic systematics (Anisoptera: Libelluloidea). Odonato- Plathemis depressa AF195762 Artiss et al. (2001) logica 24 (4), 383–506. Plathemis lydia AF037184 Kambhampati and Carle, F.L., Kjer, K.M., 2002. Phylogeny of Libellula Linnaeus (Odonata: Charlton (1999) Insecta). Zootaxa 87, 1–18. Carle, F.L., Louton, J.A., 1994. The larva of Neopetalia punctata and Plathemis AF037185 Kambhampati and establishment of fam.nov. (Odonata). Proceedings of subornata Charlton (1999) the Entomological Society of Washington 96 (1), 147–155. Libellulidae: Sympetrinae Combes, S.A., Daniel, T.L., 2003a. Flexural stiffness in wings. I. Crocothemis AF266100 Misof et al. (2001) Scaling and the influence of wing venation. Journal of Experimental Biology 206 (17), 2979–2987. erythrea Combes, S.A., Daniel, T.L., 2003b. Flexural stiffness in insect wings. II. Erythemis 12S: AY282566 Saux et al. (2003) Spatial distribution and dynamic wing bending. Journal of Experi- simplicicollis 16S: AF037191 Kambhampati and mental Biology 206 (17), 2989–2997. Charlton (1999) Combes, S., Daniel, T.L., 2005. Flexural stiffness in insect wings: effects of Pachydiplax AF037189 Kambhampati and wing venation and stiffness distribution on passive bending. American longipennis Charlton (1999) Entomotologist 51 (1), 42–45. J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 309

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