Phylogeny of the higher Libelluloidea (Anisoptera: Odonata): An exploration of the most speciose...

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Molecular Phylogenetics and Evolution 45 (2007) 289–310

Phylogeny of the higher Libelluloidea (Anisoptera: Odonata):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, USAb 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 2007Available online 4 July 2007

Abstract

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 quamconsensu 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 ribosomalRNA (rRNA), and using models that take into account non-independence of correlated rRNA sites. Ninety-three ingroup taxa andsix 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 previouslysuggested monophyletic groups were supported by each analysis. Macromiinae, Corduliidae s. s., and Libellulidae are each monophy-letic. The corduliid (s.l.) subfamilies Synthemistinae, Gomphomacromiinae, and Idionychinae form a monophyletic group, separate fromthe Corduliinae. Libellulidae comprises three previously accepted subfamilies (Urothemistinae, a very restricted Tetrathemistinae, and amodified 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 evolutionof adult and larval morphological characters is discussed here to suggest areas for future focus. This study shows the inherent problemsin using poorly defined and sometimes inaccurately scored characters, basing groups on symplesiomorphies, and failure to recognize thewidespread 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

Dragonflies are among the most recognizable of insects,even having become subjects of extensive folklore (Sarot,1958) and, moreover, have been used in a wide array ofstudies dealing with functional morphology, behavior,ecology, and evolution (Corbet, 1999). Odonata are consid-ered to be among the earliest flying insects. Their recogniz-

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doi:10.1016/j.ympev.2007.05.027

* Corresponding author. Fax: +1 732 932 7229.E-mail address: [email protected] (J. Ware).

able progenitors date to the Carboniferous (360-290million years ago) and are probably the most widely knownextinct insects. Anisoptera (in their present form) aroselater, with earliest known fossils from the Triassic (250–200 million years ago; Grimaldi and Engel, 2005). Whileclearly identifiable libelluloids are not well known fromthe Jurassic (206–142 million years ago), Jarzembowskiand Nel (1996) suggest that libelluloids were already wellestablished in the Early Cretaceous (142–65 million yearsago). Extant libelluloids include, among others, the wide-spread Macromiinae and Corduliinae and the most abun-dant and familiar dragonflies, Libellulidae. Libellulidae

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290 J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310

are readily recognizable, often with colored or patternedwings and a boot shaped series of veins (the anal loop) inthe hindwing. They are commonly seen in territorial flightaround lakes and ponds, or perched along the bank.

Among libelluloids, adult reproductive and feedingbehavior, larval behavior and ecology (Corbet, 1999),and biogeography (Carle, 1995) vary widely and have beeninvestigated intensively. While it is clear that a well-sup-ported phylogenetic hypothesis is needed in order to reachan understanding of the evolution of these traits, phyloge-netic relationships among libelluloid families remain highlycontentious, with numerous hypotheses proposed (Fig. 1and Table 1). For descriptive purposes, we use the taxon-omy of Davies and Tobin (1985) unless otherwise indi-cated, since it is widely used today (Table 2).

Morphological studies of libelluloid phylogeny haverelied heavily, although not exclusively, on wing vein char-acters (Kirby, 1890; Needham, 1903, 1908; Martin, 1907;Ris, 1909–1919; Tillyard, 1910; Needham and Broughton,1927; Fraser, 1957; Gloyd, 1959; Geijskes, 1970; Lieftinck,1971; Theischinger and Watson, 1978; Carle, 1982a; Davies

(c) Glo(a) Rambur, 1842* (b) Kirby, 1890*

(e) Fraser, 1957 (f) Carle and Louton, 1994*

(g) BLohm

Cor

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Sist

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axa

Libe

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ae

Cor

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Sist

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Mac

rom

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Cor

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Synt

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omph

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Libe

llulid

ae

Cor

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Sist

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Mac

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ae

Synt

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istid

ae

Mac

rodi

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tidae

Cor

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axa

Corduliidae

Libe

llulid

ae

Cor

dule

gast

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Sist

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axa

Mac

rom

iinae

Cor

duliin

ae

Libellulidae

Sist

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Cor

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ae

Libe

llulin

ae

Cor

dule

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Fig. 1. Hypotheses, also listed in Table 1, of relationships within Libelluloideatwo subfamilies: Corduliinae and Libellulinae. *Some who use this taxonomyfamilies (Corduliidae and Libellulidae). The Corduliidae comprises two subfamhe used the term ‘Corduliinae’; (c) three higher Libelluloid families (MacCorduliinae and Libellulinae; (d) four higher Libelluloid families (SynthemistFraser (1957) but excludes Macrodiplactidae; (e) four higher Libelluloid famused the name ‘‘Synthemidae’’ for Synthemistidae; (f) five higher Libelluloid faand Libellulidae). *This is loosely based on Carle and Louton, 1994; (g) twIdomacromiidae, Austrocorduliidae, Oxygastridae, Idionychidae, CordulepLibellulidae). This scheme is loosely based on Bechly (1996) and LohmannLibelluloid families (Synthemistidae, Corduliidae, and Libellulidae). Petaluroi�In 2005, Pfau also includes the families Cordulephyidae and Gomphomacrom

and Tobin, 1985; Carle and Louton, 1994; Carle, 1995;Bechly, 1996; Lohmann, 1996a,b; Trueman, 1996; Jarzem-bowski and Nel, 1996; Carle and Kjer, 2002; Rehn, 2003).Despite progress in understanding homologies in Odonatavenation (e.g., Carle, 1982b; Riek and Kukalova-Peck,1984), many wing vein characters may support convergentrelationships when used to the exclusion of other charac-ters (Hennig, 1969; Carle, 1982b). The 11 subfamilies ofLibellulidae recognized in Davies and Tobin (1985) andBridges (1994) were largely based on wing vein morphol-ogy. Bechly’s (1996) morphological study of the Odonataalso relied heavily on venational characters to break upthe higher Libelluloidea into numerous families: Gompho-macromiinae and Synthemistinae were split into eight fam-ilies and the remaining Corduliidae and the Libellulidaewere each divided into two families.

Some studies have focused on egg, genitalic, flight mus-culature, color, and larval characteristics (St. Quentin,1939; Gloyd, 1959; Lieftinck, 1971; Pfau, 1971; Theischin-ger and Watson, 1984; Pfau, 1991, 2005; May, 1995a;Bechly, 1996; Lohmann, 1996a; Carle and Kjer, 2002).

yd, 1959 (d) *

echly, 1996; ann, 1996

(h) Pfau, 1971, 1991, 2005

Libe

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ae

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ae

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ae

Aust

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Hem

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ae

Cor

dule

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dae

Idio

nych

idae

Pseu

doco

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llulid

ae

Aesh

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e

Peta

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idea

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ae

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lidae

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ae

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ae

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ae

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Libellulidae

. Topologies are (a) one higher Libelluloid family (Libellulidae) comprisesinclude a third subfamily, the Macromiinae; (b) Two higher Libelluloid

ilies: Corduliinae, Macromiinae. *This is loosely based on Kirby, 1890, butromiidae, Synthemistidae and Libellulidae). The Libellulidae comprisesidae, Corduliidae, Macromiidae, Libellulidae). *This is loosely based onilies Synthemistidae, Corduliidae, Libellulidae, Macrodiplactidae). Frasermilies (Synthemistidae, Gomphomacromiidae, Corduliidae, Macromiidaeelve higher Libelluloid families (Synthemistidae, Gomphomacromiidae,hyidae, Hemicorduliidae, Macromiidae, Corduliidae, Urothemistidae,(1996a,b) although the nomenclature differed slightly; (h) three higher

dea includes Cordulegastridae. Placement of Macromiidae not discussed.iidae, placed basal to Synthemistidae + Libellulidae.

Table 1A 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 familiesstudied

MCLhypothesissupported

Dataset Analysis

Carle and Kjer(2002)

9 Non-Libelluloid Anisopteranfamilies; 4 Libelluloid families

Neopetaliidae,Cordulegastridae,Corduliidae, Libellulidae

A Morphology Computer-assistedparsimony

Carle (1982a) Odonata Cordulegastridae,Corduliidae, Libellulidae

A Morphology Manual parsimony

St. Quentin (1939) 3 Libelluloid families Cordulegastridae,Corduliidae, Libellulidae

A Morphology:genetalia

Intuition

Martin (1914) 1 Libelluloid family Corduliidae A Morphology IntuitionTillyard (1917) Odonata Neopetaliidae,

Cordulegastridae,Corduliidae, Libellulidae

A Morphology Intuition

Needham (1908) 2 Libelluloid families Corduliidae, Libellulidae A Morphology IntuitionMartin (1907) 1 Libelluloid family Corduliidae A Morphology IntuitionNeedham (1903) Anisoptera Neopetaliidae,



Selys (1892) Anisoptera Cordulegastridae,Corduliidae


Kirby (1890) Odonata Cordulegastridae,Corduliidae, Libellulidae


Hagen (1861) Odonata Corduliidae, Libellulidae, A Morphology IntuitionRambur (1842) Odonata Neopetaliidae,



Misof et al. (2001) 4 non-Libelluloid Anisopteran families;4 Libelluloid families


B Molecular:16s and 12s

Computer-assistedparsimony andlikelihood

Bridges (1994) Odonata Neopetaliidae,Cordulegastridae,Corduliidae, Libellulidae

B — —

Davies and Tobin(1985)

Anisoptera Neopetaliidae,Cordulegastridae,Corduliidae, Libellulidae

B Morphology Manual parsimony

Steinmann (1997) Odonata Neopetaliidae,Cordulegastridae,Corduliidae, Libellulidae

B — —

Lieftinck (1977) 1 Libelluloid families Corduliidae B Morphology Manual parsimonyLieftinck (1971) 1 Libelluloid families Corduliidae B Morphology IntuitionTillyard (1928) 2 Libelluloid families Corduliidae, Libellulidae B Morphology IntuitionGloyd (1959) 1 Libelluloid family Corduliidae C Morphology IntuitionJarzembowski and

Nel (1996)Libelluloid fossils 4 Libelluloid families Neopetaliidae,


D Morphology Computer-assistedparsimony

Nel and Paicheler(1994)

2 Libelluloid families Cordulegastridae,Corduliidae,

D * NoLibellulidaestudied

Morphology Manual parsimony

Theischinger andWatson (1978)

1 Libelluloid families Corduliidae E Morphology Manual parsimony

Fraser (1957) Zygoptera + Anisozygoptera 3 Non-Libelluloid families 2 Libelluloidfamilies


E Morphology Intuition

Carle (1995) Libelluloid fossil: Nothomacromia 3Libelluloid Anisoptera


F Morphology Manual parsimony

Carle and Louton(1994)

4 Non-Libelluloid Anisoptera families 4Libelluloid families

Neopetaliidae,Cordulegastridae,Libellulidae

F* NoCorduliidaestudied

Morphology Manual parsimony

Lohmann (1996a,b) 4 Non-Libelluloid Anisopteranfamilies, 4 Libelluloid families


G Morphology Manual parsimony

(continued on next page)

J. Ware et al. / Molecular Phylogenetics and Evolution 45 (2007) 289–310 291

Table 1 (continued)

Author and year Taxa studied Extant Libelluloid familiesstudied

MCLhypothesissupported

Dataset Analysis

Bechly (1996) Zygoptera, 4 Non-Libelluloid Anisopteranfamilies, 4 Libelluloid families

Neopetaliidae,Cordulegastridae, Corduliidae,Libellulidae

G Morphology Manualparsimony

Pfau (2005) 3 Non-libelluloid families 4 libelluloidfamilies

Neopetaliidae,Cordulegastridae, Cordullidae,Libellulidae

H Morphology:genitalia

Manualparsimony

Pfau (1991) Zygoptera + Anisozygoptera 2 Non-libelluloid families 3 libelluloid families

Cordulegastridae, Cordullidae,Libellulidae

H Morphology:secondarygenitalia

Manualparsimony

Pfau (1971) Zygoptera + Anisozygoptera 2 Non-libelluloid families 3 libelluloid families

Cordulegastridae, Cordullidae,Libellulidae

H Morphology:secondarygenitalia

Manualparsimony


Pfau’s (2005) study of sperm transfer mechanisms lead himto an alternate phylogenetic hypothesis that placed Cord-ulegastridae, Chlorogomphidae, and Neopetaliidae withinPetaluroidea rather than Libelluloidea. Much of the cur-rent confusion over libelluloid taxonomy and phylogenymay be the result of uncertain character homology andindependence (reviewed in Carle, 1982b). An independentmolecular dataset may help resolve conflicting phylogenetichypotheses.

Several recent molecular studies (Kambhampati andCharlton, 1999; Artiss et al., 2001; Saux et al., 2003; Hovm-oller and Johansson, 2004) have included many libellulinetaxa, and some (Misof et al., 2001; Misof and Fleck,2003; Hovmoller and Johansson, 2004; Hasegawa and Kas-uya, 2006) included a broader sampling across the super-family. Most of these studies were based on a single gene(Kambhampati and Charlton, 1999; Misof et al., 2001;Artiss et al., 2001; Hovmoller et al., 2002; Misof and Fleck,2003; Saux et al., 2003). Because their question focused onsubordinal relationships, Saux et al. (2003) used Locusta

migratoria as an outgroup, which may have been too dis-tantly related to answer questions about the internal orderof the families (Farris, 1982; Lyons-Weiler et al., 1998;Graham et al., 2002). The most recent study (Fleck et al.,in press), includes a large taxon sample, primarily consist-ing of tetrathemistine and libelluline Libellulidae, and com-bines molecular data with larval morphology.

Our purpose here is to present a phylogenetic hypothesisof the higher Libelluloidea (i.e., Corduliidae and Libelluli-dae of Davies and Tobin, 1985), generated from two inde-pendent gene fragments, (mitochondrial and nuclear largeribosomal RNA subunits; 16S and 28S), structurallyaligned, using basal libelluloid outgroups, and the follow-ing methods that model the correlated rRNA sites asnon-independent. Extensive taxon sampling has allowedus to assess several regions of contention in the higherLibelluloidea and to propose historical relationships withinthis group. The phylogenetic reassessment provides a basisfor improving the taxonomy in the historically difficultLibelluloidea.

2. Materials and methods

2.1. Taxon sampling

The superfamily Libelluloidea includes the Libellulidae,with 143 genera and 969 species, the most species-rich andcommonly observed family of dragonflies worldwide, aswell as the Corduliidae Kirby, 1890 (43 genera, 406 species)(Davies and Tobin, 1985; Steinmann, 1997; number of spe-cies from Schorr et al., 2006). These are the ‘‘higher’’Libelluloidea, which are the main focus of this study. Wealso include the basal libelluloids (sensu Carle, 1995) Cord-ulegastridae Calvert, 1893 (4 genera, 49 species), Chloro-gomphidae Needham, 1903 (3 genera, 46 species), and themonotypic Neopetaliidae Tillyard and Fraser, 1940.

Taxa sequenced are listed in Table 2. Cordulegastridae,Chlorogomphidae, and Neopetaliidae served as outgroups,with the tree rooted using Neopetalia punctata (2 speciesfrom 2 cordulegastrid genera, 2 species from 2 chloro-gomphid genera and 1 species from the monotypic Neope-taliidae). We sampled as broadly as we could across each ofthe libelluloid families, extracting from individuals of everysubfamily in every libelluloid family (3 species from 3 macr-omiine genera, 14 species from 11 corduliine corduliid gen-era, 18 species from 17 other non-corduliine corduliidgenera, and 70 species from 56 libelullid genera) (Table 2).

2.2. Gene selection, DNA extraction, and PCR amplification

Freshly collected dragonflies were used when possible;other taxa were obtained from personal and museum col-lections (Table 2). We amplified the second, third, and sev-enth hypervariable (divergent) regions (D2, D3, and D7) ofthe nuclear large subunit rDNA (28S) and the third domainof the mitochondrial large subunit rDNA (16S).

Muscle tissue was extracted using a Qiagen Dneasy tis-sue kit overnight at 55 �C with 180 ll of ATL Buffer and20 ll Proteinase-K. Older specimens (collected prior to1980) were extracted with 40 ll (twice the suggestedamount) of Proteinase-K buffer for several days (a sugges-

Table 2Taxon list for the present study. Taxonomy based on Bridges (1994) andDavies and Tobin (1985)

Taxon Locality GenbankNumber

CordulegastridaeTaeniogaster obliqua USA: NJ (M. L. May) D7: EF631216

D3: EF631312D2: EF63142016S: EF631533

Pangaeagaster maculata USA: NJ (M. L. May) N/AKalyptogaster erronea USA: NJ (M. L. May) D7: EF631245

D3: N/AD2: EF63145016S: EF631561

Zoraena bilineata USA: MD (M. L. May) N/A

ChlorogomphidaeChloropetalia soarer Sequences from F.L. Carle D7: EF631248

D3: EF631339D2: EF63145316S: N/A

Sinorogomphus sp Sequences from F.L. Carle D7: EF631249D3: EF631340D2: EF63145416S: EF631564

NeopetaliidaeNeopetalia punctata Sequences from F.L. Carle D7: EF631247

D3: EF631338D2: EF63145216S: EF631563

Corduliidae: CordulephyinaeCordulephya pygmea Australia (Theischinger) D7: EF631255

D3: EF631346D2: EF63146016S: EF631570

Corduliidae: CorduliinaeAeschnosoma forcipula French Guiana (J. Huff) D7: EF631223

D3: EF631319D2: EF63142716S: EF631540

Cordulia shurtleffii Canada: Ontario(J. L. Ware, J.Huff)

D7: EF631232D3: EF631326D2: EF63143516S: N/A

Cordulia aenea Sweden (K.M. Kjer) D7: EF631286D3: EF631383D2: EF63150016S: EF631603

Dorocordulia lepida USA: NJ (M. L. May) N/AEpitheca princeps USA: NJ (J. L. Ware) D7: EF631205

D3: EF631302D2: EF63140716S: EF631521

Helocordulia uhleri USA: NJ (M. L. May) D7: EF631227D3: N/AD2: EF63143116S: EF631544

Hemicordulia tau Australia (J. L. Ware,K. M. Kjer, F. L. Carle)

D7: EF631233D3: EF631328D2: EF63143716S: EF631550

Neurocordulia obsoleta USA: FL (M. L. Mayand K. Tennessen)

D7: EF631196D3: N/AD2: EF63139516S: EF631509

Metaphya elongata New Caledonia(Tobin & Davies)

N/A

Table 2 (continued)


Pentathemis membranulata Australia (F.L. Carle) D7: EF631211D3: EF631308D2: EF63141516S: EF631528

Neurocordulia xanthosoma USA: AK (M. L. May) D7: EF631242D3: N/AD2: EF63144716S: N/A

Procordulia grayi New Zealand (R. Rowe) D7: EF631199D3: N/AD2: EF63139916S: EF631513

Procordulia smithi New Zealand (R. Rowe) D7: EF631200D3: EF631295D2: EF63140016S: EF631514

Rialla villosa Chile (Heppner) D7: EF631273D3: EF631364D2: EF63148016S: EF631590

Somatochlora tenebrosa USA: NJ (M. L. May) D7: EF631215D3: EF631311D2: EF63141916S: EF631532

Tetragoneuria cynosura USA: NJ (M. L. May) D7: N/AD3: EF631379D2: N/A16S: N/A

Tetragoneuria cynosura USA: NJ (M. L. May) D7: EF631231D3: EF631325D2: N/A16S: N/A

Williamsonia fletcheri USA: MA (M. L. May) N/A

Corduliidae: GomphomacromiinaeApocordulia macrops Australia

(G. Theischinger)N/A

Archaeophya magnifica Australia (K. M. Kjer) D7: N/AD3: EF631356D2: EF63147016S: EF631580

Austrocordulia refracta Australia(G. Theischinger)

D7: EF631243D3: EF631336D2: EF63144816S: EF631559

Austrophya mystica Australia (F. L. Carle) D7: EF631236D3: EF631332D2: EF63144116S: N/A

Gomphomacromia chilensis

& paradoxa

Chile (F. L. Carle) D7: EF631206D3: EF631303D2: EF63140816S: EF631522

Hespercordulia berthoudi Australia (F. L. Carle) D7: EF631244D3: EF631337D2: EF63144916S: EF631560

Lathrocordulia metallica Australia (F. L. Carle) D7: EF631239D3: EF631334D2: EF63144416S: EF631556

Micromidia atrifrons Australia (M. L. Mayand F. L. Carle)

D7: EF631240D3: N/AD2: EF63144516S: EF631557

Neocordulia batesi longipollex Panama (M. L. May) N/A



Table 2 (continued)


Neocordulia campana Panama (M. L. May) N/AOxygastra curtisii Spain (P. Corbet) D7: N/A

D3: N/AD2: EF63141316S: EF631526

Pseudocordulia

circularis

Australia (G. Theischinger) D7: EF631251D3: EF631342D2: EF63145616S: EF631566

Syncordulia gracilis South African (P. Grant) D7: N/AD3: N/AD2: EF63143916S: N/A

Corduliidae: IdionychinaeIdionyx selysi Hong Kong (K. Wilson) D7: EF631193

D3: EF631290D2: EF63139116S: N/A

Macromidia rapida 1 Hong Kong (K. Wilson) D7: EF631209D3: EF631306D2: EF63141116S: N/A

Macromidia rapida 2 Hong Kong (K. Wilson) D7: EF631271D3: EF631362D2: EF63147816S: EF631588

Corduliidae: IdomacromiinaeIdomacromia proavita Cameroon (CAS) N/A

Corduliidae: MacromiinaeDidymops transversa USA: NJ (M. L. May) D7: N/A

D3: EF631327D2: EF63143616S: EF631549

Macromia illionoiensis USA: IL (M. L. May) D7: EF631208D3: EF631305D2: EF63141016S: EF631524

Phyllomacromia

contumax

Uganda (T. W. Donnelly) D7: EF631197D3: EF631293D2: EF63139716S: EF631511

Corduliidae: SynthemistinaeChoristhemis

flavoterminata

Australia (M. L. May, K. M.Kjer and F. L. Carle)

D7: EF631237D3: EF631333D2: EF63144216S: EF631554

Eusynthemis brevistyla Australia (J. L. Ware, K. M.Kjer, F. L. Carle)

D7: EF631230D3: EF631323D2: EF63143416S: EF631547

Synthemiopsis

gomphomacromioides

Australia (M. L. May,K. M. Kjer and F. L. Carle);D2 and D7 sequencesfrom F. L. Carle

D7: EF631213D3: N/AD2: EF63141716S: EF631530

Synthemis eustalacta Australia (J. L. Ware,K. M. Kjer, F. L. Carle)

D7: N/AD3: EF631296D2: EF63140116S: EF631515

Synthemis leachii Australia (D. Pryce) D7: EF631201D3: EF631297D2: EF63140216S: EF631516

Corduliidae: NeophyinaeNeophya rutherfordi Liberia (J. Lempert) N/A

Table 2 (continued)


Libellulidae: TetrathemistinaeCalophlebia interposita CAS (Madagascar project) D7: N/A

D3: EF631381D2: N/A16S: N/A

Nannophlebia risi Australia (M. L. May,K. M. Kjer and F. L. Carle)

D7: EF631254D3: EF631345D2: EF63145916S: EF631569

Neodythemis pauliani CAS (Madagascar Project) D7: EF631279D3: EF631368D2: EF63148616S: N/A

Tetrathemis polleni 1 South Africa (M. L. May) D7: EF631222D3: EF631318D2: EF63142616S: EF631600

Tetrathemis polleni 2 South Africa (M. L. May) D7: N/AD3: EF631376D2: EF63149516S: EF631539

Libellulidae: BrachydiplacinaeAnatya guttata Trinidad (S. Dunkle) N/ABrachydiplax

denticauda

Australia (K. M. Kjerand F. L. Carle)

D7: EF631246D3: N/AD2: EF63145116S: EF631562

Brachydiplax

c. chalybea

Thailand (J. Michalski) N/A

Chalcostephia flavifrons Guinea-Bissau (J. Huff) D7: EF631260D3: EF631351D2: EF63146516S: EF631575

Elga leptostyla Trinidad (M. L. May) D7: EF631274D3: N/AD2: EF63148116S: N/A

Micrathyria aequalis Panama (M. L. May) D7: EF631195D3: EF631291D2: EF63139316S: EF631508

Micrathyria aequalis Belize (J. L. Ware, J. Huff) D7: EF631250D3: EF631341D2: EF63145516S: EF631565

Hemistigma albipuncta Senegal (J. Huff) D7: EF631256D3: EF631347D2: EF63146116S: EF631571

Nannophya dalei Australia (J. L. Ware, K. M.Kjer and F. L. Carle)

D7: EF631241D3: EF631335D2: EF63144616S: EF631558

Nannothemis bella USA: FL (M. L. May) D7: EF631210D3: EF631307D2: EF63141216S: EF631525

Nephepeltia phyryne Trinidad (M. L. May) N/AThermochoria

equivocata

Cameroon (Mbida Mbida) N/A

Oligoclada walkeri Trinidad (M. L. May) N/AUracis imbuta Panama (M. L. May) D7: EF631228

D3: N/AD2: EF63143216S: EF631545


Table 2 (continued)


Libellulidae: LeucorrhiniinaeBrachymesia herbida Venezuela (R.West) N/ACelithemis elisa USA: NJ (M. L. May) D7: EF631224

D3: EF631320D2: EF63142816S: EF631541

Leucorrhinia glacialis USA: NY (M. L. May) D7: EF631207D3: EF631304D2: EF63140916S: EF631523

Libellulidae: LibellulinaeAgrionoptera

longitudinalis

Australia (M. L. May,K. M. Kjer andF. L. Carle)

D7: EF631235D3: EF631331D2: EF63144016S: EF631553

Cannaphila vibex Panama (M. L. May) N/ADasythemis esmeralda Trinidad (J. Michalski) D7: N/A

D3: N/AD2: EF63138616S: N/A

Hadrothemis defecta Uganda (T. W. Donnelly) D7: EF631277D3: EF631366D2: EF63148416S: EF631592

Libellula pulchella USA: NJ (J. L. Ware) D7: N/AD3: EF631329D2: N/A16S: EF631551

Libellula luctuosa USA: NJ (J. L. Ware) D7: EF631194D3: N/AD2: EF63139216S: EF631507

Libellula

quadrimaculata 1

USA: NJ (M. L. May) D7: EF631272D3: EF631363D2: EF63147916S: N/A

Libellula

quadrimaculata 2

Sweden (K. M. Kjer) D7: N/AD3: N/AD2: EF63149716S: EF631589

Ladona julia USA: WI (M. L. May) D7: EF631219D3: EF631315D2: EF63142316S: EF631536

Lyriothemis

pachygastra

Japan (M. L. May) D7: EF631276D3: EF631365D2: EF63148316S: N/A

Misagria parana French Guiana (J. Huff) D7: EF631268D3: EF631359D2: EF63147516S: EF631585

Orthemis ferruginea 1 Dominican Republic(J. Huff)

D7: EF631265D3: EF631357D2: EF63147116S: EF631581

Orthemis ferruginea 2 Dominican Republic(J. Huff)

D7: EF631266D3: N/AD2: EF63147216S: EF631582

Orthetrum sp 1 Guinea-Bissau (J. Huff) D7: N/AD3: N/AD2: EF63139616S: EF631510

Orthetrum sp 2 South Africa (K. M. Kjer) D7: EF631261D3: EF631352D2: EF63146616S: EF631576

Table 2 (continued)


Orthetrum sp 3 South Africa (K. M. Kjer) N/A

Orthetrum sp 4 Senegal (J. Huff) N/A

Orthetrum abbotti CAS (Madagascar) D7: EF631275D3: N/AD2: EF63148216S: EF631591

Orthetrum chrysis China (X. Zhou) D7: EF631263D3: EF631354D2: EF63146816S: EF631578

Orthetrum julia 1 South Africa (K. M. Kjer) D7: EF631285D3: EF631380D2: EF63149816S: EF631601

Orthetrum julia 2 South Africa (K. M. Kjer) D7: N/AD3: EF631382D2: EF63149916S: EF631602

Orthetrum pruinosum

neglectum

CAS (Madagascar Project) D7: EF631267D3: N/AD2: EF63147316S: EF631583

Plathemis lydia USA: NJ (F. L. Carle) D7: EF631234D3: EF631330D2: EF63143816S: EF631552

Libellulidae: SympetrinaeAcisoma panorpoides Guinea-Bissau (J. Huff) D7: EF631229

D3: EF631322D2: EF63143316S: EF631546

Bradinopyga strachani Guinea-Bissau (J. Huff) D7: EF631257D3: EF631348D2: EF63146216S: EF631572

Brachythemis

leucosticta

Guinea-Bissau (J. Huff) D7: EF631258D3: EF631349D2: EF63146316S: EF631573

Crocothemis erythraea South Africa (M. L. May) D7: EF631225D3: EF631321D2: EF63142916S: EF631542

Crocothemis servilla USA: FL (M. L. May) D7: EF631192D3: EF631289D2: EF63139016S: EF631506

Crocothemis sp Senegal (J. Huff) N/ADeielia phaon Japan (M. L. May) D7: EF631226

D3: EF631543D2: EF63143016S: N/A

Diplacodes haematodes Australia (J. L. Ware,K. M. Kjer, F. L. Carle)

D7: EF631238D3: N/AD2: EF63144316S: EF631555

Erythemis simplicicollis USA: TX (M. L. May) D7: EF631191D3: EF631288D2: EF63138916S: EF631505

Erythrodiplax minuscula USA: FL (J. L. Wareand J. Huff)

D7: EF631190D3: EF631287D2: EF63138816S: EF631504



Table 2 (continued)


Pachydiplax longipennis USA: NJ (F. L. Carle) D7: EF631198D3: EF631294D2: EF63139816S: EF631512

Rhodopygia hollandi Trinidad (M. L. May) N/ASympetrum janeae USA: NJ (F. L. Carle) D7: EF631214

D3: EF631310D2: EF63141816S: EF631531

Sympetrum ambiguum USA: DE (M. L. May) D7: N/AD3: EF631324D2: N/A16S: EF631548

Libellulidae: TrithemistinaeBrechmorhoga mendax USA: TX (M. L. May) D7: EF631572

D3: N/AD2: EF63138516S: EF631502

Dythemis fugax USA: TX (M. L. May) D7: N/AD3: N/AD2: EF63138716S: EF631503

Dythemis multipunctata Panama (M. L. May) D7: EF631259D3: EF631350D2: EF63146416S: EF631574

Huonia oreophila New Guinea (J. Michalski) D7: EF631270D3: EF631361D2: EF63147716S: EF631587

Macrothemis celeno Puerto Rico (M. L. May) D7: EF631282D3: EF631370D2: EF63148916S: EF631594

Macrothemis hemichlora Panama (M. L. May) D7: N/AD3: EF631292D2: EF63139416S: N/A

Macrothemis pulmila Trinidad (M. L. May) N/APaltothemis lineatipes USA: CA (M. L. May) D7: N/A

D3: EF631373D2: EF63149216S: EF631597

Scapanea archboldi Dominican Republic (J. Huff) D7: EF631269D3: EF631360D2: EF63147616S: EF631586

Trithemis basileri South Africa (K. M. Kjer) N/ATrithemis dorsalis South Africa (M. L. May) D7: N/A

D3: EF631299D2: EF63140416S: EF631518

Trithemis monardi Guinea-Bissau (J. Huff) D7: EF631264D3: EF631355D2: EF63146916S: EF631579

Libellulidae: OnychothemistinaeOnychothemis

culminicola

Thailand (T. W. Donnelly) D7: N/AD3: EF631374D2: EF63149316S: EF631598

Onychothemis testacea 1 Thailand (T. W. Donnelly) D7: EF631278D3: EF631367D2: EF63148516S: EF631599

Table 2 (continued)


Onychothemis

testacea 2

Thailand (T. W. Donnelly) D7: EF631284D3: EF631375D2: EF63149416S: N/A

Libellulidae: PalpopleurinaePalpopleura jucunda South Africa (M. L. May) D7: N/A

D3: N/AD2: EF63141416S: EF631527

Palpopleura lucia Senegal (J. Huff) D7: EF631262D3: EF631353D2: EF63146716S: EF631577

Perithemis tenera New Jersey (J. L. Ware) D7: EF631212D3: EF631309D2: EF63141616S: EF631529

Zenithoptera fasciata 1 French Guiana (J. Huff) D7: EF631283D3: EF631371D2: EF63149016S: EF631595

Zenithoptera fasciata 2 French Guiana (J. Huff) D7: N/AD3: EF631372D2: EF63149116S: EF631596

Libellulidae: TrameinaeTramea onusta USA: NJ (M. L. May) D7: EF631281

D3: N/AD2: EF63148816S: EF631593

Tramea lacerata USA: NJ (J. L. Ware) D7: EF631221D3: EF631317D2: EF63142516S: EF631538

Rhyothemis

semihyalina 1

South Africa (M. L. May) D7: EF631204D3: EF631301D2: EF63140616S: EF631520

Rhyothemis

semihyalina 2

South Africa (C. Chaboo) D7: N/AD3: EF631358D2: EF63147416S: EF631584

Miathyria marcella USA: FL (M. L. May) N/APantala flavescens 1 South Africa (M. L. May) D7: EF631220

D3: EF631316D2: EF63142416S: EF631537

Pantala flavescens 2 Senegal (J. Huff) D7: EF631280D3: EF631369D2: EF63148716S: N/A

Hydrobasileus

brevistylus

Australia (M. L. May, K. M.Kjer and F. L. Carle)

D7: EF631252D3: EF631343D2: EF63145716S: EF631567

Idiataphe amazonica Bolivia (Mauffray) D7: N/AD3: EF631377D2: N/A16S: N/A

Tholymis tillarga Guinea-Bissau (J. Huff) D7: EF631202D3: EF631298D2: EF63140316S: EF631517

Tauriphila australis Panama (M. L. May) N/A


Table 2 (continued)


Libellulidae: UrothemistinaeAethriamanta rezia Guinea-Bissau (J. Huff) D7: EF631188

D3: N/AD2: EF63138416S: EF631501

Macrodiplax balteata USA: FL (M. L. May) N/AUrothemis assignata Senegal (J. Huff) D7: EF631217

D3: EF631313D2: EF63142116S: EF631534

Zyxomma elgneri Australia (K. M. Kjer,F. L. Carle)

D7: EF631253D3: EF631344D2: EF63145816S: EF631568

Zyxomma petiolatum Bali (A. Rowat) D7: N/AD3: EF631378D2: EF63149616S: N/A

Libellulidae: ZygonychinaeZygonyx torridus South Africa (M. L. May) D7: EF631218

D3: EF631314D2: EF63142216S: EF631535

Zygonyx natalensis South Africa (M. L. May) D7: EF631203D3: EF631300D2: EF63140516S: EF631519

See Appendix A for taxa omitted from analyses.


tion made by R. Caesar, pers. comm.). All other steps fol-lowed the manufacturer’s protocol. PCR primers, and theirsources, are given in Table 3. Programs used for amplifica-tions were (a) 96 �C, 3 min; 94 �C, 30 s; 50 �C, 30 s; 72 �C,45 s for 35–40 cycles; 72 �C, 10 min and (b) 96 �C, 3 min;94 �C, 30 s; 46 �C, 30 s, 72 �C, 45 s for 10 cycles; 94 �C,30 s; 48 �C, 40 s; 72 �C, 45 s for 30 cycles; 72 �C, 10 min.A Qiagen PCR purification kit was used to purify amplifiedproduct, which was then sequenced on an ABI 3100 capil-lary sequencer. Sequences from both strands were com-pared and edited in Sequence Navigator (AppliedBiosystems). Lowercase letters were used to indicate nucle-otides that were readable but difficult to interpret with cer-tainty in either strand (i.e., there was competingbackground peaks). These lowercase letters were changedto uppercase letters only if there was agreement in thetwo complementary strands. When there was conflict abouta single base call between reads from complementarystrands, this nucleotide was coded with an R (for ambigu-

Table 3Primers used in the present study

D2 region of the 28S D3 region of the 28S D7 reg

Forward primersequence

50TGCTTGAGAGTGCAGCCCAA30

50ACCCGTCTTGAAACACGGAC30

50CGSGTA

Reverse primersequence

50CCTTGGTCCGTGTTTCAAGAC30

50ATAGTTCACCATCTTTCGGGTCC30

50CTTAAT

ous purines), Y (for ambiguous pyrimidines), or N (for allother ambiguities).

2.3. Alignment and phylogenetic reconstruction

Initial sequence alignments were made using Clustal,and the resulting files were then aligned manually in Micro-soft Word using the structural methods described in Kjeret al. (1994), Kjer (1995), Kjer et al., 2007 and secondarystructure models based on Guttell et al. (1993). Ambigu-ously aligned regions were defined as single strandedregions with multiple insertions and deletions (indels) ofvariable length (and thus unclear nucleotide homology),bounded by hydrogen bonded base pairs. These regionswere excluded from the dataset. For parsimony analyses,these characters were recoded as single multistate charac-ters with the program INAASE (Lutzoni et al., 2000), withthe stepmatrices applied. Alignments are available on theKjer lab website, www.rci.rutgers.edu/~entomology/kjer.Although we regard manual alignment based on secondarystructure to be quite strongly supported (e.g., Kjer, 1995,2004; Titus and Frost, 1996; Schnare et al., 1996; Hicksonet al., 2000; Lutzoni et al., 2000; Mugridge et al., 2000; Ellisand Morrison, 1995; Morrison and Ellis, 1997; Gillespieet al., 2005) as the most accurate method for rDNA, werecognize that alignment methodology is a contentiousissue. For those who prefer a different alignment proce-dure, however, the original sequences are, of course, depos-ited in GenBank and thus will be available for anyreanalysis that is desired.

The data were analyzed using parsimony, maximumlikelihood, and Bayesian criteria. For the parsimony recon-struction, a tree bisection-reconnection (TBR) branchswapping heuristic search was run using PAUP 4.0b10(Swofford, 2001) with 10,000 random additions. Gaps ofuniform length were each treated as presence/absence char-acters; other gaps were treated as missing data, exceptthose encoded with INAASE as described above. To esti-mate branch support, 500 bootstrap pseudoreplicates (Fel-senstein, 1985) were performed using 10 random additionsearches per pseudoreplicate.

Prior to maximum likelihood and Bayesian analyses, weused MODELTEST 3.06 Akaike weights (Posada andCrandall, 1998; Posada and Buckley, 2004) and DT-Mod-Sel (Minin et al., 2003) to select an appropriate model ofevolution for each of the two independent gene fragments.

ion of the 28S 16S primers by E. Pilgrim 16s primers fromMisof et al., (2001)

GCGACGAGGAGGG3’

50GTAAGAGTTTAAASGTCGAACAGA30

LR-J-1288750GGAGCTCCGGTTTG

AACTCAGATC30

CAGAGCCCCTTAT30

50AGGATTAGATACCCTTTTATTTTAAATG30

LR-N-1339850CGGCCGCCTGTTAT

CAAAAACAT30

http://www.rci.rutgers.edu/~entomology/kjer


Both programs suggested a GTR + I + G model for the28S and 16S (Yang, 1994; Yang et al., 1994; Gu et al.,1995). GARLI (Zwickl, 2006; available at http://www.zo.utexas.edu/faculty/antisense/garli/Garli.html) wasused to run a rapid maximum likelihood analysis. TheGARLI bootstrap analysis was run using 100 replicatesof a 500,000-generation search; the heuristic search ran500,000 generations. Datasets were unable to be parti-tioned in GARLI, although future versions of GARLI willbe able to do so (Derrick Zwickl, pers. comm.). Because itconsiders the non-independence of hydrogen-bondedrRNA sites, we also used the RNA7A seven state modelavailable in the PHASE program (Jow et al., 2002), torun Markov-chain Monte Carlo (MCMC) analyses of par-titioned RNA data (10 million generations each) and aREV model for the loop regions. Unlike the current ver-sion of Mr. Bayes, which uses a 16 by 16 rate matrix, theRNA7A seven state model (Higgs, 2000) uses a 7 by 7 ratematrix (7 frequencies, 21 rate parameters).This biologicallyrealistic model is useful for studies of rRNA. The REVmodel is the most general loop model with the time revers-ible constraint (four frequencies, five rate parameters).

Using our PHASE trees, we tested the classifications ofDavies and Tobin (1985) and Bechly (1996) using the con-straint function in PAUP. Constraints were written foreach of Davies and Tobin’s subfamilies, and for each ofBechly’s families. We then filtered our PHASE tree fileusing these constraints and recorded the number of treesthat contained these clades.

To recheck our data for possible contaminants, and toincorporate additional available taxa, we ran a parsimonyanalysis, as described above, with 71 libelluloid sequencesdownloaded from GenBank and aligned them with ourdata (Appendix A). Most of the libelluloid sequences inGenBank are mitochondrial. Some of the data availablein GenBank included a longer fragment of mitochondrialrRNA that included a fragment of the 12S. These charac-ters were included in the analysis and coded as missingfor our taxa (for a discussion of missing data see Weins,2005).

3. Results

3.1. Molecular data collection

Ninety-three ingroup taxa and six outgroup taxa wereamplified for the 28S fragment; 78 ingroup taxa and fiveoutgroup taxa were amplified for the 16S (Appendix A)are included in the analysis. After 192 ambiguously alignedcharacters were excluded and coded in INAASE, 1418 ntsremained from the nuclear gene fragment and 430 nts fromthe mitochondrial fragment. Five hundred and forty-threecharacters were parsimony informative (nuclear = 346,mitochondrial = 189, INAASE = 8); 370 were parsimonyuninformative and 943 characters were constant. Allsequences are deposited in GenBank under AccessionNo. EF631188–EF631603.

3.2. Phylogenetic relationships

Bayesian, likelihood and parsimony analyses of thecombined data produced well-resolved phylogenetichypotheses (GARLI and PHASE results: Fig. 2; parsi-mony: Fig. 3). Minor differences occur in areas for whichthere is low branch support (<50%). Several previouslysuggested monophyletic groups were supported by all threemethods: (1) Libellulidae (100% support from all analyses)and Macromiinae (100% support from all analyses). Theseclades, plus Corduliinae, together form a monophyleticgroup, hereafter the MCL group in the PHASE andGARLI analyses (similar but not identical to the MCLgroup of Carle, 1995). (2) Corduliidae s.l is polyphyletic.Corduliinae is monophyletic (89% PHASE posterior prob-ability; 60% GARLI bootstrap; 86% parsimony bootstrap).(3) Surprisingly, Gomphomacromiinae + Synthemisti-nae + Cordulephyinae + Idionychinae (hereafter, the GSIgroup) together form a monophyletic group (100%PHASE; 79% GARLI; 71% parsimony bootstrap) that,however, is not clearly divided into the traditional(sub)families and does not nest within Corduliidae.

The results do not support most of the 17 families pro-posed by Bechly (1996) and Lohmann (1996a,b), or thesubfamilies of Corduliidae suggested by Davies and Tobin(1985) and Bridges (1994). Furthermore, the puzzling Aus-tralian species Cordulephya pygmea, variously placed as (1)a monogeneric subfamily (Fraser, 1957; Davies and Tobin,1985), (2) with Hetronaias and Libellulosoma (Bridges,1994) or (3) with Neophya (Bechly, 1996), is well nestedwithin the GSI clade as sister to the nominal gomphomacr-omiine Pseudocordulia circularis (Hetronaias, Libelluloso-

ma, and Neophya were not sequenced). The two generaof Idionychinae, Macromidia and Idionyx, form a mono-phyletic but poorly supported (35% PHASE) subcladewithin the GSI clade. Corduliinae and Macromiinae areseparate monophyletic clades in all analyses. In terms ofthe MCL interfamilial relationships, the PHASE analysisplaced the Corduliinae as sister to the Libellulidae,although with such low support that we would prefer toconsider the relationship unresolved.

Within the Libellulidae, three subfamilies were consis-tently recovered: Leucorrhiniinae Tillyard 1917 (99%PHASE; 94% GARLI, 80% parsimony), UrothemistinaeLieftinck, 1954 (96% PHASE; 79% GARLI; 85% parsi-mony), and Libellulinae Rambur 1842 (76% PHASE; lar-gely, though not entirely recovered). Otherwise, membersof the subfamilies listed in Davies and Tobin (1985) andBridges (1994) are scattered throughout the Libellulidae.Conspecifics and congenerics (with the exceptions of Libell-

ula, Cordulia, Zyxomma, and Orthetrum) form monophy-letic groups.

To assess congruence among independent data sets, wealso ran separate PHASE analyses of the mitochondrialand nuclear genes. The nuclear PHASE data lends strongsupport to monophyly of the GSI (97%) but the mitochon-drial data place them instead as a polytomy at the base of

http://www.zo.utexas.edu/faculty/antisense/garli/Garli.html

http://www.zo.utexas.edu/faculty/antisense/garli/Garli.html

0.1

Neopetalia punctataKalyptogaster erronea

Taeniogaster obliquaSinorogomphus sp

Chloropetalia soarerSyncordulia gracilis

Idionyx selysiMacromidia rapida

Macromidia rapida Oxygastra curtisii

Gomphomacromia spSynthemis leachii

Synthemis eustalactaCordulephya pygmeaPseudocordulia circularis

Lathrocordulia metallicaHesperocordulia berthoudiMicromidia atrifrons

Austrophya mysticaEusynthemis brevistyla

Austrocordulia refractaSynthemiopsis gomphomacromioides

Choristhemis flavoterminataArchaeophya magnifica

Pentathemis membranulataAeschnosoma forcipula

Epitheca princepsTetragoneuria cynosura

Tetragoneuria cynosura Neurocordulia obsoleta

Neurocordulia xanthosomaHemicordulia tau

Procordulia smithiiProcordulia grayi

Cordulia shurtleffiRialla villosa

Helocordulia uhleriCordulia aenea

Somatochlora tenebrosaPhyllomacromia contumax

Macromia illinoiensisDidymops transversa

Calophlebia interpositaTetrathemis polleni Tetrathemis polleni

Urothemis assignataAethriamanta rezia

Zygonyx natalensisZygonyx torridus

Nannophlebia risiHuonia oreophila

Onychothemis culminicolaOnychothemis testacea Onychothemis testacea

Rhyothemis semihyalinaRhyothemis semihyalina

Sympetrum ambiguumSympetrum janeae

Leucorrhinia glacialisCelithemis elisa

Hydrobasileus brevistylusTramea onusta

Tramea lacerataDasythemis esmeralda

Chalcostephia flavifronsBrachydiplax denticauda

Brachythemis leucostictaDeielia phaon

Zyxomma petiolatumTholymis tillarga Idiataphe

amazonicaZyxomma elgneriPachydiplax longipennis

Elga leptostylaMicrathyria aequalis

Micrathyria aequalisZenithoptera fasciata

Zenithoptera fasciata Dythemis multipunctata

Dythemis fugaxMacrothemis celeno

Macrothemis hemichloraScapanea frontalis

Paltothemis lineatipesBrechmorhoga mendax

Diplacodes haematodesBradinopyga strachani

Hemistigma albipunctaPalpopleura jucunda

Palpopleura luciaNannophya dalei

Uracis fastigiataAcisoma panorpoides

Erythemis simplicicollisNannothemis bella

Erythrodiplax minusculaCrocothemis erythraea

Crocothemis serviliaPerithemis tenera

Trithemis monardiTrithemis dorsalis

Pantala flavescens Pantala flavescens

Plathemis lydiaNeodythemis pauliani

Misagria speciesHadrothemis defecta

Agrionoptera longitudinalisOrthemis ferruginea

Orthemis ferrugineaLibellula pulchella

Libellula luctuosaLyriothemis pachygastra

Libellula quadrimaculata Libellula quadrimaculata

Ladona juliaOrthetrum sp

Orthetrum pruinosumOrthetrum abbotti

Orthetrum spOrthetrum chrysis

Orthetrum julia Orthetrum julia 100*

73100*

6996*

54100*

9498*

68

52

100*

76

70

100*

100*

50

60

98100*

97*

5599 100

56

88

97

9971

100*

100*

100*

93*53

53

95100*

9796

9268

100*

100* 75*

99*100*

100*

100*100*

98*87

79100*

60

96*

100*

100*94*

100*100*

628760

99*56

100*85

100*

100*70

100*

100*

89*

94

100*

100*

3099

55

73

99

5385*

72*

8683

75*

95

100*

100*

Leucorrhiniinae

UrothemistinaeLibellulidae

Libellulinae

Macromiidae

Corduliinae

'GSI'

ABC

D

E

F

G

'Higher' Libelluloids

'MCL'

H

36

43

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4135

4622

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4742

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38

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 monophylyof Macromiinae and of Libellulidae. The 16S data supportthe monophyly of the Corduliinae (80% 16S), but 28S sup-

port for this group was low (50%), probably due to theunstable position of the genera Pentathemis + Aeschnoso-

ma. Within the Libellulidae, only the Libellulinae are lar-

Chloropetalia soarerSinorogomphus spNeopetalia punctataTaeniogaster obliquaKalyptogaster erroneaPhyllomacromia contumaxMacromia illinoiensisDidymops transversaPentathemis membranulataAeschnosoma forcipulaHemicordulia tauProcordulia grayiProcordulia smithiiEpitheca princepsTetragoneuria cynosuraTetragoneuria cynosura Somatochlora tenebrosaHelocordulia uhleriCordulia aenea Neurocordulia obsoletaNeurocordulia xanthosomaCordulia shurtleffiRialla villosaSyncordulia gracilisMacromidia rapidaMacromidia rapidaIdionyx selysiOxygastra curtisiiGomphomacromia spSynthemis eustalactaSynthemis leachiiSynthemiopsis gomphomacromioidesAustrocordulia refractaChoristhemis flavoterminataArcheophya magnificaEusynthemis brevistylaAustrophya mysticaPseudocordulia circularisCordulephya pygmeaLathrocordulia metallicaMicromidia atrifronsHespercordulia berthoudiCalophlebia interpositaTetrathemis polleniTetrathemis polleni Hydrobasileus brevistylusAethriamanta reziaUrothemis assignataPerithemis teneraRhyothemis semihyalinaRhyothemis semihyalinaLeucorrhinia glacialisCelithemis elisaSympetrum janeaeSympetrum ambiguumUracis fastigiataAcisoma panorpoidesErythemis simplicicollisPachydiplax longipennisNannothemis bellaNannophya daleiBradinopyga strachaniHemistigma albipunctaPalpopleura jucundaPalpopleura luciaErythrodiplax minusculaDiplacodes haematodesCrocothemis serviliaCrocothemis erythraeaElga leptostylaZenithoptera fasciata Zenithoptera fasciata Macrothemis hemichloraMacrothemis celenoScapanea frontalisPaltothemis lineatipesBrechmorhoga mendaxDythemis fugaxDythemis multipunctataMicrathyria aequalisMicrathyria aequalisTramea onustaDasythemis esmeraldaTramea lacerataBrachydiplax denticudataChalcostephia flavifronsDeielia phaonBrachythemis leucostictaTholymis tillargaZyxomma petiolatumZyxomma elgneriIdiataphe amazonicaTrithemis dorsalisTrithemis monardiPantala flavescensPantala flavescens Zygonyx natalensisZygonyx torridusNannophlebia risiHuonia oreophilaOnychothemis culminicola Onychothemis testaceaOnychothemis testacea Misagria speciesHadrothemis defectaNeodythemis paulianiAgrionoptera longitudinalisOrthemis ferrugineaOrthemis ferrugineaLadona juliaPlathemis lydiaLibellula luctuosaLibellula pulchellaLyriothemis pachygastraLibellula quadrimaculataLibellula quadrimaculataOrthetrum speciesOrthetrum chrysisOrthetrum speciesOrthetrum pruinosumOrthetrum abbottiOrthetrum juliaOrthetrum julia

Strict94

76 100

100 99

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6610084

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6096

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78100

52 8193

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98 95AB *

D*

G

E&F

'Higher' Libelluloids

Macromiidae

Corduliinae

'GSI'

Libellulidae

Urothemistinae

Leucorrhiniinae

Libellulinae

3028

2124

22 4044

42

327

3083 61

19

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D*

Fig. 3. Phylogenetic reconstruction from 10,000 replicate parsimony heuristic search. The ‘‘*’’ indicates that this clade differs in composition in thePHASE analysis. Bootstrap support is written above the branches.


gely supported by both genes. Subtle differences occurbetween the 16S and 28S in the composition and/or posi-tion of Clades B and F (Clade B includes Hydrobasileus

in the 28S analysis while there is little support for the sub-family Urothemistinae in the 16S analysis; Clade Fincludes Rhyothemis in the 28S analysis, while Clade F is


not supported by the 16S analysis). Other differencesbetween the 16S and 28S were found only in areas with lessthan 50% support.

Our hypothesis testing revealed that neither Gompho-macromiinae nor Synthemistinae were present in any ofthe 69,009 trees (after burnin discarded) created by ourPHASE analysis. Idionychinae was recovered, in 15,736trees (23%). Bechly’s (1996) Synthemistidae, Gompho-macromiidae, Austrocorduliidae, and Oxygastridae werenever present. In addition, the monogeneric Pseudocordu-liidae and Cordulephyidae are nested well within the GSIclade and thus do not form the pectinate arrangement ofBechly (Fig. 1g) that would justify family status. Bechly’sHemicorduliidae (Hemicordulia and Procordulia), is recov-ered in 84% of the trees (Fig. 2), but its nested position doesnot suggest that family status is warranted. The libellulidsubfamilies of Davies and Tobin (1985), with the exceptionof Leucorrhiniinae and Libellulinae were never recovered.We constrained Pantala and Tramea only but found thatthey were never recovered together. Dasythemis neveroccurred within Libellulinae.

Parsimony and PHASE analyses of the dataset thatincluded GenBank sequences were largely consistent withour other analyses (Fig. 7). The taxon sample in GenBankconsists mostly of libellulids. In all cases, except for Eryth-

emis, Pachydiplax, Plathemis, Ladona, Lyriothemis, andLibellula congenerics group together. Because the supportvalues are so low within the Libellulinae, we consider rela-tionships among Plathemis, Ladona, Lyriothemis, andLibellula to be unresolved. Erythemis and PachydiplaxGenBank sequences are available for the 12S fragmentonly, a fragment we did not sequence, and so they maynot have enough information to place them with our con-generic taxa. Similarly, data may be insufficient to correctlyplace Idiataphe, for which we have only the D3 fragmentsequence: in the larger dataset, it is placed in Clade A, withlow support. The Urothemistinae, Leucorrhiniinae, andLibellulinae remain as monophyletic groups, and becausethe support along the backbone of Libellulidae is low,the other groupings (Clades A, C, D, E, F, and G) are ran-domly arranged (and present as a polytomy in the boot-strap analysis). The composition of the clades does notdiffer greatly. Not surprisingly, the differences betweenthe phylogenetic reconstructions were found in areas withless then 50% bootstrap support.

4. Discussion

4.1. Taxonomic implications

Our results agree with those of most previous dragonflysystematists in placing Synthemistinae as basal and Libell-ulidae as terminal libelluloids (e.g., Tillyard, 1917; Fraser,1957; Carle, 1995; Bechly, 1996). Pfau’s (1991, 2005) inno-vative morphological study of the odonate vesica spermalis(penis), however, differs radically in placing Cordulegastri-dae and related taxa in the Petaluroidea and the Synthe-

mistinae as sister to the Libellulidae. In the remainder ofthis section, we compare in more detail our hypothesis withprevious systematic treatment of Libelluloidea.

4.1.1. The GSI clade

Our analysis places Synthemistinae relatively basallyamong higher libelluloids, as have most earlier studies(Trueman’s (1991) study of egg morphology and early lar-val characteristics; wing venation by Tillyard (1917), Fraser(1957), Davies and Tobin (1985), Carle (1995) Lohmann(1995) and Bechly (1996)). Since Tillyard and Fraser(1940) and Fraser (1957), all authors have regarded Gom-phomacromiinae and Idionychinae as distinct from Synthe-mistinae, although with various internal subdivisions(Carle, 1995; Bechly, 1996, and see Table 1). Our analysis,however, fails to support this distinction, with the threegroups mingled within the GSI group. This is a surprisingresult, since Theischinger and Watson (1984) identified sev-eral convincing larval morphological characters favoringsuch a division. Subsequent authors (e.g., Carle and Lou-ton (1994), Carle (1995), Lohmann (1996a,b) and Bechly(1996)), using additional characters, also found supportfor the separation of these Synthemistinae and Gompho-macromiinae. In our phylogeny, support is low for manyrelationships within the GSI clade, but, Synthemistinaeand Gomphomacromiinae were never found in any of thenear optimal trees from the Bayesian treefile. Consider-ation of additional morphological or molecular data maysuggest a more traditional structure within the clade. Itwould be very difficult, however, to reconcile our conclu-sions with the hypothesis that the Gomphomacromii-nae + Idionychinae are paraphyletic with respect toCorduliinae s. s. (e.g., May, 1995b; Bechly, 1996; Loh-mann, 1996a,b).

4.1.2. Corduliidae and Macromiinae

Corduliidae s. s. and Macromiinae have long been con-sidered closely related and have been regarded as confamil-ial by many workers (Martin, 1906, 1909; Tillyard, 1917;Fraser, 1957; Lieftinck, 1971; Davies and Tobin, 1985;Steinmann, 1997). They share a number of characters, mostof which, however, are either plesiomorphies or are alsoshared with Libellulidae (see below). Gloyd (1959) pro-posed that Macromiinae be raised to family status, buther arguments were based almost entirely on autapomor-phies of Macromiinae. Nevertheless, Gloyd’s suggestionis not inconsistent with our results. Our data support themonophyly of the Macromiinae; whether this monophy-letic group deserves family status is a matter of taxonomicpreference. The relationship of these two families to Libell-ulidae is not strongly supported by our data, althoughmost morphological features suggest a corduliid–libellulidsister group relationship.

4.1.3. LibellulidaeThree putative libellulid subfamilies (Fraser, 1957;

Davies and Tobin, 1985) are supported, with some modifi-


cation, as monophyletic by all our analyses (Urothemisti-nae, a modified Libellulinae and a restricted Tetrathemist-inae). Urothemistines are recovered as monophyletic nearthe base of the libellulids (Clade B): this group was consid-ered a family, Macrodiplactidae, by Fraser (1957) and Bec-hly (1996), but their nested position within the Libellulidaein some analyses suggests caution should be used in elevat-ing the urothemistines to family status (e.g., Davies andTobin, 1985). The Libellulinae also is apparently mono-phyletic (Clade H) except for the exclusion of Dasythemis

esmeralda, which falls into Clade E in all analyses. Amonophyletic Libellulinae agrees with the results presentedby Fleck et al. (in press), including their placement ofAgrionoptera, Misagria, and most notably, Neodythemis(they did not include Dasythemis in their analysis). In addi-tion, the Leucorrhiniinae (Leucorrhinia + Celithemis,placed in Clade D) are sister to Sympetrum, which is con-sistent with Pilgrim (2006) and Fleck et al. (in press). Sub-family status for Leucorrhiniinae is probably unwarrantedbecause they are a small distinct group within a much lar-ger clade (apophyletic sensu Carle (1995), i.e., its distinctiveautapomorphies have resulted in its assignment to an exag-gerated taxonomic rank).

Three well-established taxa are clearly polyphyletic. Spe-cies usually attributed to Tetrathemistinae, commonlyregarded as the most plesiotypic of libellulid subfamilies,are scattered throughout Libellulidae, with Tetrathemis

and Calophlebia in Clade A, Nannophlebia in Clade C,and Neodythemis in Clade H. Thus, a very restricted Tetr-athemistinae might remain as the most basal Libellulidae(Clade A), but clearly its composition and delimiting char-acters are very different than previously defined. As notedalready by Dijkstra and Vick (2006) and Fleck et al. (inpress), the venational traits used heretofore to define Tetr-athemistinae are correlated with narrowing of the wingbase and thus are probably subjected to convergence.

Second, the Trameinae is also polyphyletic, althoughmost species cluster loosely in clade E. This clade alsoincludes some genera generally thought to be closelyrelated to trameines but placed by Fraser (1957) and oth-ers in the Zyxommatinae (Tholymis, and Zyxomma). Theplacement of Idiataphe in Clade E is ambiguous. Morpho-logical evidence is inconclusive about its position.Although Davies and Tobin (1985) place it in Trameinae,its position is unstable in our analyses, quite possiblybecause only the D3 fragment was sequenced for this spe-cies. In addition, its branch is suspiciously, long comparedto other nearby taxa. Rhyothemis, also previously thoughtto be closely related to trameines, is placed by PHASE(with low support) in clade D. The most surprising resultwith respect to the polyphyly of the trameines is thatPantala is very distantly related to the other trameines,falling to the base of Clade H in the smaller dataset,and nested within Clade C in the larger dataset. Again,convergent modification of the hindwing base, in this caseexpansion as an adaptation to extended periods of glid-ing, could explain the morphological similarity that has

caused Pantala and Tramea to be placed together previ-ously (Fig. 4).

Finally, Diastatopidinae is distributed among Clades F(Zenithoptera), G (Palpopleura), and H (Perithemis,although with considerable uncertainty). Several authors(e.g., Fraser, 1957) have noted that members of these gen-era have unusually patterned wings and may mimic Hyme-noptera. Possibly this convergence on wasp-like color andbehavior has resulted in similarities of morphology thatmisled previous workers. The larvae of these genera differmarkedly in lateral and dorsal abdominal spine develop-ment, abdomen shape, and epiproct length (Zenithoptera,Costa et al., 2004; Palpopleura, Fraser, 1955; Perithemis,Needham et al., 2000). Palpopleura + Hemistigma, placedtogether in our phylogeny with high support, share severallarval characteristics. They both lack dorsal spines, haveprominent eyes and share a striking pale dorsal stripe(Whiteley et al., 1999). Adults of these two genera alsoshare markedly bicolored pterostigma. None of these char-acters is definitive, but the combination tends to supportseparation of the diastatopidines and the grouping of Pal-

popleura and Hemistigma.Most subclades of Libellulidae recovered in our phylog-

eny comprise a mixture of taxa from various previouslyrecognized subfamilies. Members of Sympetrinae, Trithe-mistinae, and Brachydiplacinae are scattered throughoutLibellulidae and none of these subfamilies were presentamong our PHASE trees.

4.2. Character evolution

4.2.1. Adult characters

As in many taxa, traditional systematic treatment ofLibelluloidea has typically emphasized an essentially lineartransformation of characters, especially of wing veins, lead-ing from an ‘‘archaic’’ to a ‘‘modern’’ state. As Fraser(1957) expressed it, the superfamily ‘‘. . . exhibits an almostunbroken chain of evolution. . ..’’ Such a progression wouldonly be expected if all phylogenies were perfectly pectinateand without homoplasy. The situation is almost certain tobe less clear-cut in the real world, and such appears to bethe case based on our phylogeny.

For example, the elongation of the anal loop and thedevelopment of a midrib (Fig. 4) can be considered to pro-gress from its ‘‘absence’’ in most non-libelluloids to itsextreme (a boot-shaped structure, with a ‘‘toe’’ and a mid-rib) in Libellulidae, although with notable generic excep-tions (Needham, 1903; Tillyard, 1910; Tetrathemis,Nannothemis, Misagria, and Fylgia for example, are libellu-lids with reduced anal loops). Our reconstruction, however,suggests that either elongation of the anal loop and devel-opment of a midrib occurred in parallel, perhaps multipletimes, in the GSI and the MCL clades or this conditionwas replaced by a short broad loop independently in Syn-themistinae and Macromiinae. Moreover, the loop proba-bly has been secondarily lost several times independentlyin Libellulidae and possibly in members of GSI. A charac-

Cordulia

Libellula

Gomphomacromia*

PantalaTetrathemis

Cordulegaster

Macromia

Synthemis*

Palpopleura

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 thepresence of a ‘‘broken’’ costal side of the triangle, makingthe triangle, in fact, quadrangular; this, too, seems to haveevolved multiple times. Several other characters show aconsiderable degree of homoplasy between the GSI andMCL clades. The supposedly increasing alignment of theantenodal crossveins (these are crossveins at the leadingedge of the wings, anterior to the nodus), for example,and the proximity of the HW triangle to the arculus (a flex-ion point), both emphasized by Fraser (1957), and to someextent, by Bechly (1996), vary across the GSI and MCL(although they are most strongly expressed in the Libellu-lidae and Corduliidae, respectively). It appears possiblethat all of these changes are partially correlated. Theymay have the effect of reducing, or at least reconfiguring,chordwise flexibility of the wing base, especially in thehindwing, and perhaps consequently increasing twisting

and camber of the distal portion of the wings. Understand-ing of the details of wing kinematics and aerodynamicsduring flapping flight, however, is as yet too rudimentaryto make confident predictions of these effects (Woottonand Kukalova-Peck, 2000; Combes and Daniel, 2003a,b,2005).

Reduction in the ovipositor, convergently shared withthe Gomphidae (Carle, 1995), is a prominent feature ofLibelluloidea (and considered a synapomorphy of Gom-phidae + Libelluloidea by Bechly, 1996 and Lohmann,1996a,b; Fig. 5). The ovipositor of Aeshnoidea and Petalu-ridae (and Zygoptera) comprises three pairs of ventral pro-cesses. The first and second pairs (anterior and posteriorgonapophyses) are enclosed by the third (gonoplacs). Inlibelluloids, including Cordulegastridae, the ovipositor ismodified for exophytic oviposition (Tillyard, 1917; Carle,1995). In Cordulegastridae, the third processes (gonoplacs)

Fig. 5. Terminal abdominal segments in lateral view of (a) female Zoraena

diastatops (Cordulegastridae) and (b) Sympetrum costiferum (Libellulidae).Arrows indicate the well-developed first gonapophyses typical of Cord-ulegastridae and some Synthemistinae and Gomphomacromiinae (a) andthe nearly obsolete ovipositor, often reduced to a short extension of thesternum of S8 and/or a pair of small scales at the base of S9 in Libellulidaeand most Corduliidae (b). Figures from Needham et al. (2000).

Fig. 6. Lateral view of male secondary genitalia of (a) Zoraena diastatops

(Cordulegastridae); (b) Didymops transversa (Corduliidae, Macromiinae);(c) Libellula quadrimaculata (Libellulidae, Libellulinae). In each case theventral side is upward and the anterior direction to the right, and theabdominal terga are removed to reveal the genitalic structures. Accessorysecondary genitalia are the anterior lamina (AL), anterior hamules (AH;absent in L. quadrimaculata and other Libellulidae), anterior frame (AF),posterior hamule (PH), posterior frame (PF), and genital ligula (GL);these structures engage the female ovipositor during copulation and assistmovement of the vesica spermalis. Segments of the vesica spermalis, whichfunctions both for temporary sperm storage before copulation and forintromission, are indicated by Roman numerals I, II, III, and IV frombase to apex; these medial structures are most easily visible in A.Terminology follows Pfau (1971).


are vestigial. In the GSI clade, the third processes areabsent and at least the second processes are reduced,although in some taxa the first pair is present and nearlyas long as in Cordulegastridae. Our results do not allowus to conclude whether the latter GSI condition is plesio-morphic or secondarily (re)developed. In the MCL, thefirst processes are reduced to small flaps and the otherstructures are apparently absent except for the probablevestige of the styli emerging directly from the ninth sternite(Tillyard, 1917). In a few instances, the eighth (e.g., someSomatochlora) or eighth and ninth sternites (Uracis) aresecondarily produced to form an ovipositor in MCLspecies.

Pfau’s (2005) detailed morphological study of the vesicaspermalis (v. s.; Fig. 6), and especially of the distal ‘‘spermpump’’ implies a very different phylogeny than that foundhere or by most other workers. Most radically, Pfau sug-gests that cordulegastrids are members of a larger groupthat includes petalurids, and gomphids (Fig. 1). He reasonsthat because it appears impossible to derive the complexsperm pump apparatus of libelluloids directly from the cor-dulegastrid sperm pump, the multiple similarities of Cord-ulegastridae and libelluloids are the result of convergence.Moreover, he maintains that Corduliinae s.s. are the mostplesiomorphic libelluloid group, with synthemistids as thesister taxon to Libellulidae at the apex of the libelluloids.Despite Pfau’s (1971, 1991, 2005) conclusion, our analysisis rooted with Cordulegastridae. Given other, independentsupport, both morphological (Bechly, 1996; Gorb, 1999;Carle and Kjer, 2002), and molecular (Misof et al., 2001;

Carle and Kjer, unpublished data), we feel some confidencethat Cordulegastridae + Neopetaliidae + Chlorogomphi-dae are the closest relatives to other Libelluloidea, andappropriate outgroups.

Even if our tree were to be re-rooted, however, Pfau’shypothesis clearly cannot be reconciled with ours forhigher libelluloids. No previous molecular dataset appliesbroadly to the higher Libelluloidea, but based on ourresults we must conclude that the v. s. morphology of Gom-

phomacromia + synthemistids is convergent to that oflibellulids. It is unclear whether the synthemistid-type orthe corduliid-type v. s. is plesiomorphic for higher libellu-loids. Although the GSI is strongly supported, there is suchlow internal support that it is hard to make any conclu-

0.1

Neopetalia punctataTaeniogaster obliqua

Anotogaster sieboldii OCordulegaster picta OCordulegaster boltonii OKalyptogaster erronea

Chloropetalia soarer Chlorogomphus brunneus OSinorogomphus sp

Idionyx selysiSyncordulia gracilis

Macromidia rapidaMacromidia rapida

Oxygastra curtisiiOxygastra curtisii O

Gomphomacromia sp Synthemis leachiiSynthemis eustalacta

Cordulephya pygmeaPseudocordulia circularis

Lathrocordulia metallicaHespercordulia berthoudiMicromidia atrifronsAustrophya mysticaEusynthemis brevistyla

Choristhemis flavoterminataArcheophya magnifica

Austrocordulia refractaSynthemiopsis gomphomacromiodesPhyllomacromia contumax

Didymops transversaMacromia splendens

Macromia illinoiensisPentathemis membranulata

Aeschnosoma forcipulaEpitheca princepsTetragoneuria cynosuraTetragoneuria cynosura

Neurocordulia obsoletaNeurocordulia xanthosoma

Hemicordulia tauProcordulia smithi

Procordulia grayiCordulia shurtleffi

Rialla villosaHelocordulia uhleri

Cordulia aeneaSomatochlora tenebrosa

Macrodiplax cora OUrotnemis assignata

Aethriamanta reziaNotiothemis robertsi O

Idiataphe amazonicaTetrathemis polleni O

Calophlebia interpositaTetrathemis polleni

Tetrathemis polleniRhyothemis variegata imperatrixO


Leucorrhinia glacialisCelithemis elisa OCelithemis elisa

Sympetrum corruptumSympetrum janeae

Sympetrum vulgatum OSympetrum ambiguum





Zyxomma petiolatumZyxomma elgneri

Tholymis tillargaTholymis citrina O

Micrathyria aequalisMicrathyria aequalis

Elga leptostylaMicrathyria didyma O

Erythemis simplicicollis OPachydiplax longipennis O

Zenithoptera fasciataZenithoptera fasciata

Dythemis multipunctataDythemis fugax

Scapanea frontalisBrechmorhoga mendax

Paltothemis lineatipesMacrothemis celeno

Macrothemis hemichloraUracis fastigiata

Nannothemis bellaNannophya pygmaea O

Nannophya daleiAcisoma panorpoides

Pacnydiplax longipennisErythemis simplicicollis

Diplacodes haematodesErythrodiplax minusculaCrocothemis servilia

Crocothemis erythraeaCrocothemis erythraea O

Bradinopyga strachaniHemistigma albipuncta

Palpopleura jucundaPalpopleura lucia

Perithemis lais OPerithemis tenera


Malgassophlebia aequatoris OTrithemis monardi

Trithemis dorsalisOnychothemis culminicola

Onychothemis testaceaOnychothemis testacea O

Onychothemis testsaceaZygonyx natalensis

Zygonyx torridusNannophlebia risi

Bironides sp OHuonia epinephela O

Huonia oreophilaPlathemis depressa O

Oxythemis phoenicosceles OLadona deplaneta O

Ladona exusta OLadona juliaO

Ladona juliaLyriothemis elegantissima O

Ladona fulva OHadrothemis infesta O

Plathemis lydiaPlathemis lydia O

Platnenin subornata OOrthetrum albistylum OOrthetrum cancellatum O

Orthetrum coerulescens OOrthetrum sp

Orthetrum pruinosumLyriothemis pachygastraOrthetrum sp

Orthetrum abbottiOrthetrum chrysis

Orthetrum juliaOrthetrum julia

Orthetrum brunneum OOrthetrum brunneum OOrthetrum albistylum OOrthetrum brunneum OOrthetrum brunneum O

Libellula croceipennis OLibellula semifasciata O

Libellula composita OLibellula angelina O

Libellula quadrimaculata OLibellula quadrimaculata O

Libellula quadrimaculataLibellula quadrimaculata

Libellula flavida OLibellula axilena O

Libellula vibrans OLibellula needhami OLibellula luctuosa

Libellula pulchellaLibellula jesseana OLibellula incesta OLibellula luctuosa OLibellula auripennis O

Libellula comanche ONeodythemis pauliani

Neodythemis africana OAllorhizucha preussi O

Allorhizucha klingi OLibellula cyanea O

Agrionoptera longitudinalisAgrionoptera insignis O

Thermorthemis madagascariensis OOrthemis cultrifornis O

Orthemis discolor OOrthemis ferruginea

Orthemis ferruginea OOrthemis ferruginea

Cratilla metallica OLibellula pulchella O

Libellula nodisticta OLibellula forensis O

Micromacromia camerunica OHadrothemis defecta

Misagria paranaMisagria parana O

4871

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G

H

MacromiidaeCorduliinae

Libellulidae

Higher libelluloids

GSI

MCLUrothemistinae

Leucorrhiniinae

Libellulinae

0.1

Neopetalia punctataTaeniogaster obliqua

Anotogaster sieboldii OCordulegaster picta OCordulegaster boltonii OKalyptogaster erronea

Chloropetalia soarer Chlorogomphus brunneus OSinorogomphus sp

Idionyx selysiSyncordulia gracilis

Macromidia rapidaMacromidia rapida

Oxygastra curtisiiOxygastra curtisii O

Gomphomacromia sp Synthemis leachiiSynthemis eustalacta

Cordulephya pygmeaPseudocordulia circularis

Lathrocordulia metallicaHespercordulia berthoudiMicromidia atrifronsAustrophya mysticaEusynthemis brevistyla

Choristhemis flavoterminataArcheophya magnifica

Austrocordulia refractaSynthemiopsis gomphomacromiodesPhyllomacromia contumax

Didymops transversaMacromia splendens

Macromia illinoiensisPentathemis membranulata

Aeschnosoma forcipulaEpitheca princepsTetragoneuria cynosuraTetragoneuria cynosura

Neurocordulia obsoletaNeurocordulia xanthosoma

Hemicordulia tauProcordulia smithi

Procordulia grayiCordulia shurtleffi

Rialla villosaHelocordulia uhleri

Cordulia aeneaSomatochlora tenebrosa

Macrodiplax cora OUrotnemis assignata

Aethriamanta reziaNotiothemis robertsi O

Idiataphe amazonicaTetrathemis polleni O

Calophlebia interpositaTetrathemis polleni

Tetrathemis polleniRhyothemis variegata imperatrixO


Leucorrhinia glacialisCelithemis elisa OCelithemis elisa

Sympetrum corruptumSympetrum janeae

Sympetrum vulgatum OSympetrum ambiguum





Zyxomma petiolatumZyxomma elgneri

Tholymis tillargaTholymis citrina O

Micrathyria aequalisMicrathyria aequalis

Elga leptostylaMicrathyria didyma O

Erythemis simplicicollis OPachydiplax longipennis O

Zenithoptera fasciataZenithoptera fasciata

Dythemis multipunctataDythemis fugax

Scapanea frontalisBrechmorhoga mendax

Paltothemis lineatipesMacrothemis celeno

Macrothemis hemichloraUracis fastigiata

Nannothemis bellaNannophya pygmaea O

Nannophya daleiAcisoma panorpoides

Pacnydiplax longipennisErythemis simplicicollis

Diplacodes haematodesErythrodiplax minusculaCrocothemis servilia

Crocothemis erythraeaCrocothemis erythraea O

Bradinopyga strachaniHemistigma albipuncta

Palpopleura jucundaPalpopleura lucia

Perithemis lais OPerithemis tenera


Malgassophlebia aequatoris OTrithemis monardi

Trithemis dorsalisOnychothemis culminicola

Onychothemis testaceaOnychothemis testacea O

Onychothemis testsaceaZygonyx natalensis

Zygonyx torridusNannophlebia risi

Bironides sp OHuonia epinephela O

Huonia oreophilaPlathemis depressa O

Oxythemis phoenicosceles OLadona deplaneta O

Ladona exusta OLadona juliaO

Ladona juliaLyriothemis elegantissima O

Ladona fulva OHadrothemis infesta O

Plathemis lydiaPlathemis lydia O

Platnenin subornata OOrthetrum albistylum OOrthetrum cancellatum O

Orthetrum coerulescens OOrthetrum sp

Orthetrum pruinosumLyriothemis pachygastraOrthetrum sp

Orthetrum abbottiOrthetrum chrysis

Orthetrum juliaOrthetrum julia

Orthetrum brunneum OOrthetrum brunneum OOrthetrum albistylum OOrthetrum brunneum OOrthetrum brunneum O

Libellula croceipennis OLibellula semifasciata O

Libellula composita OLibellula angelina O

Libellula quadrimaculata OLibellula quadrimaculata O

Libellula quadrimaculataLibellula quadrimaculata

Libellula flavida OLibellula axilena O

Libellula vibrans OLibellula needhami OLibellula luctuosa

Libellula pulchellaLibellula jesseana OLibellula incesta OLibellula luctuosa OLibellula auripennis O

Libellula comanche ONeodythemis pauliani

Neodythemis africana OAllorhizucha preussi O

Allorhizucha klingi OLibellula cyanea O

Agrionoptera longitudinalisAgrionoptera insignis O

Thermorthemis madagascariensis OOrthemis cultrifornis O

Orthemis discolor OOrthemis ferruginea

Orthemis ferruginea OOrthemis ferruginea

Cratilla metallica OLibellula pulchella O

Libellula nodisticta OLibellula forensis O

Micromacromia camerunica OHadrothemis defecta

Misagria paranaMisagria parana O

4871

65100

100100

1003528

100

9478

100

936441

62

94

8899

33

69

81

58

100

60100100

100

72100

100

10085

100

749365

69

66

100

86

42

8623BA 58

819851

50

100100

10092

9310062

99

25

84100

10088

989196

100

98

66

22

91

3350

50

4354

100

99393295

96

36

35

15

6

7470

6355

5530

4710096

9610094

45

40

46

92

100

9776

10086

100

99

8999100

58

62

47

68

41

9637

9514

45

3499

164

17

100

9458

31

51

9747

100

2121

10022

43

31

100

12

12.5

453861

30

20

1964

3045

4946

15

10

4

1009225

11

3618

4

1004794

94

2848

1004115

610

6

2

1

2

12

13

48

49

41

18

5

7

100

55

100

99

C

D

E*

F

G

H

MacromiidaeCorduliinae

Libellulidae

Higher libelluloids

GSI

MCLUrothemistinae

Leucorrhiniinae

Libellulinae

Fig. 7. Phylogenetic reconstruction from a ten thousand replicate parsimony heuristic search. The ‘‘*’’ indicates a clade that differs in composition fromthe smaller PHASE dataset. The ‘‘�’’ indicates that the taxon sequence was downloaded from GenBank. Posterior probabilities are written above thebranch except on very short branches where it is written below.


sions about the evolution of characters within this group.To begin to understand the evolution of the v. s. in libellu-

loids, a more extensive taxon sampling in the GSI andMCL must be examined to uncover any intra- and interfa-


milial differences that may shed light on v. s. development.Studies of the ontogeny of the sperm pump within libellu-loids might help clarify its pattern of evolution. Pfau (1971)described its last larval instar form, but only in Cordulegas-

ter and Libellula.Several authors have noted the tendency toward reduc-

tion of the anterior hamule (a male secondary genetalicstructure; Fig. 6) in higher libelluloids. Pfau (1971) showeda loss of hamular muscle 9a in Cordulia; once 9a is lost, acorrelated loss of 9b occurs in Libellulidae. Here too, how-ever, homoplastic loss of 9a occurs in Synthemis eustalacta;other synthemistids, as well as gomphomacromiines andmacromiids that have been investigated retain 9a, and lossof 9b is a unique synapomorphy of Libellulidae, as far aswe know (May, unpublished).

4.2.2. Larval characters

The value of larval characters in understanding odonatephylogeny has long been recognized (e.g., Fraser, 1957)and often reveals relationships that are unclear from adultmorphology (Carle and Louton, 1994; Novelo-Gutierrez,1995; Fleck et al., in press). Several larval apomorphiesunite the Libelluloidea including the scoop-like prementum(lower mouthparts) with numerous setae, and an asymmet-rical proventriculus (foregut) with large tooth-like lobes(Tillyard, 1917 [Libellulidae + Cordulegastridae]; Carle,1995; Bechly, 1996 [Cavilabiata]). Dentition of the distalborder of the labial palps differs among the libelluloids,however, again forming what has been interpreted as a lin-ear transformation from plesiomorphic to apomorphic:from large, irregular teeth that lack setae in Cordulegastri-dae to more or less straight distal borders and shallow cren-ulations with setal tufts or single setae in each concavity inLibellulinae, with intermediates in Macromiidae and Cor-duliinae s. s. (Tillyard, 1917). Theischinger and Watson(1984) noted that synthemistid larvae, plus those of Gom-

phomacromia, Archeophya and Pseudocordulia (GSI) havemore cordulegastrid-like dentition. Cordulegastrid andsynthemistid larvae also share wing sheaths that extendapart from one-another, while in other higher libelluloidsthe wing sheaths lie parallel (Tillyard, 1910, 1917). Ouranalyses show no evidence of this divide. Furthermore,the molecular evidence uniting the GSI clade, which hasstrong support (Fig. 2), argues that these characters inthe gomphomacromiines (other than the three genera listedabove) are convergent with those of the Corduliidae s.s.

5. Conclusions

Based on our phylogeny, we suggest that higher Libellu-loidea comprises four families: the Gomphomacromiidae(including Gomphomacromiinae, Idionychinae, Cordule-phyinae, and Synthemistinae of Fraser, 1957, and Daviesand Tobin, 1985), the Macromiidae, the Corduliidae, andthe Libellulidae. The ‘‘Corduliidae’’ s.l. are polyphyletic.We include in Corduliidae only the taxa defined as Cordu-liinae in Fraser (1957), and Davies and Tobin (1985),

although placement of several other genera not studiedhere certainly should be examined in the future (e.g., Ido-

macromia, Neophya, Heteronias, Libellulosoma, Metaphya

and Williamsonia; the South American Navicordulia, Sant-

osia, Schizocordulia, andRialla; and the geographically dis-junct Antipodochlora of New Zealand).

Libellulidae probably comprises three previouslyaccepted subfamilies, (i.e., Urothemistinae, a veryrestricted Tetrathemistinae, similar to that suggested byDijkstra and Vick (2006), and, with some modification,Libellulinae) as well as five additional groups that are con-sistently recovered (one of these including an apophyleticLeucorrhiniinae). Closer examination of the morphologicalbasis of these groupings is needed before a definitive taxon-omy of the family can be proposed. It was beyond thescope of this study to sequence every genus in Libellulidae,but clearly the placement of nearly all the remaining generaremains uncertain.

Biogeographical studies may help to determine a patternof origin with the libellulid groups and clarify the relation-ships among major taxa within GSI and MCL. In ourtaxon sample several broad geographical patterns sug-gested. The GSI, for example, are virtually confined tothe southern Hemisphere, especially Australia (except forthe Indomalayan Idionyx and Macromidia and the Palae-arctic Oxygastra). Corduliinae and Macromiinae are pre-dominantly Holarctic and Indomalayan, with substantialradiations and/or expansions into South America by theformer and Africa by the latter. Libellulidae, as a whole,is cosmopolitan.

Future morphological work should be cautious aboutcreating families or subfamilies based on characters proneto convergence. While some of the details of our tree areweakly supported and may change with further data andanalysis, most of the framework appears sound. On a muchbroader scale, this reinforces suggestions that previousphylogenetic hypotheses have suffered from: (1) use ofpoorly defined and sometimes inaccurately scored charac-ters (O’Grady and May, 2003); (2) groups based on sympl-esiomorphies; and (3) failure to recognize the widespreadeffects of character correlation and convergence, especiallyin aspects of venation (Carle, 1982b; Dijkstra and Vick,2006; Fleck et al., in press). An increase in the use of larvaland genitalic character information is a promising step for-ward. Future work should use this phylogeny as a tool tofocus in on phylogenetically problematic areas within theLibelluloidea. Although Libelluloidea is highly speciose,often collected, and familiar, our understanding of its phy-logenetic history is only just beginning.

Acknowledgments

We thank Jeremy Huff, Kenneth S. Macdonald III, andDana Price for careful review of the manuscript. Thanksalso to Frank Carle for many discussions of phylogenyand character evolution. Thanks to Dennis Paulson forthoughtful comments on our phylogenetic reconstructions.


We are greatly indebted to those who collected or loaned usspecimens for DNA: F.L. Carle, C. Chaboo, T.W. Donnel-ly, S. Dunkle, P. Grant, J. Huff, B. Mauffray, M. Mbida, J.Michalski, D. Paulson, D.L. Price, A. Rowat, R. Rowe, K.Tennessen, G. Theischinger, R. West, the American Mu-seum of Natural History and the California Academy ofSciences. Many thanks to Dave Britton and Gunter Theis-chinger for assistance with Australian collecting permits(permit numbers WT2004-10767, and WITK02489604;loan number 1914). We are also very thankful to GeorgeBaskinger, Mary McLaughlin, and Wlodek Lapkiewiczfor their assistance in the lab. This work was supportedby NSF DEB-0423834.

Appendix A

Although we had samples for the following taxa we wereunable to amplify any of the gene fragments: Pangaeagas-

ter maculata (Cordulegastridae); Apocordulia macrops,Dorocordulia lepida, Idomacromia proavita, Metaphya

elongata, Neocordulia campana, Neocordulia batesi longi-

pollex, Neophya rutherfordi, Williamsonia fletcheri (Cordu-liidae); Anatya guttata, Brachydiplax c. chalybea,Cannaphila vibex, Crocothemis sp, Macrodiplax balteata,Miathyria marcella, Orthemis sp 1 & 2, Tauriphila australis,Thermochoria equivocata, and Trithemis basileri

(Libellulidae).In addition, we were unable to amplify the D2 portion

of the 28S for Zoraena bilineata (Cordulegastridae); Brac-

hymesia herbida, Oligoclada walkeri, and Rhodopygia hol-landi (Libellulidae). Since the D2 region provided manyof the parsimony-informative characters in our datasetwe chose to exclude these from the analysis. Some taxa suc-cessfully sequenced for the 28S gene fragment failed toamplify for the 16S gene fragment but were included inthe analysis: Chloropetalia soarer (Chlorogomphidae);Austrophya mystica, Cordulia shurtleffi, Idionyx selysii,Macromidia rapida, Syncordulia gracilis, andTetragoneuriacynosura (Corduliidae); Dasythemis esmerelda, Elga lepto-

styla, Idiataphe amazonica, Libellula quadrimaculata 2, Lyr-

iothemis pachygastra, Macrothemis celeno, Macrothemis

hemichlora, Neodythemis pauliani, Onychothemis testacea

1, Pantala flavescens 2, Rhodopygia hollandi, Tramea onu-

sta, and Zenithoptera fasciata 1, Zenithoptera fasciata 2(Libellulidae).

We included, in a separate analysis (Fig. 5), 12S and 16Ssequences for several libelluloids from GenBank (below).

Taxon name
Accession No. Author
Cordulegasteridae and Chlorogomphidae
Cordulegaster
boltoni

AF266056
Misof et al. (2001)
Cordulegaster

picta

AF266086
Misof et al. (2001)
Anotogaster

sieboldi

AB127061
Hasegawa andKasuya (2006)
Appendix A (continued)

Taxon name
Chlorogomphus

brunneus

AF266088
Misof et al. (2001)
Corduliidae: Gomphomacromiinae
Oxygastra curtisii AF266103 Misof et al. (2001)
Corduliidae: Macromiinae
Macromia splendens AF266048 Misof et al. (2001)
Libellulidae: Tetrathemistinae
Allorhizucha klingi DQ021437 Fleck et al. (in press) Allorhizucha
preussi

DQ021434
Fleck et al. (in press)
Bironides sp
DQ021432 Fleck et al. (in press) Malgassophlebia
aequatoris

DQ021433
Micromacromia

camerunica

DQ021436
Neodythemis

africana

DQ021435
Notiothemis

robertsi

DQ021431
Tetrathemis polleni
DQ021430 Fleck et al. (in press) Thermorthemis
madagascariensis

DQ021438
Libellulidae: Brachydiplacinae
Micrathyria
didyma

DQ021421
Nannophya

pygmaea

DQ021420
Libellulidae:Leucorrhiniinae
Celithemis elisa DQ021425 Fleck et al. (in press)
Libellulidae: Libellulinae
Agrionopuera
insignis

DQ021439
Cratilla metallica
DQ021441 Fleck et al. (in press) Hadrothemis
infesta

DQ021440
Ladona deplaneta
AF037187 Kambhampati andCharlton (1999)
Ladona exusta
Ladona fulva
AF266098 Misof et al. (2001) Ladona julia AF037186 Kambhampati and
Charlton (1999)
Libellula angelina AF195726 Artiss et al. (2001) Libellula auripennis AF037176 Kambhampati and
Charlton (1999)
Libellula axilena AF037175 Kambhampati and
Charlton (1999)
Libellula comanche AF037182 Kambhampati and
Charlton (1999)
Libellula composita AF195727 Artiss et al. (2001) Libellula
croceipennis

AF037183
Kambhampati andCharlton (1999)
Libellula cyanea



Taxon name
Libellula flavida
AF195728 Artiss et al. (2001) Libellula forensis AF195729 Artiss et al. (2001) Libellula incesta AF037179 Kambhampati and
Charlton (1999)
Libellula jesseana AF037174 Kambhampati and
Charlton (1999)
Libellula luctuosa 12S: AY282563 Saux et al. (2003)
16S: AF037178
Libellula needhami
AF195730 Artiss et al. (2001) Libellula nodistica AF195731 Artiss et al. (2001) Libellula pulchella AF037180 Kambhampati and
Charlton (1999)
Libellula
quadrimaculata

DQ021418
Libellula

semifasciata

AF037171
Libellula vibrans
Lyriothemis

elegantissima

DQ021442
Misagria parana
DQ021419 Fleck et al. (in press) Orthemis
cultriformis

DQ021444
Orthemis discolor
DQ021417 Fleck et al. (in press) Orthemis
ferruginea

AF195732
Artiss et al. (2001)
Orthetrum

albistylum

DQ021412
Orthetrum

albistylum

DQ021413
Orthetrum

brunneum

DQ021411
Orthetrum

brunneum

DQ021414
Orthetrum

brunneum

DQ021415
Orthetrum

brunneum

DQ021416
Orthetrum

cancellatum

AF266097
Misof et al. (2001)
Orthetrum

coerulescens

DQ021445
Oxythemis

phoenicosceles

DQ021443
Plathemis depressa
AF195762 Artiss et al. (2001) Plathemis lydia AF037184 Kambhampati and
Charlton (1999)
Plathemis
subornata

AF037185
Libellulidae: Sympetrinae
Crocothemis
erythrea

AF266100
Misof et al. (2001)
Erythemis

simplicicollis

12S: AY282566
Saux et al. (2003) 16S: AF037191 Kambhampati and
Charlton (1999)
Pachydiplax
longipennis

AF037189

Taxon name
Sympetrum

corruptum

AF037192
Sympetrum

vulgatum

DQ021426
Libellulidae: Trithemistinae
Huonia epinephela DQ021429 Fleck et al. (in press)
Libellulidae: Onychothemistinae
Onychothemis
testacea

DQ021427
Libellulidae: Palpopleurinae
Perithemis lais DQ021422 Fleck et al. (in press)
Libellulidae: Trameinae
Tholymis citrina DQ021423 Fleck et al. (in press) Rhyothemis variegata
imperatrix

DQ021428
Libellulidae: Urothemistinae
Macrodiplax cora DQ021424 Fleck et al. (in press)
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