Syst. Biol.

54(3):347–362, 2005
Copyright
c Society of Systematic Biologists
ISSN: 1063-5157 print / 1076-836X online
DOI: 10.1080/10635150590949869

Phylogenetic Relationships, Divergence Time Estimation, and Global Biogeographic

Patterns of Calopterygoid Damselflies (Odonata, Zygoptera) Inferred
from Ribosomal DNA Sequences
HENRI J. D UMONT ,1 J ACQUES R. VANFLETEREN,1 J OHAN F. D E J ONCKHEERE,2,3 AND PETER H. H. WEEKERS ,1
1
Department of Biology, Ghent University, Ledeganckstraat, 35, B-9000 Ghent, Belgium; E-mail: HenriDumont@UGent.be (H.J.D.),
Phylogeneticist@hotmail.com (P.H.H.W.)
2
Protozoology Laboratory, Scientific Institute of Public Health—Louis Pasteur, J. Wytsmanstraat 14, B-1050 Brussels, Belgium
3
Present address: Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology, Avenue Hippocrate 74-75,
B-1200 Brussels, Belgium

Abstract.—The calopterygoid superfamily (Calopterygidae + Hetaerinidae) is composed of more than twenty genera in two
families: the Calopterygidae (at least 17) and the Hetaerinidae (at least 4). Here, 62 calopterygoid (ingroup) taxa representing
18 genera and 15 outgroup taxa are subjected to phylogenetic analysis using the ribosomal 18S and 5.8S genes and internal
transcribed spacers (ITS1, ITS2). The five other families of calopterid affinity (Polythoridae, Dicteriadidae, Amphipterygi-
dae, Euphaeidae, and Chlorocyphidae) are included in the outgroup. For phylogenetic inference, we applied maximum
parsimony, maximum likelihood, and the Bayesian inference methods. A molecular phylogeny combined with a geographic
analysis produced a well-supported phylogenetic hypothesis that partly confirms the traditional taxonomy and describes
distributional patterns. A monophyletic origin of the calopterygoids emerges, revealing the Hetaerinid clade as sister group to
the Calopterygidae sensu strictu. Within Calopterygidae, seven clades of subfamily rank are recognized. Phylogenetic dating
was performed with semiparametric rate smoothing by penalized likelihood, using seven reference fossils for calibration.
Divergence time based on the ribosomal genes and spacers and fossil constraints indicate that Calopteryginae (10 genera,
approximately 50% of all Calopterygid taxa studied here), Vestalinae (1 genus), and Hetaerinidae (1 genus out of 4 studied
here) started radiating around 65 Mya (K/T boundary). The South American Iridictyon (without distinctive morphology
except for wing venation) and Southeast Asian Noguchiphaea (with distinctive morphology) are older (about 86 My) and
may be survivors of old clades with a Gondwanian range that went extinct at the K/T boundary. The same reasoning
(and an even older age, ca. 150 My) applies to the amphipterygids Rimanella and Pentaphlebia (South America–Africa). The
extant Calopterygidae show particular species and genus richness between west China and Japan, with genera originating
between the early Oligocene and Pleistocene. Much of that richness probably extended much wider in preglacial times.
The Holarctic Calopteryx, of Miocene age, was deeply affected by the climatic cooling of the Pliocene and by the Pleistocene
glaciations. Its North American and Japanese representatives are of Miocene and Pliocene age, respectively, but its impov-
erished Euro-Siberian taxa are late Pliocene-Pleistocene, showing reinvasion, speciation, and introgression events. The five
other calopterid families combine with the Calopterygidae and Hetaerinidae to form the monophyletic cohort Caloptera,
with Polythoridae, Dicteriadidae, and Amphipterygidae sister group to Calopterygoidea. The crown node age of the latter
three families has an age of about 157 My, but the Dicteriadidae and Polythoridae themselves are of Eocene age, and the
same is true for the Euphaeidae and Chlorocyphidae. The cohort Caloptera itself, with about 197 My of age, goes back to the
early Jurassic. [Biogeography; Calopteridae; dating; divergence times; damselflies; internal transcribed spacers; odonata;
phylogeny; phylogeography; 18S and 5.8S ribosomal DNA.]

Calopterygoid damselflies sensu strictu (= the fam-
ilies Calopterygidae and Hetaerinidae) are cosmopoli-
tan excluding only Australia, New Zealand, and the
polar zones. Across this vast territory, they are a re-
markably uniform in habitat choice (running waters,
which they rarely leave) and in morphology, but the
males present a wide array of precopulatory displays
(Buchholtz, 1955; Heymer, 1972). Calopterygids can be
distinguished by size, body, and wing color (almost in-
variably showing bright metallic sheens) and behavior,
but rarely by morphology (Asahina, 1976). The phyloge-
netic relationships among genera and species are there-
fore unclear. Morphology is not a robust criterion for clas-
sifying taxa in which reproductive isolation is achieved
by color-flashing and premating displays rather than by
mechanical (lock-antilock) mechanisms (Dumont et al.,
1987). In search of new technical approaches to taxon-
omy, electrophoretic analysis of allele frequencies was at-
tempted (Maibach, 1985), but this approach is restricted
to water-soluble enzymes and requires fresh specimens.
In Calopteryx spp., quantitative wingspot analysis has
been used for studying the taxonomy and range of
species (Dumont et al., 1993). This technique is limited to
347
taxa that have a wingspot, which is the case in less than
half of the genera.
The internal transcribed spacers, in which substitu-
tions are more frequent than in the structural rRNAs, are
useful to distinguish between related species that other-
wise show little genetic divergence (Porter and Collins,
1991; Fritz et al., 1994; Tang et al., 1996). To date, a
handful of studies have addressed the phylogenetic re-
lationship of the Odonata at the molecular level (Artiss
et al., 2001; Chippendale et al., 1999; Kambhampati and
Charlton, 1999; Misof et al., 2000, 2001; Weekers et al.,
2001). Misof et al. (2000) sequenced fragments of the mi-
tochondrial 16S rDNA to provide a partial phylogeny
of the Calopterygids, with special emphasis on species
groups within Calopteryx sensu strictu. Weekers et al.
(2001) used the combined ITS1 and ITS2 to resolve the
western and central European taxa of Calopteryx at the
species-group, species, and, where possible, subspecies
level. They confirmed most of Misof’s conclusions and
found evidence for incipient speciation in west Mediter-
ranean taxa. Lindeboom (1996) used a combination of
wing venation characters and male ligula structure to
derive a hypothetical tree for 13 genera. Both Misof et al.
348 SYSTEMATIC BIOLOGY
(2000) and Lindeboom’s (1996) phylogeny are partial and
require further testing. Recently, Rehn (2003) published
a morphology-based phylogeny of the order Odonata,
which was particularly detailed for the suborder Zy-
goptera, placing the Calopterygoidea near the base of
the Zygoptera.
Here, we sequence and analyze the rDNA genes (18S,
5.8S) and internal transcribed spacers (ITS1, ITS2) of 62
Calopterygidae (all extant genera) and Hetaerinidae, and
15 outgroup taxa. The latter include the families Poly-
thoridae, Dicteriadidae, Amphipterygidae, Euphaeidae,
and Chlorocyphidae, supposed to form a clade of higher
order, the cohort Caloptera. Other outgroup families
included in the study were Megapodagrionidae, Pro-
toneuridae, Platycnemidae, and Diphlebiidae. We com-
bined molecular, geographical, and fossil data to inves-
tigate phylogenetic relationships, to relate these to their
geographic ranges, and to link them to geological events.
M ATERIALS AND M ETHODS
DNA Extraction, PCR Amplification,
and Sequencing Reactions
The origins of the samples used in this study are
listed in Table 1. Single samples of taxa that were ex-
pected to be well demarcated were analyzed, whereas
several populations were sampled in the case of taxa
that were a priori expected to be closely related. Mus-
cular tissue was isolated from the thorax and total
DNAs were prepared according to the protocol of the
Puregene DNA isolation kit type D-5000A (Gentra Sys-
tems, Inc., BIOzym, Landgraaf, The Netherlands). The
complete region of the ribosomal spacers (ITS1 and
ITS2) and the ribosomal 18S, 5.8S, and part of the 28S
genes was amplified using the polymerase chain reac-
tion (PCR) with Qiagen DNA polymerase (Westburg,
Leusden, The Netherlands). Eukaryote-specific exter-
nal primers complementary to the 5
-terminus of
the 18S rDNA gene (5
-TYCCTGGTTGATYYTGCCAG-
3
) and the 5
-terminus of the 28S rDNA gene (5
-
TCCTCCGCTTABTDATATGCTTAA-3
) were used to
amplify the entire 18S-ITS1-5.8S-ITS2 and part of the 28S
region. PCR amplifications, purification of the PCR prod-
ucts, and DNA sequencing was done according to stan-
dard procedures (Samraoui et al., 2003). External (see
above) and internal primers in conserved regions of the
18S and 5.8S rDNA were used for sequencing (Weekers
et al., 1994; Samraoui et al., 2003).
Sequence Alignment and the Construction of Datasets
Because we have nucleotide sequences from different
genes (18S, 5.8S) and internal transcribed spacers (ITS1,
ITS2) that evolve at different rates, several methods can
be applied for treatment of partitioned data: the com-
bined data, separate analysis, and conditional combina-
tion approaches (Huelsenbeck et al., 1996).
The DNA sequences covering the complete 18S-
ITS1-5.8S-ITS2-28S (partial) region were aligned with
CLUSTALW 1.64b (Thompson et al., 1994) using de-
fault settings, resulting in an initial data set. A second
data set was created by fine-tuning the alignment of
VOL. 54
the initial data set based on secondary structural in-
formation, using DCSE 3.4 (Dedicated Comparative Se-
quence Editor program; De Rijk and De Wachter, 1993)
or GeneDoc 2.6.002 (Nicholas et al., 1997). The align-
ment of the 18S gene region was manually optimized
with published 18S rDNA sequences based on the con-
servation of both primary sequence data and inferred
secondary structural features (the rRNA WWW Server:
http://www-rrna.uia.ac.be/ssu/index.html) (The Ri-
bosomal Database Project: http://rdp.cme.msu.edu/
download/SSU rRNA/alignments/). The small and
highly conserved 5.8S gene region and the small por-
tion of the 28S gene were easy to align and were used
to position the highly variable ITS1 and ITS2 regions.
The boundaries of the ITS1 and ITS2 were determined
by comparison of the aligned data set with previously
published ITS sequences of calopterygids (Weekers et al.,
2001), and other hexapod taxa available in the EMBL
databank (e.g., Pseudococcus sp., Beuning et al., un-
published data; Luehdorfia sp., Makita et al., unpub-
lished data; Anopheles sp., Beebe et al., 1999; Adalia sp.,
Schulenburg et al., 2001). The ITS regions were manu-
ally optimized based on conservation of both primary se-
quence data and inferred secondary structural features.
The secondary structures of the ITS1 and ITS2 regions
were predicted using the Mfold webserver for nucleic
acid folding and hybridization prediction (Zuker, 2003)
(http://www.bioinfo.rpi.edu/applications/mfold) and
were compared with published data (Fritz et al., 1994;
May and Coleman, 1997; Morgan and Blair, 1998;
Gottschling et al., 2001).
Sequence and Phylogenetic Analyses
First, the likelihood-ratio test (LRT) and Akaike Infor-
mation Criteria (AIC) in ModelTest 3.06 (Possada and
Crandall, 1998) were used to select an appropriate sub-
stitution model of DNA evolution. The data set was an-
alyzed with the Bayesian inference algorithm (MrBayes,
version 3.0b4; Huelsenbeck and Ronquist, 2001), and the
maximum parsimony (MP) and the maximum likelihood
(ML) algorithms in PAUP∗ 4.0b10 (Swofford, 2003) to re-
solve the phylogenetic relationships. The Bayesian es-
timates of posterior probability and bootstrap analyses
were included to assess support. The model with corre-
sponding nucleotide frequencies, substitution rates and
types, and Ti/Tv ratios was selected by ModelTest 3.06
(Posada and Crandall, 1998) and was used for MP and
ML algorithms in PAUP∗ , and in the Bayesian analysis.
Pairwise sequence divergence data between taxa
were computed for the complete 18S-ITS1-5.8S-ITS2-
28S (partial) region. Absolute distance values and dis-
tances based on a maximum-likelihood distance matrix
(PAUP∗ ), with appropriate parameters for the DNA evo-
lution model (ModelTest), were calculated.
The Bayesian analysis was performed with MrBayes
version 3.0b4 (Huelsenbeck and Ronquist, 2001), speci-
fying the appropriate model structure for each partition
(18S, ITS1, 5.8S, ITS2) in the data set. The appropriate
mixed models for the heterogeneous data were speci-
fied, thus applying site-specific models of rate variation
2005 DUMONT ET AL.—RIBOSOMAL DNA PHYLOGENY, DATING AND BIOGEOGRAPHY OF CALOPTERYGOIDS
TABLE 1. Taxon names, geographical origin, collectors name, and EMBL accession number of the ribosomal DNA sequences (18S, ITS1, 5.8S,
ITS2, partial 28S) that are used in this study. Collector’s institutional affiliations are provided in Acknowledgments.
Species/subspecies name
Calopteryx splendens
Calopteryx splendens ancilla
Calopteryx splendens johanseni
Calopteryx splendens taurica
Calopteryx splendens caprai
Calopteryx intermedia persica
Calopteryx orientalis
Calopteryx exul
Calopteryx syriaca
Calopteryx virgo virgo
Calopteryx virgo
Calopteryx xanthostoma
Calopteryx xanthostoma
Calopteryx haemorrhoidalis
Calopteryx haemorrhoidalis
Calopteryx japonica japonica
Calopteryx japonica altaica
Calopteryx aequabilis
Calopteryx maculata
Calopteryx cornelia
Calopteryx amata
Atrocalopteryx atrata
Archineura incarnata
Matrona cyanoptera
Matrona nigripectus
Matrona basilaris
Matrona basilaris
Matronoides cyaneipennis
Neurobasis chinensis
Neurobasis chinensis
Neurobasis chinensis
“Leucopteryx” hetaerionoides
Echo modesta
Mnais pruinosa
Mnais tenuis
Mnais andersoni (hyaline wing tip)
Mnais andersoni (orange wing tip)
Mnais mneme
Mnais yunosukei
Psolodesmus mandarinus dorothea
Psolodesmus mandarinus mandarinus
Psolodesmus mandarinus kuroiwae
Vestalis gracilis
Vestalis lugens
Vestalis smaragdina
Vestalis amoena
Vestalis anne
Caliphaea angka
Caliphaea confusa
Noguchiphaea yoshikoae
Iridictyon myersi
Umma saphirina
Umma longistigma
Sapho cf. ciliata
Sapho ciliata
Sapho gloriosa
Sapho bicolor
Phaon iridipennis
Phaon madagascarensis
Hetaerina americana
Hetaerina titia
Hetaerina medinai
Euthore fasciata
Heliocharis amazona
Rimanella arcane
Geographical origin
Ljubljana, Strojanova voda, Slovenia
Pripja, Belarus
Krasnogoskoje, Altai, Russia
Crimea Peninsula, Ukraine
Pontecorvo, Frosinone, Italy
Shiraz, Iran
Alamdeh, Iran
Ifrane, Middle Atlas, Morocco
North-Jordan River, Jordan
Laon, France
River Kolpa, Kostel Kocevje, Slovenia
Liguria, Italy
Fleuve l’Argens, Chateauvert, France
Ifrane, Middle Atlas, Morocco
Oued Bou Redin, Algeria
Kamogawe River, Japan
S. Antybas, Russia
Wisconsin, USA
Oklahoma, USA
Yuragawe River, Japan
New Brunswick, Canada
Dokigawe River, Japan
North Guangdong, China
Wanli, Tapei Co, North Taiwan
Chiang Mai, Doi Inthanon, Thailand
Hainan, China
Chechuan, Omei Shan, China
Sabah, Mount Kinabalu, Borneo
Kanchanaburi river, Thailand
Asan Lake, India
Hainan, China
Lak Sao, Laos
Kanchanaburi, Thailand
Sakaigawe River, Japan
Neishwangsi, Tapei, North Taiwan
Chiang Mai, Doi Inthanon, Thailand
Chiang Mai, Doi Inthanon, Thailand
Hainan, China
Chiang Mai, Doi Inthanon, Thailand
Unknown, South Taiwan
Unknown, North Taiwan
Mount Omoto, Ishikagi, Japan
Kanchanaburi, Thailand
West-Sumatra prov., Sumatra
Chiang Mai, Doi Inthanon, Thailand
Krabi, Khao Phanom Bencha, Thailand
Ranong, Klong Nakha, Thailand
Chiang Mai, Doi Inthanon, Thailand
Unknown, Bhutan
Chiang Mai, Doi Inthanon, Thailand
Sierra De Lema, Venezuela
Budongo Forest, Uganda
Mbalmago Forest, Cameroon
Ivory Coast
Mabi, Sierra Leone
Mbalmago Forest, Cameroon
Mbalmago Forest, Cameroon
Mbalmago Forest, Cameroon
Unknown, Madagascar
Arkansas, USA
Arkansas, USA
Sierra De Lema, Venezuela
Cumbre, Venezuela
Env. De Saisl, French Guyana
Sierra De Lema , Venezuela
349
EMBL accession no.
Collector’s name (sequenced region)
A. Brancelj X98502 (18S, ITS1, 5.8S, ITS2)
L. Nagorskaja AJ459187 (18S, ITS1, 5.8S, ITS2)
H. J. Dumont AJ459188 (18S, ITS1, 5.8S, ITS2)
Y. Zaitsev Y12894 (18S), AJ458979 (ITS & 5.8S)
A. Cordero-Rivera AJ458966 (18S), AJ308371 (ITS & 5.8S)
H. Heidari AJ459191 (18S, ITS1, 5.8S, ITS2)
H. Heidari AJ459192 (18S, ITS1, 5.8S, ITS2)
H. J. Dumont Y12891 (18S), AJ308346 (ITS & 5.8S)
A. Budieri AJ459190 (18S, ITS1, 5.8S, ITS2)
H. J. Dumont AJ458968 (18S), AJ308359 (ITS & 5.8S)
A. Brancelj X98503 (18S, ITS1, 5.8S, ITS2)
M. Pavesi AJ458971 (18S), AJ308353 (ITS& 5.8S)
M. Papazian AJ458972 (18S), AJ308354 (ITS& 5.8S)
H. J. Dumont Y12892 (18S), AJ308347 (ITS& 5.8S)
B. Samraoui AJ458976 (18S), AJ308362 (ITS& 5.8S)
K. Inoue Y12893 (18S), AJ458980 (ITS& 5.8S)
H. J. Dumont AJ459193 (18S, ITS1, 5.8S, ITS2)
S. W. Dunkle Y12888 (18S), AJ308360 (ITS & 5.8S)
S. W. Dunkle AJ459198 (18S, ITS1, 5.8S, ITS2)
K. Inoue Y12890 (18S), AJ458981(ITS & 5.8S)
S. W. Dunkle AJ458977 (18S), AJ308361 (ITS & 5.8S)
K. Inoue Y12889 (18S), AJ458982 (ITS & 5.8S)
K. O. P. Wilson AJ459202 (18S, ITS1, 5.8S, ITS2)
W. C. Yeh AJ459205 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459206 (18S, ITS1, 5.8S, ITS2)
K. O. P. Wilson AJ459203 (18S, ITS1, 5.8S, ITS2)
Su. Rong AJ459204 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459201 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen Y12899 (18S), AJ458983 (ITS & 5.8S)
H. J. Dumont AJ459199 (18S, ITS1, 5.8S, ITS2)
K. O. P. Wilson AJ459200 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ746327 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen Y12895 (18S), AJ458984 (ITS & 5.8S)
K. Inoue & I. Wakana Y12898 (18S), AJ458985 (ITS & 5.8S)
W. C. Yeh AJ459212 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459210 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459211 (18S, ITS1, 5.8S, ITS2)
K. O. P. Wlison AJ459213 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459214 (18S, ITS1, 5.8S, ITS2)
W. C. Yeh AJ459207 (18S, ITS1, 5.8S, ITS2)
W. C. Yeh AJ459208 (18S, ITS1, 5.8S, ITS2)
K. Watanabe (via Yeh) AJ459209 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen Y12901 (18S), AJ458986 (ITS & 5.8S)
M. Hämäläinen AJ459218 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459217 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459215 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459216 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459219 (18S, ITS1, 5.8S, ITS2)
A. Mitra AJ459220 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459228 (18S, ITS1, 5.8S, ITS2)
H. J. Dumont AJ459227 (18S, ITS1, 5.8S, ITS2)
V. Clausnitzer AJ459223 (18S, ITS1, 5.8S, ITS2)
G. Chiambeng AJ459224 (18S, ITS1, 5.8S, ITS2)
W. Schneider Y12902 (18S), AJ458988 (ITS & 5.8S)
W. Schneider Y12903 (18S), AJ458987 (ITS & 5.8S)
G. Chiambeng AJ459221 (18S, ITS1, 5.8S, ITS2)
G.Chiambeng AJ459222 (18S, ITS1, 5.8S, ITS2)
G. Chiambeng AJ459225 (18S, ITS1, 5.8S, ITS2)
M. J. Parr AJ459226 (18S, ITS1, 5.8S, ITS2)
S. W. Dunkle Y12896 (18S), AJ458989 (ITS & 5.8S)
S. W. Dunkle Y12897 (18S), AJ458990 (ITS & 5.8S)
H. J. Dumont AJ459229 (18S, ITS1, 5.8S, ITS2)
J. Demarmels AJ746325 (18S, ITS1, 5.8S, ITS2)
G. Fleck AJ746326 (18S, ITS1, 5.8S, ITS2)
J. Demarmels AJ746323 (18S, ITS1, 5.8S, ITS2)
(Continued on next page)
350 SYSTEMATIC BIOLOGY
TABLE 1. Continued
Species/subspecies name Geographical origin
Pentaphlebia stahli Mount Cameroon, Cameroon
Euphaea impar Perak, Malaysia
Dysphaea walli Kanchanaburi, Thailand
Sundacypha petiolata Pahang, Krau, Malaysia
Chlorocypha curta Unknown, Cameroon
Heteragrion mitratum Tachira, Venezuela
Philogenia cassandra Cumboto, Venezuela
Prodasineura autumnalis Krabi, Khao Phanom Bencha, Thailand
Elattoneura analis Pahang Krau, Malaysia
Coeliccia loogali Chiang Mai, Thailand
Platycnemis pennipes Damvallei, Belgium
Philoganga vetusta Shingmum, Honkong
for each partition. In a first analysis, the length of the
Bayesian run was tested in order to be certain of con-
vergence. The Markov chain Monte Carlo (MCMC) pro-
cess was set so that four chains ran simultaneously for
5,000,000 generations, with trees being sampled every
100 generations for a total of 50,000 trees in the initial
sample. For the final analysis, five independent Bayesian
runs were performed in order to confirm that there was
adequate convergence and mixing. Each MCMC process
started from random starting points, and was set so that
four chains ran simultaneously for 1,000,000 generations,
with trees sampled every 100 generations for a total of
10,000 trees in the initial sample. Variation in the ML
scores in the samples was examined by inspecting the
MrBayes log file, and the position where the ML scores
stopped improving was determined. The portion of the
trees before the position (tree number) where the ML
score stopped improving dramatically and only fluctu-
ated around a plateau was discarded. The posterior prob-
ability of the phylogeny and its branches was determined
for all those trees in the plateau phase with near the best
ML scores.
Equally weighted MP analyses were performed with
PAUP∗ . Heuristic search settings were stepwise taxon
addition, tree bisection-reconnection branch swapping,
multiple trees retained, no steepest descent, rearrange-
ments limited to 10,000, and accelerated transformation.
Treating gaps as characters as in Swofford (1996) or
Lutzoni et al. (2001) would have provided more informa-
tion from these sites, but we treated gaps as missing data
so that the MP analysis could be directly compared to
the ML analyses. The nonparametric bootstrap analysis
used 1000 replicates to assess the reliability of individual
branches in the phylogenetic trees obtained by heuris-
tic search with stepwise sequence addition (Felsenstein,
1985).
For ML analysis, the substitution model of DNA evo-
lution with corresponding parameters that best fitted
the data was determined by the LRT and the AIC, us-
ing ModelTest 3.06 (Posada and Crandall, 1998). Heuris-
tic search settings were stepwise taxon addition, TBR
branch swapping, MulTrees option in effect, no steepest
descent, and rearrangements limited to 10,000. The non-
parametric bootstrap analysis with 100 replicates was
used to assess the reliability of individual branches in
VOL. 54
EMBL accession no.
Collector’s name (sequenced region)
H. J. Dumont AJ746324 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ746322 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ746321 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ746320 (18S, ITS1, 5.8S, ITS2)
G. Chiambeng AJ746319 (18S, ITS1, 5.8S, ITS2)
J. Demarmels AJ746317 (18S, ITS1, 5.8S, ITS2)
J. Demarmels AJ459232 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ459231 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ746315 (18S, ITS1, 5.8S, ITS2)
M. Hämäläinen AJ746316 (18S, ITS1, 5.8S, ITS2)
H. J. Dumont AJ459230 (18S, ITS1, 5.8S, ITS2)
K. O. P. Wlison AJ746318 (18S, ITS1, 5.8S, ITS2)
the phylogenetic trees obtained by heuristic search with
stepwise sequence addition (Felsenstein, 1985). Trees
were displayed with TREEVIEW 1.6.6 (Page, 1996).
Divergence Time Estimation
To date, many methods are available for phylogenetic
dating (e.g., Britton et al., 2002; Thorne and Kishino,
2002; Yang and Yoder, 2003). Here, dating was done with
the r8s program (Sanderson, 2002, 2003) by semiparamet-
ric rate smoothing using a penalized likelihood approach
applied to the distances inferred from the phylogenetic
trees (ML, MrB). It combines a model-based likelihood
approach with a roughness penalty that prevents too
much rate variation across the tree. The size of the rough-
ness penalty is specified by a smoothing parameter ob-
tained by a cross-validation procedure. Cross validation
was performed for the trees with branch lengths, ob-
tained by multiplying the per site values as reported by
PAUP∗ with the number of sites. This procedure provides
an objective method for model selection and choice of op-
timal smoothing value (Sanderson, 2002, 2003). Compar-
ison of six independent dating analyses (ML trees with
smoothing factor 31, 100, 316; MrB trees with smooth-
ing factor 100, 316, 1000), using optimal (= lowest) and
suboptimal smoothing factors, was used as a measure of
confidence; average node date and confidence intervals
were calculated.
We used seven reference fossils (Table 2) to estimate
divergence times. All fossil calibration points were used
simultaneously, using the fixage command of r8s. Alter-
natively, we tested dating each with one fossil as calibra-
tion point using the fixage command, and the other six
as constraints specifying minimum ages using the con-
straints command.
R ESULTS
Sequence Analysis and Alignments
We obtained the complete, unambiguous DNA se-
quence for the 18S, 5.8S, partial 28S and internal tran-
scribed spacers (ITS1, ITS2) of 62 calopterygoid (ingroup)
and 15 outgroup taxa (Table 1). The length of the ribo-
somal genes (18S, 5.8S) and internal transcribed spacers
(ITS1, ITS2) and their GC content are listed in Table 3.
2005 DUMONT ET AL.—RIBOSOMAL DNA PHYLOGENY, DATING AND BIOGEOGRAPHY OF CALOPTERYGOIDS
TABLE 2. Reference fossils used in the calibration. Minimum and maximum ages in My are listed; the average is used as calibration point in
the analyses. All fossils were reported by Nel and Paicheler (1993), except Calopteryx atrata, reported by Nel and Brisac (1994).
Genus Family
Steleopteron deichmulleri + Amphypterigidae
Ambiella cruciqera
Prothore explicata Polythoridae
Litheupaea carpenteri + Euphaeidae
Eodichroma mirifica
Umma sp. Calopterygidae
Sapho sannoisiensis Calopterygidae
Sapho armissani Calopterygidae
Calopteryx andancensis Calopterygidae
Calopteryx atrata Calopterygidae
Genetic Distances
Pairwise sequence comparison of 18S-ITS15.8S-ITS2-
28S (partial) region using distance measurements by ML
with settings corresponding to the GTR+G+I model
showed remarkable differences in interspecific sequence
diversity (Table 4; simple data matrix, data matrix for 77
taxa not shown). The tropical and subtropical taxa dis-
played higher interspecific sequence diversity than those
of temperate Eurasia: viz. 0 to 5.5 and 0 to 0.8 substitu-
tions per site. The holarctic genus Calopteryx showed lit-
tle genetic variation (0 to 2.04 substitutions per site) and
divides into North American, Euro-Siberian, and east
Asian taxa. The Euro-Siberian taxa showed least genetic
variation (<0.08 substitutions per site) (Table 4; simple
data matrix, data matrix for 77 taxa not shown).
Phylogenetic Analysis
The alignment contained 2745 aligned positions; 737
sites were variable, 603 of which were parsimony in-
formative. Maximum parsimony, maximum likelihood,
minimum evolution LogDet, and Bayesian inference
analyses were used to construct phylogenetic trees cov-
ering all Calopterid taxa and to compare support among
phylogenetic trees. These analyses resulted in well-
resolved, strongly supported trees, showing consistent
and corroborated topologies among phylogenetic meth-
ods (Figs. 1 to 3).
Based on the results of ModelTest LRT and AIC evalu-
ations, ML analysis was performed with the GTR+G+I
model (for parameters see Fig. 1) and resulted in a tree
(Ln likelihood = −18454.19013) with high ML bootstrap
support for most of the clades. The MP analysis with
heuristic search resulted in 94 most parsimonious trees
(MPTs) of 3071 steps; the bootstrap 50% majority-rule
consensus tree (Fig. 2) shows a similar topology to the
ML tree with only a few minor topological changes. The
Bayesian inference analysis ran for 5,000,000 generations
and showed that a stable likelihood value was reached
after 15,000 generations. Therefore, further multiple in-
dependent Bayesian analyses were confidently run with
a maximum of 1,000,000 generations. All five indepen-
dent Bayesian analyses resulted in identical topologies,
showing a similar topology as the ML tree (Fig. 3). We
351
Age (in My)
Period Maximum Minimum Average
Late Jurassic 159.4 142.0 150.7
Eocene 55.0 33.7 44.4
Middle Oligocene + 41.3 23.8 32.6
Late Eocene
Late Oligocene 28.5 23.8 26.2
Late Oligocene 28.5 23.8 26.3
Oligo–Early Miocene 33.7 16.4 25.1
Late Miocene 7.1 5.3 6.2
Late Pliocene 3.6 1.8 2.7
also tested for variation in nucleotide bias among taxa
by running an ME LogDet+I analysis in PAUP∗ with the
appropriate model, in order to see if the nucleotide bias
altered the phylogeny. LogDet distance analyses (ME)
resulted in a tree topology similar to the ML, MP, and
MrB results, clustering the same groups in the same
way. The only difference was that for some nodes the
bootstrap values increased or decreased a little (data not
shown). The monophyly of cohort Caloptera is strongly
supported (ML = 90, MP = 89, MrB = 100), and most
major and minor clades within it are well supported,
too (>70). The branch point separating Euphaeidae and
Chlorocyphidae from the other Caloptera is also well
supported (ML = 92, MP = 90, MrB = 100). The next
branch points separating Calopterygidae, Hetaerinidae,
Polythoridae, Dicteriadidae, and Amphipterygidae are
not consistently supported. The Calopterygoidea (clus-
ters 1 to 7) are supported by ML (ML = 72) and Bayesian
analysis (MrB = 99), but not by MP analysis (MP = 44).
The monophyly of the true Calopterygidae (clusters 1 to
6) with the Hetaerinidae (cluster 7) as sister group is sup-
ported by all analyses (ML = 99, MP = 88, MrB = 100). A
majority of the major clades in Calopterygidae (clusters
1 to 6) are monophyletic with high support. One of them,
grouping clusters 1 to 3, is considered to include those
genera that together form the Calopteryginae (ML = 90,
MP = 91, MrB = 99). The crown of the tree is composed of
two major monophyletic clades; one for the Neurobasis-
Matrona group (ML = 97, MP = 93, MrB = 100) and one
for the Calopteryx group (ML = 98, MP = 98, MrB = 100).
Within each major clade, the topology of most taxa is
well resolved, confirmed by high support, clustering the
genera. Only the topology within the Calopteryx clade
suffers from poor resolution, with only a few species-
groups well resolved, with high support.
Dating Analysis
Application of the seven fossil calibration points in the
penalized likelihood procedure applied to the ML and
MrB trees provided us with a range of data. Initial results
were obtained with the default settings for dating anal-
ysis in the r8s program, with cross validation function
enforced. The rate smoothing parameters with optimal
(= lowest) and suboptimal cross-validation scores were
352 SYSTEMATIC BIOLOGY VOL. 54

TABLE 3. Length and G+C content of the ribosomal 18S, ITS1, 5.8S, and ITS2 DNA regions for the taxa used in this study.

18S ITS-1 5.8S ITS2

Gene G+C Spacer G+C Gene G+C Spacer G+C

Species/subspecies name length (%) length (%) length (%) length (%)
Ingroup taxa
C. splendens ancilla (Pr, Belarus) 1863 51.85 190 49.47 166 56.02 212 71.70
C. splendens johanseni (Russia) 1863 51.85 190 49.47 166 56.02 212 71.70
C. syriaca (Jordan) 1863 51.85 190 48.95 166 56.02 212 71.70
C. splendens (Slovenia) 1862 51.83 190 49.47 166 56.02 212 71.70
C. intermedia persica (Iran) 1863 51.91 190 49.47 166 56.02 212 71.70
C. orientalis (Iran) 1863 51.91 190 49.47 166 56.02 212 71.70
C. splendens taurica (Crimea) 1863 51.80 190 49.47 166 56.02 212 71.70
C. splendens caprai (Fr, Italy) 1863 51.80 191 50.26 166 56.02 212 71.70
C. japonica japonica (Japan) 1863 51.85 190 49.74 166 56.02 212 70.75
C. japonica altaica (Russia) 1863 51.85 190 49.74 166 56.02 212 71.70
C. xanthostoma (France) 1863 51.91 190 49.21 166 56.02 212 71.70
C. xanthostoma (Italy) 1863 51.91 190 48.69 166 56.02 212 70.28
C. exul (Morocco) 1862 51.88 190 51.31 166 55.42 212 71.70
C. haemorrhoidalis (Algeria) 1863 51.85 190 50.79 166 56.02 212 71.70
C. haemorrhoidalis (Morocco) 1862 51.88 191 51.31 166 56.02 212 71.70
C. virgo (Slovenia) 1862 51.83 192 51.04 166 56.02 212 71.23
C. virgo virgo (France) 1863 51.85 192 51.04 166 56.02 212 71.23
C. cornelia (Japan) 1863 51.85 187 48.13 166 54.82 211 69.67
C. maculata (USA) 1863 51.80 183 50.82 166 56.63 212 71.70
C. aequabilis (USA) 1863 51.80 195 48.21 166 56.02 212 70.28
C. amata (Canada) 1863 51.80 195 47.69 166 56.02 211 69.67
N. chinensis (Thailand) 1863 51.85 177 43.50 165 56.36 212 69.81
N. chinensis (India) 1863 51.85 177 44.07 165 56.36 212 69.81
N. chinensis (China) 1863 51.85 177 43.50 165 56.36 212 69.81
M. cyaneipennis (Borneo) 1863 51.80 163 52.15 165 55.15 216 68.98
A. atrata (Japan) 1863 51.80 188 47.87 165 56.36 211 69.19
A. incarnata (China) 1863 51.80 175 48.00 165 56.36 213 68.54
M. basilaris (China) 1863 51.69 190 48.95 165 56.97 206 67.48
M. basilaris (China) 1863 51.69 187 49.73 165 56.97 213 67.61
M. cyanoptera (Taiwan) 1863 51.64 192 47.40 165 57.58 214 69.16
M. nigripectus (Thailand) 1863 51.80 187 49.73 165 56.97 213 68.08
P. dorothea (Taiwan) 1863 51.80 195 52.31 165 56.97 222 65.77
P. mandarinus (Taiwan) 1863 51.80 195 52.31 165 56.97 222 65.77
P. mandarinus (Japan) 1863 51.80 196 52.04 165 56.97 229 65.50
M. andersoni (Thailand) 1863 51.80 203 52.22 166 56.63 220 72.27
M. andersoni (Thailand) 1863 51.80 203 52.22 166 56.63 220 72.27
M. tenuis (Taiwan) 1863 51.80 200 50.00 166 56.63 220 72.27
M. mneme (China) 1863 51.80 193 54.40 166 56.63 221 71.95
M. pruinosa (Japan) 1863 51.80 192 52.08 165 56.97 218 72.48
M. yunosukei (Thailand) 1863 51.80 201 54.23 166 56.63 220 71.30
E. modesta (Thailand) 1860 51.77 199 53.27 164 57.32 213 60.09
L. hetaerinoides (Laos) 1863 51.95 189 48.68 163 57.67 214 74.30
V. amoena (Thailand) 1863 51.74 187 53.48 166 56.02 207 74.40
V. anne (Thailand) 1863 51.74 186 52.69 166 56.02 207 75.36
V. gracilis (Thailand) 1861 51.69 189 55.56 166 56.02 208 73.56
V. smaragdina (Thailand) 1863 51.80 213 49.77 166 56.63 208 68.27
V. lugens (Sumatra) 1863 51.74 171 50.29 166 56.02 205 71.22
C. angka (Thailand) 1863 51.91 202 45.54 166 56.63 220 68.18
C. confusa (Bhutan) 1863 51.91 202 45.05 166 56.63 220 68.64
S. bicolor (Cameroon) 1863 51.64 233 52.79 166 56.63 233 67.81
S. gloriosa (Cameroon) 1863 51.64 225 53.33 166 56.63 220 73.64
S. cf. ciliata (Ivory Coast) 1861 51.80 216 54.63 166 56.63 201 71.64
S. ciliata (Sierra Leone) 1861 51.59 217 56.22 166 56.63 203 71.92
U. saphirina (Uganda) 1863 51.85 215 58.14 166 56.02 196 69.90
U. longistigma (Cameroon) 1863 51.91 211 56.40 166 56.63 195 63.59
P. iridipennis (Cameroon) 1863 52.07 200 58.00 166 56.63 218 73.85
P. madagascarensis (Madagascar) 1863 52.01 200 57.50 163 56.44 215 71.16
I. myersi (Venezuela) 1863 51.80 191 58.12 165 57.58 208 69.08
N. yoshikoae (Thailand) 1863 52.01 178 54.49 164 57.32 207 69.52
H. medinai (Venezuela) 1863 51.69 192 46.35 166 54.22 206 63.59
H. titia (USA) 1860 51.56 203 48.77 166 53.61 212 62.26
H. americana (USA) 1860 51.61 195 42.05 166 53.61 210 60.95
(Continued on next page)
2005 DUMONT ET AL.—RIBOSOMAL DNA PHYLOGENY, DATING AND BIOGEOGRAPHY OF CALOPTERYGOIDS 353

TABLE 3. Continued

18S ITS-1 5.8S ITS2

Gene G+C Spacer G+C Gene G+C Spacer G+C

Species/subspecies name length (%) length (%) length (%) length (%)
Outgroup taxa
Euthore fasciata (Polythoridae) 1863 51.74 222 32.43 169 56.80 228 43.86
Heliocharis amazona (Dicteriadidae) 1863 51.85 169 59.76 166 56.63 215 72.09
Rimanella arcane (Amphipterigidae) 1863 51.80 189 51.85 168 54.76 200 68.50
Pentaphlebia stahli (Amphipterigidae) 1863 52.12 202 48.51 166 56.02 234 68.80
Euphaea impar (Euphaeidae) 1863 51.80 172 48.84 168 55.36 210 67.62
Dysphaea walli (Euphaeidae) 1863 52.01 174 61.49 167 57.49 207 70.53
Sundacypha petiolata (Chlorocyphidae) 1863 51.96 156 60.26 168 56.55 197 68.53
Chlorocypha curta (Chlorocyphidae) 1863 52.07 196 50.51 170 55.88 209 62.68
Heteragrion mitratum (Megapodagrionidae) 1863 51.85 192 47.40 164 56.71 212 66.04
Philogenia cassandra (Megapodagrionidae) 1863 51.85 240 59.17 166 56.63 228 74.56
Ellatoneura analis (Protoneuridae) 1863 51.91 190 58.95 168 55.95 224 68.30
Prodasineura autumnalis (Protoneuridae) 1863 51.85 204 55.39 166 57.23 223 69.06
Coeliccia loogali (Platycnemididae) 1863 51.96 200 40.50 165 55.15 180 59.44
Platycnemis pennipes (Platycnemididae) 1863 51.91 203 50.25 164 56.71 225 71.11
Philoganga vetusta (Diphlebiidae) 1863 52.01 170 50.59 167 56.29 224 70.54

selected, and the dating procedure was then repeated.
The result of the time divergence estimation is shown
in Figure 4, and age estimates for all internal nodes are
shown in Table 5. The analyses using alternative tree
topologies (ML, MrB) and different smoothing factors,
resulting from optimal and suboptimal cross-validation
scores, yielded small deviations in age estimates. The use
of several reference fossils is expected to reduce variation
due to error. The age constraints imposed by the seven
fossils in different parts of the tree are likely to restrict
variation caused by a variety of other factors. The ages
found vary from 238 My (deviation of Caloptera from
outgroup) to virtually zero.
D ISCUSSION
Sequence Variation in Genes and Spacers
The length of the 18S (1860 to 1863 bp) and 5.8S
genes (163 to 170 bp) of the Caloptera under examina-
tion is in the range of previously described odonate taxa
(Samraoui et al., 2002, 2003) and other insect taxa (Gen-
bank/EMBL). The internal transcribed spacers (ITS1,
156 to 233 bp; ITS2, 195 to 234 bp) are among the
shortest known among eukaryotes. The GC content of
the ribosomal genes and internal transcribed spacers is
within normal range among eukaryotes (data from Gen-
bank/EMBL). However, the GC content in the ITS2 is
much higher than in the ITS1 and differs from other ITS
sequences described in literature (Torres et al., 1990). A
balanced GC content between ITS1 and ITS2 (Torres et al.,
1990) is thus not found in Caloptera.
Genetic Distances
It is interesting to compare genetic distances within
and among taxa to determine whether these particular
sequences for a given group of Caloptera have diverged,
on average, more or less than others. The minimum and
maximum divergence values among Calopterygoidea
(Calopterygidae + Hetaerinidae) vary greatly (Table 4).
The highest genetic distance is found between Phaon,
Noguchiphaea, Iridictyon, and Hetaerina. All these, plus
Caliphaea and Vestalis, have at times been given subfam-
ily rank in the literature (e.g., Bechly, 1996). This is here
accepted, as is the family status of the Hetaerinidae. Of
course, the genetic distance with the five “outgroup”
families is even higher. A point that deserves analysis
is the fact that tropical and subtropical taxa (clusters 2
to 7) display higher interspecific sequence diversity than
those of the temperate region (cluster 1): respectively, 0 to
5.5 and 0 to 2.0 substitutions per site. The lowest variation
in genetic distance is between Calopteryx taxa (cluster 1 in
Fig. 1), even though these can be subdivided into a North
American, an Euro-Siberian and an East Asian group. Es-
pecially the Euro-Siberian taxa reveal little genetic vari-
ation (<0.8 substitutions per site) (Table 4; simple data
matrix, data matrix for 77 taxa not shown).
Phylogenetic Relationships and Biogeographic Patterns
All tree topologies suggest that cohort Caloptera is
monophyletic (ML = 90, MP = 89, MrB = 100) (Fig. 1
to 3) and about 200 My old (Fig. 4). Rehn’s (2003) topolo-
gies differ from ours in two main respects: he places
Amphipterygidae outside of Caloptera and shows an
ambiguous topology for the Chlorocyphidae, Dicteri-
adidae, and Euphaeidae. In addition, he considers the
hetaerinids inside Calopterygidae, with Caliphaea and
Vestalis as basal sister groups. Here, we show the mono-
phyly of the Calopterygidae sensu strictu (clusters 1 to 6),
supported by all analyses (ML = 99, MP = 88, MrB = 100)
(Figs. 1 to 3), with Hetaerinidae as basal sister group to
Calopterygidae. However, like in Rehn’s analyses (2003),
the monophyly of the Calopterygoidea (clusters 1 to 7)
has ML bootstrap support (72) and Bayesian probability
support (100) (Figs. 1, and 3), although no MP bootstrap
support (44) (Fig. 2).
The branching order among the 18 genera of
Calopterygoidea (1 newly created, 2 reinstated from syn-
onymy) shows a consistent tree topology. The super-
family arose some 175 Mya; from it, the Calopterygi-
dae and Hetaerinidae started diversifying 151 Mya. Our
354
TABLE 4. Pairwise sequence comparison of the 18S-ITS1-5.8S-ITS2-28S (partial) region for the taxa used in this study. Above the diagonal are absolute nucleotide differences.
Below the diagonal are distances in substitutions per 100 sites. Distance measurements are by maximum likelihood with setting corresponding to the GTR+G+I model, as determined
by ModelTest. Number of substitution types is 6, substitution rate matrix (1.1764, 1.2785, 1.7839, 1.3789, 1.8306), assumed nucleotide frequencies (A = 0.2466, C = 0.2523, G = 0.2702,
T = 0.2309); among-site rate variation (proportion of invariable sites = 0.7097, distribution of rates at variable sites = gamma [continuous] with shape parameter [alpha] = 1.2727).
Minimum and maximum values are given for the monophyletic clusters as shown in Figure 1. Abbreviations conform to the species and location descriptions in Table 1.

C. ma/ama/ Ma/Ar/At/ Mna/Ech/ Sappho/ Iridicti/

Cluster Calopteryx sp. aeq/cor Ne/Ma Leuc/Psol Umma Caliphaea Vestalis Noguchi Phaon Hetaerina
(No.) (1a) (1b) (2) (3) (4) (5a) (5b) (6a) (6b) (7) Outgroup

Calopteryx sp. (1a) 0–19 26–42 42–102 57–85 76–107 86–95 68–87 106–115 94–106 146–154 98–178
———-
0.0–0.80
C. macul/amat/aequa/ 5–46 49–114 69–89 77–106 92–99 74–94 109–117 96–102 151–156 103–179
cornelia (1b) ———–
1.11–1.86 0.21–2.04
Matrona, Archineura, 0–108 70–128 83–138 93–117 76–107 97–128 90–120 144–167 98–180
Atrocalopteryx, Neurobasis, ———–
1.87–5.15 2.42–9.94 0.0–5.54
Matronoides (2)
Mnais, Echo, Psolodesmus, 0–67 61–95 85–96 65–93 95–107 86–102 132–150 100–189
Leucopteryx (3) ———–
2.63–4.18 3.29–4.37 3.37–6.83 0.0–3.26
Sapho, Umma (4) 8–87 86–111 73–104 99–128 85–122 138–166 110–202
————
3.70–5.58 3.76–5.54 3.83–7.66 3.02–5.81 0.32–4.22
Caliphaea (5a) 8 88–95 110–121 105–120 143–155 116–197
——-
4.24–4.94 4.56–5.01 4.67–6.22 4.14–4.75 4.24–5.82 0.033
Vestalis (5b) 3–95 94–106 85–108 131–147 105–183
————
3.25–4.36 3.57–4.79 3.70–5.61 3.07–4.61 3.53–5.37 4.36–4.65 0.12–3.91
Noguchiphaea, Iridictyon (6a) 95 96–110 137–161 118–195
——
5.62–6.02 5.80–6.30 5.64–7.20 4.85–6.01 5.18–7.05 5.74–6.43 4.80–5.56 4.83
Phaon (6b) 42 135–153 119–198
——
4.84–5.60 4.94–5.33 5.28–6.33 4.58–5.52 4.25–6.56 5.53–6.43 4.22–5.67 4.89–5.86 1.87
Heteaerina (7) 73–97 141–189
————
8.38–9.00 8.78–9.06 8.28–10.14 7.41–8.97 7.85–9.83 7.90–8.85 7.20–8.37 7.60–9.40 7.73–8.80 3.48–4.91
Outgroup 49–214
————-
5.15–11.22 5.46–11.29 5.16–11.38 5.12–11.89 5.67–13.36 6.06–12.64 5.59–11.46 6.44–12.77 6.35–12.90 8.11–11.97 2.21–14.25
2005 DUMONT ET AL.—RIBOSOMAL DNA PHYLOGENY, DATING AND BIOGEOGRAPHY OF CALOPTERYGOIDS 355

FIGURE 1. Maximum likelihood estimate of the calopterygoid phylogeny based on combined ribosomal gene (18S, 5.8S) and internal tran-
scribed spacer (ITS1, ITS2) data. The ML analysis with GTR+G+I, gamma correction, R = (1.1764, 1.2785, 1.7839, 1.3789, 1.8306), Pinv = 0.7097,
and gamma-shape parameter = 1.2727 generated a tree with a log likelihood of −18454.19013. Branch lengths are corrected according to the
settings mentioned above. The scale bar represents 0.1 substitutions/site. Bootstrap support, calculated from 100 replicates, is expressed as
percentage.
356 SYSTEMATIC BIOLOGY VOL. 54

FIGURE 2. Maximum parsimony estimate of the calopterygoid phylogeny based on combined ribosomal gene (18S, 5.8S) and internal
transcribed spacer (ITS1, ITS2) data. The MP analysis generated 94 most parsimonious trees (MPTs) of 3071 steps. Bootstrap method with
heuristic search, stepwise taxon addition, TBR branch swapping, MULTREES option, no steepest descent, rearrangements limited to 10,000, and
accelerated transformation. Gaps were treated as missing data. Bootstrap support, calculated from 1000 replicates, is expressed as percentage.
The scale bar represents 10 steps.
2005 DUMONT ET AL.—RIBOSOMAL DNA PHYLOGENY, DATING AND BIOGEOGRAPHY OF CALOPTERYGOIDS 357

FIGURE 3. Bayesian probability estimate of the calopterygoid phylogeny based on combined ribosomal gene (18S, 5.8S) and internal tran-
scribed spacer (ITS1, ITS2) data. Settings of the ML parameters in MrBayes were determined by ModelTest for each individual data partition.
Parameters for the appropriate substitution model were specified for each data partition to enable site-specific rate variation, allowing a mixed
model approach for the heterogeneous data set, with P. vetusta as outgroup. Five independent Bayesian runs where performed, and in each run
one out of every 100 trees was sampled for 1,000,000 generations. The point of stationary ML scores (“burnin”) was after 200 trees; these first
200 trees were discarded and the posterior probability of the phylogeny was determined from 9800 trees. A 50% majority-rule consensus tree
with Bayesian probability values was calculated in PAUP∗ using the 9800 trees with lowest ML scores, rooted with the Diphlebiidae (Philoganga
vetsusta), Megapodagrionidae (Philogenia cassandra, Heteragrion mitratum), Protoneuridae (Prodaisineura autumnalis, Elattoneura analis), and Platy-
cnemididae (Platycnemis pennipes, Coeliccia loogali) as outgroup. Branch lengths are corrected for ML distances using the GTR+G+I model. The
scale bar represents 0.1 substitutions/site. The numbers along the branches are support indicated by Bayesian probability analysis, which is
expressed as percentage.
358 SYSTEMATIC BIOLOGY VOL. 54

FIGURE 4. Dated phylogenetic tree of Calopterygoid damselflies obtained from semiparametric rate smoothing (penalized likelihood;
Sanderson 2002, 2003) of a 77-taxon phylogenetic tree, with maximum likelihood branch lengths from ribosomal genes (5.8S, 18S) and spacers
(ITS1, ITS2), and calibrated with seven reference fossils. Reference fossils are listed in Table 2, and further information on age estimates is found
in Table 5. The time scale shows ages in million years (My) before present. The bar represents a stratigraphy showing geological era, periods
and epoches. Abbreviations: Neo = Neogene; P = Paleocene; E = Eocene; O = Oligocene; M = Miocene; e = early; m = middle; l = late; K/T =
Cretaceous/Tertiary boundary.
2005 DUMONT ET AL.—RIBOSOMAL DNA PHYLOGENY, DATING AND BIOGEOGRAPHY OF CALOPTERYGOIDS 359

TABLE 5. Stem node ages (sna) and crown node ages (cna) in My for was of Cretaceous age. Its present location is a small area
mayor clusters and families of Calopterygids obtained from semipara- of tropical forest situated in the ancient Serra Pacaraima,
metric rate smoothing (penalized likelihoods; Sanderson 2002, 2003)
of a 77-taxon Calopterygoid tree, with maximum likelihood branch northeast South America. This, and its relatedness to
lengths from ribosomal genes (5.8S, 18S) and spacers (ITS1, ITS2), and Noguchiphaea, can only be understood in terms of con-
calibrated with seven reference fossils. Ages with asterisks are aver- tinental drift and both are likely living fossils, relicts
age fossil dates (see Table 2), set to fixed ages in the divergence time of a much richer fauna that is presently almost extinct.
estimation.
Typical calopterygid traits are also found in the African
sna cna
Phaon (support = 100) but the Thai endemic Noguchiphaea
displays several unusual traits: it has petiolated wings,
Group (node) Cluster Age SD Age SD
distinctive male appendages, and peculiar outgrowths
Outgroup — — 238.9 19.5 on the pronotum. The common ancestor it shared with
Caloptera 238.9 19.5 197.0 11.3 Phaon and Iridictyon around 152 Mya originated in the
A 197.0 11.3 174.9 5.8
Chlorocyphidae 110.9 5.8 56.5 3.5 late Jurassic, before the separation of India from Africa
Euphaeidae 110.9 5.8 32.6∗ 0.0 and South America.
Amphipterygidae 157.0 0.9 150.7∗ 0.0 The Phaoninae, with a stem node age of 125 My, are
Polythoridae 157.0 0.9 44.4∗ 0.0 old, and therefore, the presence of Phaon on Madagas-
Dicteriadidae 157.0 0.9 44.4∗ 0.0
Calopterygoidea 1–7 174.9 5.8 151.5 5.8
car, where it is the only calopterygid on record, could
Hetaerinidae 7 151.5 5.8 61.1 1.2 qualify the Malagasy taxon as a Gondwanian relict.
Calopterygidae 1–6 151.5 5.8 125.2 5.7 However, it speciated only some 32 Mya, and is of
Phaon 6b 125.2 5.7 31.9 3.4 Oligocene age. About 95% of the zygopteran odonates
B 125.2 5.7 118.5 6.1 of Madagascar are endemic, but the island has two
Iridiction, Noguchiphaea 6a 118.5 6.1 86.5 4.9
C 118.5 6.1 94.7 4.9 types of endemics, “young” and “old” (Dijkstra and
Vestalis 5b 94.7 4.9 68.6 6.1 Clausnitzer, 2004). Furthermore, the aquatic fauna of the
D 94.7 4.9 89.2 4.6 island lacks rheophilic species, as well among dragon-
Caliphaea 5a 89.2 4.6 4.5 0.5 flies (including the Calopteran families) as among fish
E 89.2 4.6 81.3 3.9
Umma, Sapho 4 81.3 3.9 51.3 2.1
(Kiener and Richar-Vindard, 1972). It is, in general, im-
Umma 51.3 2.1 26.2∗ 0.0 poverished (its total number of dragonfly species is only
Sapho 51.3 2.1 25.6∗ 0.0 175, about half of what would be expected from its sur-
Calopteryginae 1–3 81.3 3.9 60.4 3.2 face area). This suggests a phase of extinction, followed
Mnais, Psolodesmus 60.4 3.2 36.1 4.7 by a recolonization from the African mainland at a time
F 60.4 3.2 56.2 3.2
G 56.2 3.2 46.1 3.8 when the distance to Madagascar was still bridgeable
H 46.1 3.8 35.8 4.3 (Briggs, 2003). Although Phaon is rheophilic and not mi-
Matr, Psol, Neur, Atro, Arch 2 35.8 4.3 32.1 4.0 gratory, it covers a much wider range in Africa than the
Calopteryx 1a + 1b 35.8 4.3 21.0 3.0 strict forest-dwelling Saphoinae, and hence may have
North American Calopteryx 1b 21.0 3.0 16.1 2.1
Eurasian Calopteryx 1a 21.0 3.0 6.2∗ 0.0
some dispersal abilities.
Caliphaea (cluster 5a) and Vestalis (cluster 5b) from trop-
ical Asia are separated and well supported, although the
topology varies between the three analyses (Figs. 1 to
taxon sampling of the Hetaerinidae is much less com- 3). The geographic ranges of both taxa overlap, and the
plete than that of the Calopteryigidae. The speciose condition “stalked wings” is seen to have arisen in the
genus Hetaerina radiated 61 Mya, slighty after the ancestor to Caliphaea independently from Noguchiphaea,
K/T boundary (65 Mya), but some of its three other where stalked wings also occur and which have a similar
genera (Garrison, 2005) may be as old as the oldest stem node age (89 versus 87 My, respectively).
Calopterygidae. Iridictyon (surviving only in Venezuela), Caliphaea with a stem node age of 89 My branched
Noguchiphaea (presently only in North Thailand), and off around the same time as the common ancestor of
the amphipterygids Rimanella (only in Venezuela and Iridictyon and Noguchiphaea and is considered as a sub-
Guyana) and Pentaphlebia (limited to the Cameroon family on account of its stalked wings. We here accept
rainforest) distinctly predate the K/T boundary (about this position, meaning that claims of a subfamily sta-
86 and 150 My, respectively). They probably be- tus for taxa deeper than Caliphaeinae, viz. Vestalinae,
long to clades that were common and geographically Phaoninae, Iridictyoninae, and Noguchiphaeinae (e.g.,
widespread until the K/T events eliminated most of their Bechly, 1996) are also accepted, whether they have a dis-
representatives. These Gondwanian relicts consequently tinctive wing venation and morphology (as in Caliphaea
qualify as living fossils. Aside from these basic clades, it and Noguchiphaea), or mainly a distinctive wing vena-
is of interest to define the status of some other within- tion (as in Vestalis, Iridictyon, and Phaon). The Saphoinae,
calopterygid clades. forest-dwelling African calopterygids, with the morpho-
Among Calopterygidae, clusters 6a and 6b accom- logically well-defined genera Sapho and Umma (cluster
modate the South American Iridictyon, the Asian 4), form a monophyletic clade (support = 100), split off
Noguchiphaea, and African Phaon. Iridictyon has con- from Calopteryginae some 81 Mya (cluster 4). Both gen-
served a typical calopterygid habitus and structure, even era have an age of about 26 My, corroborated by the find-
though its common ancestor with extant calopterygids ing, in France, of well-preserved wings of Umma and
360 SYSTEMATIC BIOLOGY
Sapho of Oligo-Miocene age (Nel and Paicheler, 1993).
Thus, in the subtropical climate that characterized most
of Eurasia during the Cenozoic, at least these calopter-
ine genera already coexisted in Europe, far from their
present geographic ranges. Their current confinement to
the forest zone of tropical Africa reflects the cooling of
the planet since the Cenozoic.
The Calopteryginae (clusters 1 to 3), grouping 10 gen-
era, includes more than half of all extant calopterygids,
East Palaearctic, but extending into the Oriental with the
largest number of genera and species found between
west China and Japan. Only the Hetaerinidae rivals
Calopteryginae in species richness. Like the former, they
are seen to have originated soon after the K/T bound-
ary, and represent another example of radiation follow-
ing the K/T events 65 Mya. Presumably, many drag-
onflies, calopteran and noncalopteran in nature, went
extinct at that time, and left the old world open to the
Calopteryginae.
Tropical and subtropical calopterygids (clusters 2 to
7) display higher interspecific sequence diversity than
those of the temperate region (Table 4), revealing a
dichotomy of the Calopterygoids in taxa with short
branches (presumably young speciation events) and taxa
with long branches (presumably old speciation events).
The “young” group almost unequivocally corresponds
to Holarctic taxa that populate North Africa, Europe,
continental Asia as far as 60◦ N, and North America.
Old taxa are predominantly tropical or straddle the
temperate-tropical transition zone of East Asia, from the
pacific coast of Russia (“Primoriye”) to south China and
Indochina-India.
Young genera, like Mnais, and the complex Matrona-
Atrocalopteryx-Archineura are typically 2.7 to 10.0 My old,
whereas old genera have stem node ages from 95 to 89
My old (Vestalis, Caliphaea). Mnais is of late Miocene age
(ca. 10 My) and has a number of species in Japan that are
notoriously “difficult” to distinguish, reminiscent of the
C. splendens group. In fact, it is only marginally older than
the entire cluster 1a, grouping all Eurasian Calopteryx.
Like Calopteryx, Mnais is apparently still speciating. It is
also noteworthy that the distinctive Chinese Archineura
forms a subcluster with Atrocalopteryx and Matrona that
is hardly 2.7 My old. Archineura thus appears to be a
Pleistocene taxon, unrelated to the Laotian “Leucopteryx”
hetaerinoides (estimated age 46 My), which is sometimes
cited as the “second Archineura.”
The east Palaearctic Atrocalopteryx (2.7 My old) ac-
commodates the former “Calopteryx” atrata, and possi-
bly a few other little-known Chinese “Calopteryx.” For C.
atrata, Selys-Longchamps and Hagen (1854) had created
a special “section” with the following characters: wings
compatively long and narrow, without a pterostigma in
both sexes; postocular tubercles very small to absent;
the wing veins R2 and IR3 detach together form R4+5
(in Calopteryx, R2 detaches from R+M). We here rename
this “section” Atrocalopteryx n.g., with atrata as the type
species.
The genus Calopteryx arose around 35 Mya and started
to speciate approximately 21 ± 3 Mya. It is shared be-
VOL. 54
tween Eurasia and North America, and may have ex-
tended across the Beringian and Thulean routes. The
North Atlantic (Thulean) route was available to ther-
mophilic organisms like swallowtail butterflies until
about 35 Mya (Zakharov et al., 2004), but because of pro-
gressive cooling, and the transformation of the Thulean
route into a chain of islands, younger and thermally
less demanding organisms like Calopteryx (Tiffney, 1985)
may have taken advantage of the Beringian route. North
American Calopteryx started radiating less than 16 Mya;
the Eurasian group began its radiation only around 6.2
Mya. The first product of this radiation, around 5.3 Mya,
was the C. virgo-group (including the Japanese C. cor-
nelia); the C. splendens-group s.l., with only some 3.7 My,
is even younger. At that time, corresponding to the onset
of the Pliocene, the climatic deterioration that culminated
in the Pleistocene glaciations seems to have been suffi-
ciently advanced to start causing significant extinctions.
This condition, which has been repeatedly addressed
in the literature, and was recently reviewed by Hewitt
(2000): the regions of the globe that were directly hit
by the Pleistocene glaciations were impacted by huge
genetic losses, and were later repopulated by a limited
number of taxa with reduced genetic variation. Japan, the
Pacific fringe, China, and North America, less or not af-
fected by mass extinctions and easier to repopulate than
Europe, show this effect to a lesser extent, and have con-
served identified species-groups and even genera (like
Mnais). In Europe, the west Mediterranean refugium al-
lowed C. xanthostoma-exul and C. haemorrhoidalis to sur-
vive the glaciations. Both are between 2.2 and 2.4 My old,
and thus originated just before the onset of the major cli-
matic cooling. The origin of C. splendens s.s. is hard to
identify, and its numerous subspecific tax are all of very
recent origin, and hybridize freely. Even C. xanthostoma is
currently suffering from introgression by an advancing
edge of C. splendens coming from the east, and its range is
both shrinking and breaking up into disjunct fragments
(Weekers et al., 2001).
CONCLUSIONS
Combined analyses of the ribosomal genes (18S, 5.8S)
and their internal transcribed spacers (ITS1, ITS2) pro-
duced a fairly satisfactory reconstruction of the phy-
logeny of a major group of the Zygoptera, the co-
hort Caloptera, with particular emphasis on the family
Calopterygidae. The group is found to be of early Meso-
zoic age. The Calopterygidae (Old World plus the an-
cient Serra Pacaraima upland in South America) form
a clade, whereas a second clade contains the neotropical
Hetaerinidae. We found several Gondwanaland disjunc-
tions in taxa with relict distributions such as Iridictyon,
Noguchiphaea, and the Amphipterygid genera Rimanella
and Pentaphlebia. Our results also provide support to the
concept that taxa in the temperate zone, having been af-
fected by a series of glaciations because the Pleistocene
have not recovered from that climatic vagary. If sequence
variation can be assumed to be relatively steady, then all
speciation events in the Calopteryx splendens group and
2005 DUMONT ET AL.—RIBOSOMAL DNA PHYLOGENY, DATING AND BIOGEOGRAPHY OF CALOPTERYGOIDS
in Mnais are recent. In Europe, particularly, introgres-
sion between previously disjunct taxa is continuing. The
Tropics, in contrast, have conserved the old taxa cited
above, some of which occur in such extremely relict ge-
ographic ranges that they qualify as living fossils.
ACKNOWLEDGEMENTS
The following individuals kindly provided specimens: V. Alekseev
(St. Peterburg, Russia), A. Brancelj (Ljubljana, Slovenia), A. Budieri
(Amman, Jordan), G. Chiambeng (Limbe, Cameroon), V. Clausnitzer
(Marburg, Germany), A. Cordero-Rivera (Pontevedra, Spain), J. De-
marmels (Caracas, Venezuela), J. Dommanget/V. Bosc (Bois d’Arcy,
France), S. W. Dunkle (Plano, TX, USA), G. Fleck (Strassbourg, France),
M. Hämäläinen (Helsinki, Finland), H. Heidari (Teheran, Iran), K. Inoue
and I. Wakana (Osaka, Japan), A. Mitra (Calcutta, India), L. Nacelli-
Flores (Palermo, Sicily), L. Nagorskaja (Minsk, Russia), M. Papazian
(Marseille, France), M. J. Parr (Somerset, United Kingdom), M. Pavesi
(Milan, Italy), S. Rong (Hohhot, China), B. Samraoui (Annaba, Algeria),
W. Schneider (Darmstadt, Germany), K. Van Damme (Ghent, Belgium),
K. Watanabe (via Yeh) (Taipei, Taiwan), K.O.P. Wilson (Hong Kong,
China), W.C. Yeh (Taipei, Taiwan), and Y. Zaitsev (Odessa, Ukraine)
(for full addresses see: http://www.bechly.de/ododir.htm). We also
wish to thank Matti Hämäläinen, Michael May, Karl Kjer, Chris Simon,
and an anonymous reviewer for their critical reading and excellent
suggestions. Funding for this research was provided by Belgian Na-
tional Foundation for Scientific Research to HJD and JFDJ (FWO grant
G.0066.96) and JVF (FWO grant G.0292.00). PHHW was funded in 1996
by a visiting postdoctoral fellowship from the Belgian National Foun-
dation for Scientific Research (FWO) and in 1997 by a grant from Ghent
University. All experimental procedures and phylogenetic and DTE
analyses were performed by PHHW.
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First submitted 28 March 2002; reviews returned 9 August 2002;
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Editor: Chris Simon
Associate Editor: Karl Kjer