counting the spots: a molecular and morphological phylogeny of the spotted darner boyeria (odonata:...

Systematic Entomology (2014), 39, 190–195 DOI: 10.1111/syen.12049

Counting the spots: a molecular and morphologicalphylogeny of the spotted darner Boyeria (Odonata:Anisoptera: Aeshnidae) with an emphasison European taxa

M A N P R E E T K A U R K O H L I 1, T H O M A S S C H N E I D E R 2,O L E M U L L E R 3 and J E S S I C A L . W A R E 1

1Department of Biology, Rutgers University, Newark, NJ, U.S.A., 2Berlin, Germany and 3Libbenichen, Germany

Abstract. Boyeria irene and Boyeria cretensis are species of spotted dragonfliesbelonging to the ‘darner’ family, Aeshnidae. In 1991, Peters classified Boyeria fromCrete as B. cretensis, based on adult morphological characters. In this study, weused molecular evidence to determine if indeed B. irene and B. cretensis are differentspecies. DNA was sequenced from samples of B. irene (from France, Switzerland,Tunisia, Spain and Italy) and B. cretensis (from Crete). These species were recoveredas two different clades with strong support. We conclude that B. irene and B. cretensisare different species, with evidence based on molecular and morphological differences.In addition, we present the first phylogenetic hypothesis for Boyeria for which we havesequenced all but three species. Lastly, we discuss different scenarios that may haveled to the present-day distribution and speciation patterns of Mediterranean Boyeria .

Introduction

Boyeria McLachlan (1896) is a Holarctic genus of drab-coloured spotted dragonflies, often seen patrolling alongstreams flying low above the water, commonly know as thespotted darners. Currently the genus has eight described speciesdistributed across the temperate regions of North America,Eurasia and northern Africa: Boyeria irene Fonscolombe, Boy-eria cretensis Peters, Boyeria jamjari Jung, Boyeria karubeiYokoi, Boyeria maclachlani Selys, Boyeria sinensis Asahina,and Boyeria grafiana Williamson and Boyeria vinosa Say.

The Mediterranean B. irene occurs in the south of Europe inPortugal, Spain, France, Italy, Switzerland, and Germany. Innorthern Africa, B. irene has been found in Morocco, Algeriaand Tunisia (Boudot et al., 2009). This species has been wellstudied in an attempt to understand its life history, and ispotentially useful for inference about other Odonata (Ferreras-Romero & Garcia-Rojas, 1995; Ferreras-Romero, 1997).

A morphologically similar species, also found in Europe isB. cretensis . This species is endemic to Crete (Greece), a c.

Correspondence: Manpreet Kaur Kohli, 195 University Ave, BoydenHall 431, Rutgers University, Newark, NJ 07102, U.S.A. E-mail:[email protected]

8300 km2 island that is believed to have completely separatedfrom mainland Europe around 5 Ma (Cellinese et al., 2009).Currently the IUCN Red List of Threatened Species classifiesB. cretensis as a vulnerable species (Boudot, 2010) with themost likely cause of endangerment being habitat destruction(Schneider & Muller, 2006).

The Boyeria on Crete were first considered a differentspecies by Peters (1991), having been previously assumed tobe B. irene (Schmidt, 1965; Battin, 1989). Peters (1991) used50 morphological characters to differentiate Cretan Boyeriaspecimens from B. irene of western Europe, including: (i) thetotal length of the body; (ii) length of the hindwing; (iii) ratioof the length of pterostigma (a coloured elongated cell at thetip of the wing) to the length of hindwing; (iv) relative posi-tion of the hindwing; (v) ratio of position of pterostigma tothe hindwing; (vi) length of the superior appendages (cuticu-lar structures which stick out from the tip of the abdomen);and (vii) length of the inferior appendages. The basis forthis description, however, focused on just five adult maleindividuals collected in Crete. Nonetheless, differences werefound among this small number of individuals in charac-ters such as total length, hindwing length and pterostigmalength.

190 © 2013 The Royal Entomological Society

Phylogeny of Boyeria 191

Boudot (1998) described differences in colour patterns ofadult B. cretensis and B. irene males. In general, species fromCrete were darker, and the light-yellow to greyish-green spotson their abdomen are generally smaller. The basis of thesedescriptions, however, were two pictures taken by the author.In a recent study of larvae, it has been further suggestedthat some very subtle differences might also exist among thelength of the larval abdomen, paraprocts (anal appendages) andalso in the paraproct : epiproct ratio between the two species(Muller et al., 2012). Even though these characters do suggestdifferences amongst Boyeria from mainland Europe and theIsland of Crete, the subtlety of the characters brings in questionthe validity of the two species.

No prior phylogenetic analysis has been undertaken totest the monophyly of Boyeria or its intrageneric relation-ships. In this present work, we determine whether B. ireneand B. cretensis are indeed different species, and also evaluatespecies relationships within the genus Boyeria . We sequencedthree gene fragments (mitochondrial and nuclear) for a molec-ular phylogenetic analysis. We also performed morphometricanalyses based on wing and body characters. In the contextof our results we discuss the biogeographical history of Boy-eria cretensis and its taxonomy and we also present the mostcomprehensive phylogenetic analysis for the genus Boyeria .

Materials and methods

Taxon sampling

Boyeria irene samples were collected from France, Switzer-land, Spain, Italy, Portugal and Tunisia. All B. cretensis sam-ples were from Crete. Besides B. irene and B. cretensis , theNorth American species B. vinosa and B. grafiana and theAsian species B. maclachlani were also included in the anal-ysis. Aeshna and Anax were selected as outgroups based onvon Ellenrieder’s (2002) morphological revision of Aeshnidae.See Table S2 for specimen details.

DNA sequencing

DNA was extracted from samples using a Qiagen DNAextraction kit (Qiagen, Valencia, CA). Legs from either thelarva or adult of each sample was used to extract DNA. Theseventh hypervariable (divergent) region (D7) of the nuclearlarge subunit rDNA (28S ), mitochondrial cytochrome Oxidase1 (COI ) and the nuclear Histone 3 (H3 ) genes were amplifiedusing PCR and previously published primers were used (TableS1). These genes provided good resolution for our researchquestion, with the majority of the signal coming from the COI .We tried to ensure that all taxa had been amplified for theCOI , but attempts to amplify all specimens for the H3 and D7regions were less successful (Table S2). The PCR protocol usedfor amplifications is as follows: 96◦C, 2 min; then 30 cyclesof 94◦C, 30 s; X1

◦C, 30 s; 72◦C, 30 s; 94◦C, 30 s; X2◦C, 30 s;

72◦C, 30 s and a final step of 72◦C for 10 min. Two different

temperatures were used for the annealing step in an attempt toincrease the yield of amplified DNA. X1 was always the lowerof the two temperatures and X2 was at least one degree lessthan the lowest of the melting temperatures of the two primers(up and down) used. Values of X1 and X2 for all the primersare given in Table S1. Successfully amplified samples weresequenced at Macrogen and Genewiz.

Sequence alignment and phylogenetic reconstruction

Contig sequences were evaluated and aligned using the soft-ware tool sequencher 5.0 (Genecode, 2012) and clustalX(Larkin et al., 2007), respectively. Aligned sequences weremanually corrected in mesquite (Maddison & Maddison,2011), and ribosomal sequences were aligned with referenceto secondary structure (Kjer, 1995).

Aligned sequences were analysed using two criteria.garli–part (Zwickl, 2006) was used to evaluate our datasetusing maximum likelihood, while mrbayes (Huelsenbeck &Ronquist, 2001) was used for Bayesian analysis. DNA waspartitioned into three partitions (H3 , COI and D7). DNAevolution models for the three partitions were determinedto be GTR + I + � using jmodeltest (Posada, 2008). Ingarli–part we simulated 1000 bootstrap replicates to obtainbranch support estimates. The software tool sumtrees in den-dropy (Sukumaran & Holder, 2010), was used to obtain aconsensus tree from garli–part. For Bayesian analysis inmrbayes, four MCMC chains – one cold and three hot – weresimulated. The dataset was partitioned into three fragments asin the garli–part analysis. We used GTR + I + � as the sub-stitution model for all the three partitions. All priors were setto default settings [e.g. branch lengths (Brlenspr), topology(Topologypr)]. The rate at which samples were taken from theanalysis (samplefreq) and the rate at which they were printed(printfreq) were set to 1000. The burnin fraction was deter-mined using the software tool are we there yet (AWTY)(Wilgenbusch et al., 2004) and tracer (Drummond & Ram-baut, 2007) after checking for convergence.

Consensus and best trees obtained form garli–part andmrbayes were viewed in figtree (http://tree.bio.ed.ac.uk/software/figtree/), a software tool for viewing and editing phylo-genetic trees.

Morphology

Morphological measurements were made on dried specimenstreated with acetone. All measurements were made based onthe literature (Peters, 1991), and recorded using digital calipers.Statistical analyses were run using microsoft excel. Thecharacters we examined were as follows:

1. Body length: Adult body lengths were measured from thetip of the abdomen to the frons (Peters, 1991).

2. Hindwing length: Length of the wing from the base ofthe wing at the basal sclerite to the tip off the wing wasmeasured.

© 2013 The Royal Entomological Society, Systematic Entomology, 39, 190–195

192 M. K. Kohli et al.

3. Pterostigma length: Pterostigma were measured from themost extreme edge of each of their longer sides.

4. M3 length: Number of cells in the M3 counted. (Peters,1991).

5. Discoidal field width at base: Number of cells at the baseof the discoidal field were recorded (Peters, 1991).

6. Anal field : Number of cells counted (Peters, 1991).7. Crossveins: Number of crossveins (between costa and

subcostal, in the antenodal region) counted (Peters, 1991).

Morphological matrix is available upon request.

Results

jmodeltest suggested using the GTR + I +� model for eachpartition in the Bayesian and Maximum Likelihood analyses.Our dataset contained 1738 nucleotides, 140 of which wereparsimony informative.

Topology

Overall, Boyeria has two main clades: a European/Africanand North America/Asian clade. Figure 1 shows the besttree obtained from maximum likelihood analysis with thenumbers on the branches indicating the bootstrap percentagesfollowed by posterior probabilities (from Bayesian analysis) aspercentages. North American taxa (B. vinosa and B. grafiana)

form a strong monophyletic clade. This clade is sister toB. maclachlani from Japan. Surprisingly, a single B. irene fromGalicia (Fig. 1), Spain groups as sister to the clade includingB. vinosa, B. grafiana and B. maclachlani . A second extractionfrom the same specimen was also recovered in this topologicalposition suggesting it not be a contaminant.

The European Boyeria are recovered in a separate clade:B . cretensis forms a large clade distinct from B . irene with asupport of 91/100%. Within B. irene, populations from Tunisiaappear to be distinct from rest of the B. irene populations.Populations from France, Spain, Portugal, Switzerland andItaly appear as a monophyletic polytomy.

Morphology results

The morphological characters used by Peters (1991) todifferentiate between the two Boyeria species were deter-mined to be variable with the larger sample size studiedhere, i.e. not distinct. We did, however, find significant dif-ferences in an unpaired t-test among B. cretensis and B. irenein their pterostigma length (4.26 ± 0.062 (mean ± SE) and3.8 ± 0.034, respectively; P < 0.0001), number of discoidalcells (4.8 ± 0.133 and 4.1 ± 0.131, respectively; P = 0.0012),number of M3 cells (28.8 ± 0.355 and 25.5 ± 0.450, respec-tively; P < 0.0001), and hindwing length (44.8 ± 0.320 and43.37 ± 0.166, respectively; P < 0.0001) (Fig. 2). However, anF test for variance suggested that the hindwing length characterhad unequal variance (P = 0.05).

Fig. 1. Garli partitioned best tree with branch support represented as: bootstrap support/posterior probability. North American and Japanese Boyeriaare recovered in one clade and Boyeria from Europe – i.e. Boyeria cretensis and Boyeria Irene – are recovered together in another clade.



Significance (P<0.001)

0

10

20

30

40

50

60

70

80

(1) Bodylength

(2) Hindwinglength

(3)Pterostigma

length

Len

gth

(mm

)

Boyeria cretensis Boyeria irene

2

3

4

5

0

5

10

15

20

25

30

35

(4) M3length

(5) Discoidalfield width

at base

(6) Analfield

(7)Crossveins

Num

ber

of c

ells

Fig. 2. Histogram showing the seven morphological characters that were measured between Boyeria irene and Boyeria cretensis . The bars representthe mean for the character and the error bars represents the standard error of the mean. Characters which were significantly different between B. ireneand B. cretensis are marked by . Character 7 is a count of number of crossveins between costal and subcostal region and not the number of cells.

Discussion

Boyeria is divided into two main groups of taxa. Both of thesegroups can be loosely defined as Laurasian. North Americantaxa cluster with Japanese taxa while Mediterranean taxa area monophyletic clade. Whether the missing three species ofBoyeria (B. jamjari , Korea; B. karubei , parts of China, Laos,northeastern Vietnam; and B. sinensis , Southern China) wouldgroup with the Mediterranean or North American/Japanesetaxa remains to be tested. Based on the molecular analysishere Boyeria cretensis is a unique taxon with respect toBoyeria irene as suggested by Peters (1991) and Boudot(1998). In the following text we discuss the implications of ourfindings.

An anomaly

One intriguing anomaly is the position of a single B. irenesample from Spain (Fig. 1), which is strongly supported withNorth American species. This sample was collected in Galicia,Spain, but is not recovered with the other Spanish B. irenespecimens. This has been evaluated for contamination andfound to be a valid B. irene sequence; no ambiguities werefound in the alignment of these sequences. More Galiciansamples are required to evaluate this result, but morphologicaldata suggest this taxon is indeed Boyeria irene and notB. vinosa or B. grafiana .

Taxonomic implication and morphological evaluation

The morphological data used by Peters (1991) did notallow separation of the two taxa when we sampled across alarge distribution. Hence, for the subset of characters (Peters,1991) we examined, several are not diagnostic for the speciesB. cretensis and should not be used as such (e.g. total bodylength, number of cells in anal triangle). Others such ashindwing length and pterostigma length were different in ourseries of both taxa (B. cretensis and B. irene: n = 15, n = 21,respectively; Fig. 2). It may be that morphological differenceswould be even further reduced as we sample more broadlyacross these heterogeneous B. irene populations. From oursamples we could confirm differences in the colour patternof males as suggested by Boudot (1998). However, we foundno difference in female colour pattern although females ofB. cretensis are confirmed to have shorter cerci (Schneider& Muller, 2006). Thus, while there is still morphologicalsupport for our molecular hypothesis of the separation ofthese two taxa, it is not overwhelming. Still, the distinctionof B. cretensis as a unique species is warranted with supportfrom two independent datasets (molecules and morphology).

Possible biogeographical explanations for Mediterranean taxa

The population from Tunisia is clearly distinct based onmolecular data but is still not sufficiently morphologically


194 M. K. Kohli et al.

different to be recognized as a separate species from mainlandEuropean Boyeria at this time. There may be variation ingenetic structure across Europe, due to historical glaciationperiods [an amova in microsoft excel package GenALExv6.5 (Peakall & Smouse, 2006, 2012) suggests 70% molecularvariance among populations and 30% within populations wheneach country was treated as a population].

Dispersal ability and dispersal routes in and around theMediterranean have not been well established for Boyeria ,although other odonate taxa have been shown to disperseacross the sea to Europe from Africa (Boudot et al., 2009); forexample, the 9-mile gap of the Strait of Gibraltar is a feasibledistance for most aeshnids to cross. Hence, individuals mayhave easily migrated from Europe to Africa or vice versa, atleast occasionally. However, B. irene movement might havebeen affected by the barriers presented by the Alps and thePyrenees, especially during episodes of maximal glaciation.Furthermore, there seems to be extreme genetic isolation onCrete. How did B. cretensis colonize and become isolated onCrete?

Hypothesis of colonization of Boyeria on Crete during the lastglacial maxima

In the latest ice ages (110 000 to 10 000 years ago), Cretewas continuously separated from the mainland, as reflected bythe island’s Tertiary plant species (Bottema, 1980). However,during the last glacial maxima, 26 500 to 19 000 years ago(Clark et al., 2009), sea levels were 120–135 m lower thantoday’s (Clark & Mix, 2002), which means that the island wasperhaps less isolated in the past. Dragonflies can easily traveldistances such as that from mainland Europe or Africa to Crete(Corbet, 1999). But did Boyeria from Europe or from Africa(or both) colonize Crete and speciate into B. cretensis? It canbe assumed that all Mediterranean islands could be reached bymost of those anisopteran species from the mainland whosemigration might have been supported by strong winds, whichplay a major role in the displacement of migratory flyinginsects. For example, winds originating in Europe aroundthe last glacial maxima could have carried dragonflies toCrete. In the last glacial maxima, the Mediterranean wasaffected by strong westerlies driven by the icecaps in Europe(Bush & Philander, 1999; Hughes et al., 2006; Rodrigo-Gamizet al., 2011). These winds might have blown dragonflies fromEurope (e.g. France and Portugal) to the Mediterranean islands.However, at the time of glaciation, the temperatures on Cretewere comparatively lower than the present day (Robinsonet al., 2006), and may have been too cold for successfulestablishment of Boyeria .

Hypothesis of isolation after the Messinian Salinity Crisis(MSC, end of Miocene, 5.33 Ma)

In the upper Miocene (Messinian, 6 Ma) the Mediterraneanbasin was separated from the Atlantic Ocean. In only a few

thousand years, the Mediterranean almost completely driedout (Mai, 1989). At this time, today’s Mediterranean islandswere mountain peaks with alpine flora; large territories oftoday’s sea floor were covered with terrestrial ecosystems.With the sea level rising again, Boyeria’s isolation on Cretemay have begun. Divergence estimation of these taxa mayprovide evidence in support of either of these hypotheses, andshould be undertaken in future studies.

Conclusions

Boyeria is a genus currently containing eight distinct species,with B. cretensis now confirmed based on both molecular andmorphological evidence. This study revealed several distinctmorphological differences that demarcate B. cretensis: thelengths of cerci in females, hindwing length, and pterostigmalength. Our understanding of speciation in Boyeria and itsrelationship to historical climatic events is in its infancy. Ourwork has supported recent taxonomical changes, and suggeststhat further biogeographical and divergence time analyses areneeded.

Supporting Information

Additional Supporting Information may be found in the onlineversion of this article under the DOI reference:10.1111/syen.12049

Table S1. Primer sequences used in this work.

Table S2. Taxon sample.

Acknowledgements

We would like to acknowledge Adolfo Codero Rivera, andMichael May for additional specimens used in the presentstudy. Thanks also to: Oliver Flint for help with museumsamples; Gareth Russell and Kimberly Russell for thoughtfulcomments that improved the manuscript; Melissa SanchezHerrera for insightful discussions on odonate dispersal andphylogenetic methodology; Dr. Martin Perlmutter and Dr.Robert Mitchum for their helpful inputs on paleoclimaticconditions in the Mediterranean; and Dominic Evangelista,William Kuhn, Jayshree Patel and Dr. Ankur Agarwal forcomments on improving the manuscript. This work was fundedby Ware’s start-up grant from Rutgers University, Newark.Lastly, thanks to Jasvinder Kohli and Bhupinder Kohli forlogistical support.

References

Battin, T. (1989) Uberblick uber die Libellenfauna der InselKreta (Insecta: Odonata). Zeitschrift der ArbeitsgemeinschaftOsterreichischer Entomologen , 41, 52–64.

Bottema, S. (1980) Pollen analytical investigations on Crete. Reviewof Palaeobotany and Palynology , 31, 193–217.



Boudot, J.P. (1998) Differences in male colour patterns betweenBoyeria cretensis , 1991 and B. irene (Fonscolombe, 1838) (Odonata:Aeshnidae). Opuscula Zoologica Fluminensia , 161, 1–3.

Boudot, J.P. (2010) Boyeria cretensis . In: IUCN 2012. IUCN Red Listof Threatened Species, Version 2012.2 . [WWW document]. URLhttp://www.iucnredlist.org.

Boudot, J. P., Kalkman, V. J., Azpilicueta Amorin, M. et al., 2009.Atlas of the Odonata of the Mediterranean and North Africa.Libellula Supplement , 9, 1–256.

Bush, A.B.G. & Philander, S.G.H. (1999) The climate of the LastGlacial Maximum: results from a coupled atmosphere-ocean generalcirculation model. Journal of Geophysical Research [Atmospheres] ,104, 24 509–24 525.

Cellinese, N., Smith, S.A., Edwards, E.J., Kim, S.-T., Haberle, R.C.,Avramakis, M. & Donoghue, M.J. (2009) Historical biogeographyof the endemic Campanulaceae of Crete. Journal of Biogeography ,36, 1253–1269.

Clark, P.U. & Mix, A.C. (2002) Ice sheets and sea level of the lastglacial maximum. Quaternary Science Reviews , 21, 1–7.

Clark, P. U., Dyke, A. S., Shakun, J. D. et al., 2009. The last glacialmaximum. Science 325, 710–714.

Corbet, P.S. (1999) Dragonflies: Behavior and Ecology of Odonata .Comstock Publishing Associates/ Cornell University Press, Ithaca,NY.

Drummond, A.J. & Rambaut, A. (2007) Tracer V1.4 [WWWdocument]. URL http://beast.bio.ed.ac.uk/Tracer [accessed on 20November 2013].

von Ellenrieder, N. (2002) A phylogenetic analysis of the extantAeshnidae (Odonata: Anisoptera). Systematic Entomology , 27,437–467.

Ferreras-Romero, M. (1997) The life history of Boyeria irene(Fonscolombe, 1838) (Odonata: Aeshnidae) in the Sierra MorenaMountains (southern Spain). Hydrobiologia , 345, 109–116.

Ferreras-Romero, M. & Garcia-Rojas, A.M. (1995) Life-historypatterns and spatial separation exhibited by the odonates from aMediterranean inland catchment in southern Spain. Vie et Milieu ,45, 157–166.

Genecode (2012) Sequencher® Version 5.1 Sequence Analysis Soft-ware. Gene Codes Corporation, Ann Arbor, MI [WWW docu-ment]. URL http://www.genecodes.com [accessed on 20 November2013].

Huelsenbeck, J.P. & Ronquist, F. (2001) MRBAYES: bayesianinference of phylogenetic trees. Bioinformatics , 17, 754–755.

Hughes, P.D., Woodward, J.C. & Gibbard, P.L. (2006) Quaternaryglacial history of the Mediterranean mountains. Progress in PhysicalGeography , 30, 334–364.

Kjer, K.M. (1995) Use of rRNA secondary structure in phylogeneticstudies to identify homologous positions: an example of alignmentand data presentation from the frogs. Molecular Phylogenetics andEvolution , 4, 314–330.

Larkin, M. A., Blackshields, G., Brown, N. P. et al., 2007. Clustal Wand Clustal X version 2.0. Bioinformatics 23, 2947–2948.

Maddison, W.P. & Maddison, D.R. (2011) Mesquite: A ModularSystem for Evolutionary Analysis. Version 2.75 [WWW document].URL http://www.mesquiteproject.org [accessed on 18 November2013].

Mai, D.H. (1989) Development and regional differentiation of theEuropean vegetation during the Tertiary. Plant Systematics andEvolution , 162, 79–91.

Muller, O., Taron, U., Jansen, A. & Schneider, T. (2012) Descriptionof the larva of Boyeria cretensis Peters and comparison withB. irene (Fonscolombe) (Anisoptera: Aeshnidae). Odonatologica ,41, 47–54.

Peakall, R.O.D. & Smouse, P.E. (2006) Genalex 6: genetic analysisin Excel. Population genetic software for teaching and research.Molecular Ecology Notes , 6, 288–295.

Peakall, R. & Smouse, P.E. (2012) GenAlEx 6.5: genetic analysis inExcel. Population genetic software for teaching and research – anupdate. Bioinformatics , 28, 2537–2539.

Peters, G. (1991) The Brook Darner from Crete (Boyeria cretensisSpec-Nov) and the monophyly of genus Boyeria McLachlan,1896 (Odonata Anisoptera, Aeshnidae). Deutsche EntomologischeZeitschrift , 38, 161–196.

Posada, D. (2008) jModelTest: phylogenetic model averaging. Molec-ular Biology and Evolution , 25, 1253–1256.

Robinson, S.A., Black, S., Sellwood, B.W. & Valdes, P.J. (2006) Areview of palaeoclimates and palaeoenvironments in the Levant andEastern Mediterranean from 25,000 to 5000 years BP: setting theenvironmental background for the evolution of human civilisation.Quaternary Science Reviews , 25, 1517–1541.

Rodrigo-Gamiz, M., Martínez-Ruiz, F., Jimenez-Espejo, F.J., Gallego-Torres, D., Nieto-Moreno, V., Romero, O. & Ariztegui, D. (2011)Impact of climate variability in the western Mediterranean during thelast 20,000 years: oceanic and atmospheric responses. QuaternaryScience Reviews , 30, 2018–2034.

Schmidt, E. (1965) Uber den Wanderweg der Boyeria aus Kreta(Odonata, Aeschnidae). Nachrichtenblatt der Bayerischen Ento-mologen , 14, 43–46.

Schneider, T. & Muller, O. (2006) The endemic Boyeria cretensis:observations on the behavioral biology of the adults (Odonata:Aeshnidae). Libellula , 25, 135–146.

Sukumaran, J. & Holder, M.T. (2010) DendroPy: a python library forphylogenetic computing. Bioinformatics , 26, 1569–1571.

Wilgenbusch, J.C., Warren, D.L. & Swofford, D.L. (2004) AWTY:A System for Graphical Exploration of MCMC Convergencein Bayesian Phylogenetic Inference [WWW document]. URLhttp://ceb.csit.fsu.edu/awty [accessed on 18 November 2013].

Zwickl, D. (2006) Genetic algortihm approaches for the phylogenticanalysis of lage biological sequence datasets under the mamimumlikelihood criterion . PhD Dissertation, University of Texas atAustin, Austin, Texas.

Accepted 23 October 2013First published online 16 December 2013


counting the spots: a molecular and morphological phylogeny of the spotted darner boyeria (odonata:...

Documents