research article phylogenetic information content of...

Research ArticlePhylogenetic Information Content of Copepoda Ribosomal DNARepeat Units ITS1 and ITS2 Impact

Maxim V Zagoskin1 Valentina I Lazareva2 Andrey K Grishanin23 and Dmitry V Mukha1

1 Vavilov Institute of General Genetics Russian Academy of Sciences Gubkin Street 3 Moscow 119991 Russia2 Papanin Institute for Biology of Inland Waters Russian Academy of Sciences Borok 152742 Russia3 Dubna International University for Nature Society and Man Universitetskaya Street 19 Dubna 141980 Russia

Correspondence should be addressed to Maxim V Zagoskin zagoskinmvgmailcom Andrey K Grishaninandreygrishaninmailru and Dmitry V Mukha dmitryVmukhagmailcom

Received 23 April 2014 Revised 8 July 2014 Accepted 8 July 2014 Published 18 August 2014

Academic Editor Peter F Stadler

Copyright copy 2014 Maxim V Zagoskin et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing fourfamilies nine genera and two orders of the subclass Copepoda (Crustacea) Fragments approximately 2000 bp in length containingthe ribosomal DNA (rDNA) 18S and 28S gene fragments the 58S gene and the internal transcribed spacer regions I and II (ITS1and ITS2) were amplified and analyzed The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used toanalyze the saturation of nucleotide substitutions this test revealed the suitability of both the 28S gene fragment and the ITS1ITS2rDNA regions for the reconstruction of phylogenetic trees Distance (minimum evolution) and probabilistic (maximum likelihoodBayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferringphylogenetic relationships among families of copepods andwithin theCyclopidae family and associated genera Split-graph analysisof concatenated ITS1ITS2 rDNA regions of cyclopoid copepods suggested that theMesocyclopsThermocyclops andMacrocyclopsgenera share complex evolutionary relationships This study revealed that the ITS1 and ITS2 regions potentially represent differentphylogenetic signals

1 Introduction

Copepods are important components of zooplankton andthe food chain in marine and freshwater ecosystems Thesubclass Copepoda is believed to contain approximately13000morphospecies however the actual number of speciesin this subclass might bemuch greater [1]Themajority of thefreshwater copepod species belong to the order Cyclopoidawhich includes the free-living species (approximately 800)in the family Cyclopidae The other two free-living fami-lies (Oithonidae and Cyclopinidae) contain mainly marinespecies except for a few species in Oithonidae [2]

Systematic analyses of cyclopoid copepods (orderCyclopoida) have primarily focused on morphological char-acteristics [2ndash8] and the majority of molecular studies havetargetedmarine copepods [9ndash41]The phylogenetic history offreshwater cyclopoid copepods is not well understood A few

studies on Cyclopidae have used molecular and morpholog-ical analyses on the Mesocyclops genus (Crustacea Cyclopi-dae) [42] E serrulatus group [43] Acanthocyclops vernalis-robustus species complex [44 45] and 11 populations ofMacrocyclops albidus [46] other phylogenetic analyses havefocused only on molecular markers inDiacyclops spp whichare found in western Australia [47] and Lake Baikal [48]

Molecular markers such as genomic DNA fragments areused for phylogenetic analyses to elucidate the evolutionaryhistory of living organisms and the region of genomic DNAanalyzed is critical Mitochondrial DNA fragments (genesencoding the cytochrome c oxidase subunit I (COI) 16SrRNA and cytochrome b) [9ndash21 39 40 49ndash52] andornuclear rDNA regions have been used for the phylogeneticanalysis of cyclopoid copepods [22ndash37 41 53ndash58]Mitochon-drial DNA fragments might be less useful for the analysisof copepod phylogeny compared to the phylogeny of other

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 926342 15 pageshttpdxdoiorg1011552014926342

2 BioMed Research International

IGS IGS18S 18S58S

ITS1 ITS228S 28S

DAMS18 DAMS28

Figure 1 Organization of eukaryotic tandemly repeated rDNAclusters 18S 58S and 28S ribosomal RNA genes ITS1 and ITS2internal transcribed spacers IGS intergenic spacer Arrows indicatethe locations of the DAMS18 and DAMS28 primers

taxa furthermore amplification of COI is difficult in somecopepods [59ndash61] However these DNA fragments mightbe informative for analyses of population differentiationor cryptic speciation [9 11ndash15 38ndash40 50 52] Thereforecomparison of nuclear rDNA regions might be informativefor the phylogenetic analysis of copepods

In most eukaryotes rRNA genes are located in a multi-gene family of genomic clusters of repeated sequencesWithin these clusters the 18S 58S and 28S rRNA genes areseparated by internal transcribed spacers (ITS1 and ITS2)and an intergenic spacer [62] (Figure 1) Ribosomal DNA(rDNA) is a reliable and informative phylogenetic marker[63] that contains sequences with different rates of evolution-ary variability In most eukaryotes the most evolutionarilyconserved genes are the rRNA genes comparison of theirsequences allows estimation of the evolutionary distances atintergeneric and higher taxonomic levels Comparison of themore evolutionarily variable spacer sequences enables thestudy of phylogenetic relationships at the species and popula-tion levels [63ndash65]Therefore comparison of different regionsof rDNA enables the phylogenetic analysis of organisms overextended evolutionary distances

Phylogenetic relationships among cyclopoid copepods athigher systematic levels (ordinal familial and generic) havebeen resolved using the 18S and 28S nuclear rRNA genes [2829 55ndash57] and the relationships at the lower taxonomic levels(species and populations) have been resolved using the ITS2of the nuclear rDNA gene cluster [30 40 42 52 58]

Notably analysis of the evolutionary history of livingorganisms based on only one molecular marker can uncoverbifurcating phylogenetic trees revealing branched evolutionHowever it recently becomes evident that evolution is notalways tree-like Comparisons of gene trees based on differentgenetic loci often reveal conflicting tree topologies Thesediscrepancies are not always due to the problems withthe sampling and the gene tree reconstruction methodsReticulation events such as horizontal gene transfer (HGT)and hybridization may be responsible for contradictionsin lineages During an HGT event a DNA segment istransferred from one organism to another which is not itsoffspring whereas hybridization describes the origin of a newspecies through an interspecies mating Both processes yieldgenomes that are mixtures of DNA regions derived fromdifferent species Consequently evolutionary relationshipsbetween species whose past includes reticulation can oftenbe better represented by using phylogenetic networks ratherthan trees [65ndash67]

In view of the above comparing phylogenetic treesbased on different molecular markers may be used for theanalysis of evolutionary events caused by reticulate evolutionPhylogenetic signals from various molecular markers arepotentially divergent during reticulate evolution resultingin phylogenetic trees with alternative positions for the indi-vidual branches [68ndash71] Comparative analysis of the rDNAITS1 and ITS2 sequences is suitable for studying phylogeneticrelationships in terms of branching and reticulate evolution[63 68 70 72ndash82]

Reticulate evolution is primarily driven by hybrid speci-ation which is common among plants [83] but also occursamong animals particularly including fish [84] amphibiansand several invertebrates [85ndash87] In both mammals [88] andarthropods [89 90] a single instance of hybrid speciationhas been well described Interspecies hybridization typicallyresults in complicated relationships within species com-plexes characterized by indistinct species borders Reticulateevolution among crustaceans has been observed only withinspecies complexes of daphnids [91ndash94]

In this study we analyzed the phylogenetic relationshipswithin a small group of cyclopoid copepods representing sev-eral genera of freshwater (CyclopsThermocyclopsDiacyclopsMegacyclops Macrocyclops and Mesocyclops) and marine(Oithona and Paracyclopina) organisms Specific freshwaterspecies were selected for analysis because these species areimportant for the maintenance of food chains in Russianfreshwater ecosystems The aim of this study was to analyzethe sequence characteristics of the rDNA 28S gene ITS1 andITS2 regions as phylogenetic markers for the selected groupof organisms

2 Materials and Methods

21 Samples Collection Nine freshwater species of theCyclopidae family were collected near the Borok settle-ment in the Yaroslavskaya region of Russia Mesocyclopsleuckarti (Claus 1857) Cyclops strenuus (Fischer 1851)and Cyclops insignis (Claus 1857) (population no 1) fromthe Barskiy Pond (58∘31015840593510158401015840N 38∘151015840101610158401015840E) Thermo-cyclops oithonoides (Sars 1863) from the Sunoga pond(58∘21015840346610158401015840N 38∘141015840412910158401015840E) Thermocyclops crassus (Fis-cher 1853) Macrocyclops distinctus (Richard 1887) Macro-cyclops albidus (Jurine 1820) Diacyclops bicuspidatus (Claus1857) and Megacyclops viridis (Jurine 1820) (populationno 1) from the Ikhteologichesky Canal (58∘31015840556210158401015840N38∘151015840210510158401015840E) and Megacyclops viridis (Jurine 1820) (pop-ulation no 2) from a pond in the flood zone of the RybinskReservoir (58∘4101584047010158401015840N 38∘151015840398810158401015840E)

Cyclops kolensis (Lilljeborg 1901) and Cyclops insignis(Claus 1857) (population no 2) were collected from theAndreevsky small pond in Vorobrsquoevy Gory Moscow Russia(55∘421015840354010158401015840N 37∘34101584066110158401015840E) Two marine species Oncaeasp (Claus) and Oithona similis (Claus 1866) were collectedfrom the Norwegian Sea (68∘521015840366710158401015840N 3∘81015840219110158401015840E)

Individuals of each species were collected for furtheranalysis at the specified locations over a 05- to 1-hour period

BioMed Research International 3

No specific permission was required to collect samples atthese locations None of the studied species is endangered orprotected

A fragment of the 28S gene from each of the four marinespecies Paracyclopina nana (Smirnov 1935) (GenBank acces-sion number FJ214952) Oithona nana (Giesbrecht 1893)(GenBank accession number FM991727) Oithona simplex(Farran 1913) (GenBank accession number AF385458) andOithona helgolandica (Claus 1863) (GenBank accession num-ber FM9917241) was also used for the analysisThe DNAwasextracted from either samples preserved in 70 ethanol orraw materials (Moscow populations)

22 DNA Extraction PCR Amplification and SequencingThe genomic DNA was isolated from 10ndash20 individuals ofeach collected species using the DNeasy Blood amp Tissuekit (QIAGEN Hilden Germany) according to the manu-facturerrsquos instructions and was frozen at minus20∘C The rDNAregion (approximately 2000 bp) was amplified from thegenomic DNA by polymerase chain reaction (PCR) using theuniversal eukaryotic rDNA primers DAMS18 and DAMS28[95ndash97] (Figure 1) The amplified rDNA regions containedthe ITS1 (261ndash388 bp) and ITS2 (188ndash262 bp) regions the58S gene (157 bp) and approximately 200 and 1000 bp ofthe 18S and 28s genes respectively The amplification wasperformed in 50120583L reactions using a PCR Master Mix (2X)(Fermentas Vilnius Lithuania) according to the manufac-turerrsquos instructions the reactions were performed in a Primus25 advanced Thermocycler (PEQLAB Erlangen Germany)using previously published rDNA-specific parameters [98]The PCR products were resolved on 10 agarose gelsand DNA was extracted from the observed unique bandsusing the QIAquick Gel Extraction Kit (QIAGEN HildenGermany) The extracted products were cloned into thepGEM-T Easy vector (Promega USA) and the resultingplasmids were used to transform Escherichia coli JM109competent cells (Promega USA) according to the manufac-turerrsquos instructions For each species the amplified productand five clones were sequenced Automated sequences weregenerated on an ABI PRISM 310 Genetic Analyzer accordingto Sanger et al [99] with a BigDye Termination kit (AppliedBiosystems USA) The sequences generated in this studywere deposited in GenBank under the accession numbersKF153689ndashKF153701

23 Phylogenetic Analyses The rDNA sequences werealigned using ClustalW 21 [100 101] with some manualadjustments The boundaries of the ITS1 and ITS2 regionsand the 28S gene were identified by comparing the primer-delimited sequences against sequences in the GenBankdatabase using BLAST analysis The boundaries of theconserved sequences were considered to represent the 58S18S and 28S gene flanking regions if theywere 100 similar tothe boundaries of rDNA sequences in the GenBank databaseThe initial sequence alignment flanked byDAMS18DAMS28primers was divided into ITS1 and ITS2 alignments TherDNA genetic distances were estimated using the MEGAV52 software [102] DAMBE (Data Analysis in MolecularBiology and Evolution) software was used to analyze

substitution saturation [103ndash105] This method computes theentropy-based index of substitution saturation and its criticalvalue If the index of substitution saturation (Iss) approaches1 or if the Iss is not smaller than the critical Iss value(Issc) then sequences are considered to contain substantialsaturation As is known the substitution saturation decreasesphylogenetic information contained in sequences and hasplagued the phylogenetic analysis involving deep branches Inthe extreme case when sequences have experienced full sub-stitution saturation the similarity between the sequences willdepend entirely on the similarity in nucleotide frequencieswhich often does not reflect phylogenetic relationships [106]

The rDNA-based phylogenetic trees were estimated usingprobabilistic (maximum likelihood (ML) Bayesian) anddistance (minimum evolution (ME)) methods [107ndash110] MLand ME analyses of ITS1 ITS2 and 28S data were per-formed using the program MEGA V52 Branch support wasassessed using the bootstrap method [111] (1000 replicates)with the close-neighbor-interchange (CNI) algorithm at asearch level of 1 for ME analysis and heuristic search forML analysis The Bayesian information criterion (BIC) asimplemented in MEGA V52 was used to identify the best-fit model of sequence evolution for the trees estimated usingML The evolutionary history was inferred using the MLmethod based on the general time reversible with the gammadistribution shape parameter (GTR+G) model for 28S andthe Hasegawa-Kishino-Yano with gamma distribution shapeparameter (HKY+G)model [112] for the ITS1 ITS2 and con-catenated ITS1ITS2 alignments In addition to these meth-ods ITS1 and ITS2 alignments were constructed using theMAFFT version 7 (httpmafftcbrcjpalignmentserver)[113] and Gblocks version 091b (httpwwwphylogenyfrversion2 cgione taskcgitask type=gblocks) software pro-grams [114ndash116] to eliminate poorly aligned and highlydivergent regions Default parameters were used for both ofthese methods The Tamura 3-parameter model (T92) [117]andHKYwith evolutionary invariable (HKY+I) for Gblocks-treatedMAFFT ITS1 and ITS2 data respectively were used toinfer evolutionary history inference using the ML method

The Bayesian analysis was performed using MrBayesversion 312 software [118 119] Two replicate analyses of 1million generations each were performed for each datasetwith sampling every 10 generations The hierarchical likeli-hood ratio test (hLRT) implemented in MrModeltest version23 software [120] was used to identify the model of bestfit (Hasegawa-Kishino-Yano with invariant sites and gammadistribution shape parameter (HKY+I+G) [112] for ITS1 andthe HKY+G model for ITS2) Trees from the first 53000 and118000 generations were discarded as burn-in for ITS1 andITS2 respectively The Bayesian tree was estimated from themajority-rule consensus of the post-burn-in trees

A reticulogram [121] was constructed using the T-REX version 401a software [122] with the distance matrixcomputed using the Kimura 2-parameters model (ignoringmissing bases) the weighted least-squares method was usedfor tree reconstruction [123] and addition of reticulationbranches stopped when 119870 = 1 branches were added

Network reconstruction was performed using Splits Tree4 version 4113 software [65] The neighbor-net network


method and uncorrected p-distances were used to analyzeand visualize reticulate relationships All gaps were excludedfor analysis Network robustness was tested using 1000bootstrap replicates

3 Results and Discussion

31 Characteristics andAnalysis of an rDNASequenceDatasetIn each species the nucleotide sequences of the amplifiedrDNA region and five clones were not significantly differentfrom each other The frequency of variable nucleotides didnot exceed the average rate of nucleotide substitutions causedby DNA polymerase errors which is approximately onesubstitution per 1000 nucleotides The compared sequencescontained both relatively evolutionarily conserved (frag-ments of 18S and 28S rDNA and the complete 58S rDNA)and evolutionary variable genomic regions (ITS1 and ITS2)For different taxa the ITS1 and ITS2 sequences vary signif-icantly among individuals at the inter- and intrapopulationlevels furthermore these sequences can exhibit intrage-nomic variability [25 41 53 54] Recently a high level ofintrapopulation polymorphism of the 28S rDNA sequenceswas observed within Oithona spp [22] However there areinstances of strong evolutionary conservation of the 28S andITS sequences [15 23 30 34] Notably the M leuckartiITS2 sequence obtained in this study did not exhibit anynucleotide substitutions compared to the M leuckarti ITS2sequence described previously (GenBank accession numberGQ848499) [42] Therefore the strong evolutionary conser-vation of ITS1 and ITS2 sequences is a characteristic featureof the copepod species analyzed in this study

In this study the applicability of different segmentsof rDNA containing the ITS1 and ITS2 regions the 58SRNA gene and fragments of the 18S and 28S rRNA geneswas examined for reconstruction of the phylogenetic rela-tionships among freshwater cyclopoid copepods The 58Sgene and the analyzed fragment of the 18S gene were notconsidered for phylogenetic reconstruction due to their shortlength and strong evolutionary conservation only a fewnucleotide substitutions were detected by comparing thesesequenceswith evolutionary distant species (data not shown)

For 15 specimens ofCyclopoida species (including the twomarine species) the average length of the 28S gene fragmentsequenced was 1051 bp We trimmed these sequences to 703bp and compared them with the 28S gene sequences ofmarine species available in GenBank These 703 bp of 28SrDNA sequences were aligned and 342 variable sites wereobserved Oncaea sp (Oncaeidae family) was used as the outgroup

The ITS1 sequence lengths varied from 267 to 388 bpamong the 13 Cyclopidae specimens The ITS1 sequencealignments possessed 442 characters and among them 283were variable The ITS2 sequence lengths varied from 188 to262 bp among the 13 Cyclopidae specimens ITS2 sequencealignment possessed 302 characters and 190 were variable

All alignment sets were examined for homogeneity ofbase frequencies and substitution saturation The averagebase frequencies of the 28S gene fragment (119860 = 2052

Table 1 False test of substitution saturation

Alignment Iss Issc Std error28S 0200 0739 0019ITS1 0427 0679 0044ITS2 0376 0665 0043Testing whether the observed Iss is significantly (119875 lt 0001 two-tailed 119905-test) lower than the Issc for a symmetrical tree

119862 = 2533 119866 = 3275 and 119879 = 2140) differed from theITS1 (119860 = 1427 119862 = 3068 119866 = 2755 and 119879 = 2750)and ITS2 (119860 = 1308 119862 = 2964 119866 = 3008 and 119879 =2719) regions Gaps were excluded while estimating theaverage base frequencies of the ITS sequences Using the chi-squared test no significant differences were observed in thebase compositions of the 28S (1205942 = 3977 119889119891 = 54 and119875 = 093) ITS1 (1205942 = 2204 119889119891 = 36 and 119875 = 097) andITS2 (1205942 = 2265 119889119891 = 36 and 119875 = 096) sequences amongdifferent taxa

To analyze whether the divergence of 28S ITS1 and ITS2rDNA fragments among species was saturated we performeda substitution saturation test and generated saturation plotsUsing DAMBE the substitution saturation test revealed anIss value that was significantly (119875 lt 0001) lower than theIssc in all cases (Table 1) This result indicated the suitabilityof the data for phylogenetic analysis The total numbersof transition and transversion substitutions were plottedindividually against model-corrected maximum-likelihoodpairwise distances for the 28S ITS1 and ITS2 sequences (seeSupplementary Figure 1 in Supplementary Material availableonline at httpdxdoiorg1011552014926342) Using linearregression analysis on the 28S ITS1 and ITS2 saturationgraphs the coefficients of determination (1198772)were calculatedfor both classes of substitutions for transitions the 1198772values were 079 068 and 074 for the 28S ITS1 and ITS2sequences respectively for transversions the 1198772 values were095 093 and 091 for the 28S ITS1 and ITS2 sequencesrespectively The 1198772 values indicated that no less than 70of the total variation in pairwise transitions and transver-sions could be explained by the linear relationship betweenpairwise distances and the total number of transitions andtransversions All saturation plots showed significant lin-ear correlations (Supplementary Figure 1) Therefore bothtransitions and transversions steadily accumulated as thecorrected pairwise divergence increased indicating that sat-uration was not reached

32 Distance Analyses and Phylogenetic Tree ReconstructionPhylogenetic analysis of the cyclopoid copepods speciesbased on rDNA showed that the 28S rDNA sequencesare informative for the phylogeny of both higher-level andclosely related Copepoda species whereas the ITS1 and ITS2sequences are highly informative for reconstruction of theevolutionary history of closely related species The ITS1 andITS2 sequences are known to evolve more rapidly than theribosomal RNA genes Consistent with this observation in


Oncaea sp

Oithona helgolandica

Oithona similis

Oithona nana

Oithona simplex

Paracyclopina nana

Cyclops kolensis

Cyclops strenuus

Cyclops insignis (Moscow)

Cyclops insignis (Borok)

Diacyclops bicuspidatus

Thermocyclops oithonoides

Thermocyclops crassus

Mesocyclops leuckarti

Macrocyclops albidus

Macrocyclops distinctus

Megacyclops viridis (Borok1)


100100

100100

100100

100100

7976

9999

9999

8189 9394

57628684

5467

Figure 2 Phylogenetic relationships of Cyclopoida based onsim700 bp of the 28S rRNA gene The consensus cladogram inferredfrom the 28S ribosomalDNA fragment sequence data of 16 Podopleasuperorder species using maximum likelihood (ML) analysis underthe HKY+G model and minimum evolution (ME) analysis Thenumbers above branches indicate bootstrap percentages The valuesare listed for MLME

this study the pairwise ITS1ITS2 p-distances were signif-icantly higher thanthe 28S p-distances (compare Tables 2and 3) These data are consistent with other studies showingconsiderable variation in ITS1 and ITS2 divergence levelsamong different groups of copepods [40 42 52 124 125]In this study fragments of 28S rDNA sequences were usedfor the analysis of marine and freshwater cyclopoid cope-pods species whereas ITS1 and ITS2 sequences were usedexclusively for the analysis of freshwater cyclopoid copepodsspecies

The cladogram based on comparison of the 28S rDNAsequences reflected the evolutionary history of the analyzedspecies (Figure 2) Oncaea sp was used as the out groupSimilar topologies and levels of support at most nodes wereobtained for all 28S phylogenetic trees constructed usingthe ML and ME methods The specimens belonging to theorder Cyclopoida with high bootstrap support (MLME 99)formed two major clades on the tree (Figure 2) One cladecombined the marine cyclopoid copepods species whereasthe freshwater species specimens formed the second cladeThe p-distance between these two clades varied in the rangeof 0171ndash0245 (Table 2) The 28S phylogenetic tree revealeddetailed relationships among the Oithona spp with highbootstrap support (ML 79 100 and ME 76 100) However

Cyclops kolensis

Cyclops strenuus









9998

99100

99100

96918291

9699

100100Megacyclops viridis (Borok1)


Figure 3 Phylogenetic relationships of Cyclopoida based onsim500 bp of concatenated ITS1ITS2 rDNA sequencesThe consensuscladogram inferred from the ITS1-ITS2 ribosomal DNA fragmentsequence data of 10 species of theCyclopidae family usingmaximumlikelihood analysis under the HKY+G model and minimum evolu-tion (ME) analysis The numbers above branches indicate bootstrappercentages The values are listed for MLME

P nana (Cyclopettidae family) and Oithona spp (Oithonidaefamily) were poorly resolved Notably this study is thesecond on the molecular phylogenetics of the Oithona sppthe previous study described the phylogenetic relationshipsbetween three Oithona spp O similis O atlantica and Onana [22]

The cladogram based on the comparison of the concate-nated ITS1ITS2 sequences is shown in Figure 3 Notablythe 28S and ITS1ITS2 cladograms had several commonfeatures reflecting the evolutionary history of the analyzedfreshwater cyclopoid copepods species Both cladogramsrevealed that D bicuspidatus and specimens of the Cyclopsgenus with high bootstrap values (gt80) are separated fromother studied freshwater copepods in a distinct clade The p-distance between D bicuspidatus and Cyclops spp calculatedbased on ITS1ITS2 analysis varied in the range of 0232ndash0250 whereas the p-distance between D bicuspidatus andThermocyclops spp varied in the range of 0298ndash0333 andthe p-distance between D bicuspidatus and other analyzedfreshwater species varied in the range of 0310ndash0405 Thisresult is consistent with a previous phylogenetic study basedon 18S rDNA sequence analysis [48] Notably the systematicposition of this species based solely on the analysis ofmorphological characteristics remained unclear Diacyclopsbicuspidatus is considered to be evolutionarily closer toThermocyclops spp [8]

Another important conclusion from the analysis of 28Sand ITS1ITS2 cladograms relates to the systematic positionof C strenuus The Cyclops genera subclade was divided into


Table2Pairw

iseun

correctedgenetic

distance

amon

g18

sequ

enceso

fCyclopo

idab

ased

oncomparis

onof

28SrRNAgene

Speciesn

ame

12

34

56

78

910

1112

1314

1516

171

Oncaeasp

2Pa

racyclo

pina

nana

0229

3Oith

onasim

ilis

0230

0148

4Oith

onahelgo

land

ica0227

0143

0011

5Oith

onanana

0233

0181

0155

0146

6Oith

onasim

plex

0244

0152

0175

0166

0177

7Cy

clops

kolen

sis0209

0223

0197

0195

0244

0212

8Cy

clops

strenuu

s0207

0220

0197

0195

0242

0207

0005

9Cy

clops

insig

nisM

oscow

0216

0223

0204

0203

0245

0201

0037

0032

10C

insig

nisB

orok

0209

0216

0198

0197

0241

0197

0027

0023

0014

11Diacyclo

psbicuspidatus

0186

0200

0183

0180

0224

0194

0090

0085

0098

0087

12Th

ermocyclops

oithonoides

0220

0203

0191

0186

0216

0184

0128

0123

0120

0116

0098

13Th

ermocyclops

crassus

0218

0203

0181

0178

0220

0204

0127

0125

0127

0125

0093

0084

14Mesocyclops

leuckarti

0198

0183

0174

0171

0215

0181

0133

0128

0123

0122

0090

0087

0079

15Macrocyclo

psalbidu

s0221

0198

0197

0191

0215

0178

0145

0143

0137

0134

0099

0131

0114

0105

16Macrocyclo

psdistinctus

0230

0216

0192

0192

0218

0191

0152

0148

0143

0142

0113

0127

0143

0131

0123

17Megacyclops

virid

isBo

rok1

0213

0207

0195

0192

0230

0191

0137

0134

0134

0123

0105

0116

0117

0114

0130

0139

18MviridisB

orok2

0207

0210

0206

0203

0236

0198

0123

0122

0127

0119

0085

0114

0110

0105

0125

0130

0050

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop

tion)Finaldataset657po

sitions


Table3Pairw

iseun

correctedgenetic

distance

amon

g12

Cyclo

pidaes

equences

basedon

comparis

onof

concatenated

ITS1IT

S2rRNAsequ

ences

Speciesn

ame

12

34

56

78

910

111

Cyclo

pskolen

sis2

Cyclo

psstrenuu

s000

63

Cyclo

psinsig

nisM

oscow

0054

004

84

Cinsig

nisB

orok

004

8004

2000

65

Diacyclo

psbicuspidatus

0250

0244

0232

0232

6Th

ermocyclops

oithonoides

0333

0330

0330

0330

0313

7Th

ermocyclops

crassus

0301

0298

0295

0295

0262

0188

8Mesocyclops

leuckarti

0324

0321

0310

0310

0286

0280

0241

9Macrocyclo

psdistinctuslowast

0414

0411

040

8040

50393

0429

040

80369lowast

10Macrocyclo

psalbidu

s0393

0390

0381

0378

0357

0369

0366

0336

0321

11Megacyclops

virid

isBo

rok1lowast

0381

0384

0396

0390

0387

0387

0387

0390lowast

0455

0438

12MviridisB

orok2lowast

0375

0378

0384

0378

0363

0366

0357

0381lowast

0438

0429

0149

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop

tion)F

inaldatasets

336po

sitions

forc

oncatenatedITS1IT

S2Th

enam

esof

thethreetaxa

mostevolutio

narilydista

ntfro

mMesocyclops

andthev

alueso

fthe

correspo

ndinggenetic

dista

nces

ares

hownwith

asteris

k


Cyclops kolensis

Cyclops strenuus Cyclops insignis (Moscow)Cyclops insignis (Borok)



Thermocyclops crassusMesocyclops leuckarti

Macrocyclops albidusMacrocyclops distinctus



13

14

15

16

17

18

19

20

21

22

Figure 4 Reticulogram for concatenated ITS1ITS2 sequences of 10 species of the Cyclopidae family The red dashed line indicates thereticulation event connectingM leuckarti to theMacrocyclops clade node The number of internal vertices begins with 119899 + 1 where 119899 is thenumber of leaves The order of internal vertices distribution corresponds to the increasing lengths of the 22 reticulogram edges

the C kolensis C strenuus subsubclade and the C insignissubsubclade (Figures 2 and 3) The p-distance betweenC strenuus and C kolensis calculated based on ITS1ITS2analysis is 0006 whereas the p-distance between C strenuusand C insignis varied in the range of 0042ndash0054 ThereforeC strenuus is more closely related to C kolensis than to Cinsignis Notably the phylogenetic relationships between thestudied Cyclops species could not be elucidated solely on thebasis of morphological characteristics

The only difference between the 28S and ITS1ITS2cladograms within freshwater copepods was the position ofM leuckarti (Figures 2 and 3) The cladogram based oncomparison of the 28S rDNA sequences showed that theM leuckarti and Thermocyclops cluster together to form aseparate subclade (Figure 2) This result is consistent withthe previous observation that the Mesocyclops and Thermo-cyclops genera are phylogenetically closely related which wasconfirmed by the similarity of morphological characteristics

and using molecular data [42] ITS1ITS2 analysis revealedthat M leuckarti is located separately from Thermocyclopsand other clades (Figure 3) Using phylogenetic networks weanalyzed whether the M leuckarti position in the ITS1ITS2cladogram was caused by different contributions of ITS1 andITS2 sequences to the phylogenetic signal

33 Phylogenetic Networks A reticulogram-based phyloge-netic network inference approach was used to verify thereticulate evolution of the studied copepods ConcatenatedITS1ITS2 sequences of 10 species from the Cyclopidae familywere used for reticulogram reconstructionThe reticulogramrevealed a network with Mesocyclops and Thermocyclopsclustered together and a reticulation (lateral branch) connect-ing M leuckarti to the Macrocyclops clade node (Figure 4)Therefore the reticulogram indicated the reticulation inMesocyclops evolution


001

100

100100

100

100

100100

100

100100

100

65

999

999

802

998

512 97

779

703

703743

676

882

98

M viridis (Borok2)

M viridis (Borok1)

M distinctus

M albidus

M leuckartiT crassus

T oithonoides

D bicuspidatus

C insignis (Moscow)C insignis (Borok)

C strenuus

C kolensis

Figure 5 Split networks for concatenated ITS1ITS2 sequences of 10 species of the Cyclopidae family Split network based on concatenatedITS1ITS2 sequences the split separatingM leuckarti T oithonoides and T crassus is indicated in bold red the split separatingM leuckartiM distinctus and M albidus is indicated in bold blue purple indicates the M leuckarti reticulate relationship with Thermocyclops andMacrocyclops The values on the branches indicate bootstrap percentages

A split network represents incompatible edges of trees as aband of parallel edges Parallel edges split a network into twosets of nodes Split-graph analysis of concatenated ITS1ITS2sequences of 10 species from the Cyclopidae family revealeda reticulate relationship betweenMesocyclopsThermocyclopsand Macrocyclops with high reliability (Figure 5) All princi-pal splits werewell supported Two splits were observed in theITS1ITS2 split network The first split (parallel edges high-lighted with bold red) separated M leuckarti T oithonoidesand T crassus with 802 bootstrap support The secondsplit (parallel edges highlighted with bold blue) separatedMleuckartiM distinctus andM albidus with 650 bootstrapsupport

In addition to the network data we performed phy-logenetic reconstruction based on independent ITS1 andITS2 analyses using probabilistic and distance methodsIrrespective of the method used the main difference betweenthe topologies of the ITS1 and ITS2 phylogenetic trees was asfollows based on the ITS1 analysis M leuckarti is clusteredwith Thermocyclops whereas the ITS2 analysis revealed thatM leuckarti clustered withMacrocyclops (Figures 6(a)ndash6(d))

The impact of the chosenDNA sequence on the clusteringofM leuckartimight reflect the different evolutionary histo-ries of ITS1 and ITS2 which indicates the potential hybridorigin ofM leuckarti However the values of bootstrap sup-port for the clustering ofMesocyclops andThermocyclops andof Mesocyclops and Macrocyclops depended on the methodused for phylogenetic tree reconstruction and varied over awide range (Figures 6(a)ndash6(d))

Phylogenetic trees can be inconsistent due to the so-called long-branch attraction (LBA) phenomenon whichoccurs when two nonadjacent taxa share many homoplasticcharacter states along long branches andor fromuncorrected

sequence alignments Interpretation of the observed similar-ity depends on the method used for phylogenetic analysisand this similarity can often be interpreted as homologyModel-based methods are most resistant to LBA but thesemethods can exhibit LBA if their assumptions are seriouslyviolated or if there are insufficient taxa in the analysis toaccurately estimate the parameters of the evolutionary model[126] Taxon sampling is a crucial factor for avoiding LBAin phylogenetic analysis [127] The inclusion of additionaltaxa in phylogenetic analysis increases the accuracy of theinferred topology by dispersing homoplasty across the treeand reducing the effect of LBA The LBA effect might also berevealed by exclusion of the long-branched taxon from theanalysis [127]

To reduce the possible effects of LBA and correctness ofthe sequence alignment we used the following approaches(1) three taxa the most evolutionarily distant from Mleuckarti (M distinctus M viridis Borok1 and M viridisBorok2) were removed from the list of species used forITS1 and ITS2 phylogenetic tree reconstruction (the taxaselection was based on the data presented in Table 3) and (2)ITS1 and ITS2 sequences were aligned using new multiplesequence alignment programs to eliminate poorly alignedand highly divergent regions (see Section 2) The final ITS1and ITS2 alignments are shown in Supplementary Figures2(a) and 2(b) and the resulting phylogenetic trees are shownin Figures 6(e) and 6(f) Based on the ITS1 analysis Mleuckarti clustered with Thermocyclops whereas the ITS2analysis revealed that M leuckarti clustered with Macrocy-clops (Figures 6(e) and 6(f)) Notably the topology of the newphylogenetic trees had high bootstrap support 84 (ML)77(ME) for ITS1 and 77 (ML)72 (ME) for ITS2 (Figures 6(e)and 6(f))


Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899

100100 Megacyclops viridis (Borok1)


(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)

Figure 6 Phylogenetic relationships of Cyclopidae based on ITS1 and ITS2 sequences The ITS1 consensus Clustal tree of ten Cyclopidaespecies constructed using (a) maximum likelihood and minimum evolution and (b) Bayesian inference The ITS2 consensus Clustal treeof ten Cyclopidae species constructed using (c) maximum likelihood and minimum evolution and (d) Bayesian inference The consensusGblocks-treated MAFFT trees of eight Cyclopidae species constructed using maximum likelihood and minimum evolution (e) ITS1 (f)ITS2The numbers above the branches indicate bootstrap percentages and Bayesian posterior probabilities The values are listed for MLMEMissing or weakly supported nodes (lt50 or 05) are denoted by a ldquomdashrdquo


We think that one of the most intriguing explanations forthe observed differences in the clustering ofM leuckarti is theinterspecific hybridization between extinct taxa (presumablyclosely related) that were ancestral to both MesocyclopsMacrocyclops andThermocyclops However a rigorous proofof this hypothesis requires further analysis of a larger numberof species This will be the subject of our further research

4 Conclusion

We evaluated the utility of a sim2000 bp fragment of rDNA(easily amplified by universal primers) for the phylogeneticreconstruction of the relationships of Copepoda species Ourdata showed that the 28S rDNAand the ITS1 and ITS2 regionsare highly informative for the phylogeny of both higher-leveland closely related Copepoda species Comparative analysisof the ITS1 and ITS2 nucleotide sequences among closelyrelated Copepoda species revealed an unusual evolutionaryhistory of these spacer sequences therefore the ITS1 andITS2 regions might contain different phylogenetic signals

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Grace A Wyngaard forcriticisms and comments on earlier versions of this paperThis project was supported in part by a Russian Foundationfor Basic Research Grant no 12-04-32032-a to Maxim VZagoskin Grants nos 06-04-48474-a and 10-04-01376-a toAndrey K Grishanin and a Russian Academy of Sciencesgrant ldquoWild life current status and development problemsrdquo(subprogram ldquoDynamics and Conservation of Gene Poolsrdquo)to Dmitry V Mukha

References

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[2] B Dussart and D Defaye World Directory of Crustacea Cope-poda of Inland Waters IImdashCyclopiformes Backhuys LeidenThe Netherlands 2006

[3] F Kiefer ldquoVersuch eines Systems der Cyclopidenrdquo ZoologischerAnzeiger vol 73 no 11-12 pp 302ndash308 1927

[4] R Gurney British Fresh-Water Copepoda III Cyclopoida RaySociety London UK 1933

[5] V M Rylov Cyclopoida presnykh vod Freshwater CyclopoidaV3 Leningrad Moscow Russia 1948

[6] H C Yeatman ldquoFree-living Copepoda Cyclopoidardquo in Fresh-water Biology W T Edmondson Ed pp 795ndash815 John Wileyamp Sons New York NY USA 1959

[7] B Dussart Les Copepodes des Eaux Continentales de EuropeOccidentals Vol 2 Cyclopoides et Biologie N Boubee ParisFrance 1969

[8] V I Monchenko ldquoGnathostomata cyclopoida cyclopidaerdquoThe Fauna of Ukraina Naukova Dumka Kiev USSR vol 27no 3 pp 1ndash452 1974 httpscholargooglerucitationsviewop=view citationamphl=ruampuser=b97TEWUAAAAJampcitationfor view=b97TEWUAAAAJhqOjcs7Dif8C

[9] A Bucklin B W Frost and T D Kocher ldquoDNA sequence vari-ation of the mitochondrial 16S rRNA in Calanus (CopepodaCalanoida) intraspecific and interspecific patternsrdquo MolecularMarine Biology and Biotechnology vol 1 no 6 pp 397ndash4071992

[10] A Bucklin T C LaJeunesse E Curry J Wallinga and KGarrison ldquoMolecular diversity of the copepod Nannocalanusminor genetic evidence of species and population structure inthe North Atlantic Oceanrdquo Journal of Marine Research vol 54no 2 pp 285ndash310 1996

[11] R S Burton ldquoIntraspecific phylogeography across the pointconception biogeographic boundaryrdquo Evolution vol 52 no 3pp 734ndash745 1998

[12] C C Caudill and A Bucklin ldquoMolecular phylogeography andevolutionary history of the estuarine copepodAcartia tonsa ontheNorthwestAtlantic coastrdquoHydrobiologia vol 511 pp 91ndash1022004

[13] S Edmands ldquoPhylogeography of the intertidal copepod Tigrio-pus californicus reveals substantially reduced population differ-entiation at northern latitudesrdquoMolecular Ecology vol 10 no 7pp 1743ndash1750 2001

[14] S Eyun Y Lee H Suh S Kim and Y S Ho ldquoGeneticidentification and molecular phylogeny of Pseudodiaptomusspecies (Calanoida Pseudodiaptomidae) in Korean watersrdquoZoological Science vol 24 no 3 pp 265ndash271 2007

[15] E Goetze ldquoPopulation differentiation in the open sea insightsfrom the pelagic copepod pleuromamma xiphiasrdquo Integrativeand Comparative Biology vol 51 no 4 pp 580ndash597 2011

[16] S Laakmann and S Holst ldquoEmphasizing the diversity of NorthSea hydromedusae by combined morphological and molecularmethodsrdquo Journal of Plankton Research vol 36 no 1 pp 64ndash762014

[17] P K Lindeque R P Harris M B Jones and G R SmerdonldquoDistribution of Calanus spp as determined using a geneticidentification systemrdquo Scientia Marina vol 68 supplement 1pp 121ndash128 2007

[18] W Minxiao S Song L Chaolun and S Xin ldquoDistinctivemitochondrial genome of Calanoid copepod Calanus sinicuswith multiple large non-coding regions and reshuffled geneorder useful molecular markers for phylogenetic and popula-tion studiesrdquo BMC Genomics vol 12 no 1 article 73 2011

[19] P D Rawson and R S Burton ldquoMolecular evolution at thecytochrome oxidase subunit 2 gene among divergent popula-tions of the intertidal copepod Tigriopus californicusrdquo Journalof Molecular Evolution vol 62 no 6 pp 753ndash764 2006

[20] H Y Soh E Ok Park B A Venmathi Maran and S YongMoon ldquoA new species of Acartia subgenus Euacartia (Cope-poda Calanoida Acartiidae) from Korean estuaries based onmorphological and molecular evidencerdquo Journal of CrustaceanBiology vol 33 no 5 pp 718ndash729 2013

[21] C SWillett and J T Ladner ldquoInvestigations of fine-scale phylo-geography in Tigriopus californicus reveal historical patterns ofpopulation divergencerdquo BMC Evolutionary Biology vol 9 no 1article 139 2009

[22] G D Cepeda L Blanco-Bercial A Bucklin C M Beron andM D Vinas ldquoMolecular systematic of three species of Oithona


(Copepoda Cyclopoida) from the Atlantic ocean comparativeanalysis using 28S rDNArdquo PLoS ONE vol 7 no 4 Article IDe35861 2012

[23] J Hirai S Shimode and A Tsuda ldquoEvaluation of ITS2-28Sas a molecular marker for identification of calanoid copepodsin the subtropical western North Pacificrdquo Journal of PlanktonResearch vol 35 no 3 pp 644ndash656 2013

[24] J Kim and W Kim ldquoMolecular phylogeny of poecilostomecopepods based on the 18S rDNA sequencesrdquo Korean Journalof Biological Sciences vol 4 no 3 pp 257ndash261 2000

[25] A P Shinn B A Banks N Tange et al ldquoUtility of 18S rDNAand ITS sequences as population markers for Lepeophtheirussalmonis (Copepoda Caligidae) parasitising Atlantic salmon(Salmo salar) in Scotlandrdquo Contributions to Zoology vol 69 no1-2 pp 89ndash98 2000

[26] S J Adamowicz S Menu-Marque S A Halse et al ldquoTheevolutionary diversification of the Centropagidae (CrustaceaCalanoida) a history of habitat shiftsrdquo Molecular Phylogeneticsand Evolution vol 55 no 2 pp 418ndash430 2010

[27] L Blanco-Bercial J Bradford-Grieve and A Bucklin ldquoMolecu-lar phylogeny of the Calanoida (Crustacea Copepoda)rdquoMolec-ular Phylogenetics and Evolution vol 59 no 1 pp 103ndash113 2011

[28] E Braga R Zardoya A Meyer and J Yen ldquoMitochondrial andnuclear rRNA based copepod phylogeny with emphasis on theEuchaetidae (Calanoida)rdquoMarine Biology vol 133 no 1 pp 79ndash90 1999

[29] A Bucklin B W Frost J Bradford-Grieve L D Allen and NJ Copley ldquoMolecular systematic and phylogenetic assessmentof 34 calanoid copepod species of the Calanidae and Clauso-calanidaerdquoMarine Biology vol 142 no 2 pp 333ndash343 2003

[30] A Bucklin and BW Frost ldquoMorphological andmolecular Phy-logenetic analysis of evolutionary lineageswithinClausocalanus(Copepoda Calanoida)rdquo Journal of Crustacean Biology vol 29no 1 pp 111ndash120 2009

[31] A Cornils and L Blanco-Bercial ldquoPhylogeny of the para-calanidae giesbrecht 1888 (Crustacea Copepoda Calanoida)rdquoMolecular Phylogenetics and Evolution vol 69 no 3 pp 861ndash872 2013

[32] S M Dippenaar ldquoEstimated molecular phylogenetic relation-ships of six siphonostomatoid families (Copepoda) symbioticon elasmobranchsrdquo Crustaceana vol 82 no 12 pp 1547ndash15672009

[33] D F Figueroa ldquoPhylogenetic analysis of Ridgewayia (Cope-poda Calanoida) from the Galapagos and of a new speciesfrom the Florida keys with a reevaluation of the phylogeny ofCalanoidardquo Journal of Crustacean Biology vol 31 no 1 pp 153ndash165 2011

[34] E Goetze ldquoCryptic speciation on the high seas global phylo-genetics of the copepod family Eucalanidaerdquo Proceedings of theRoyal Society B Biological Sciences vol 270 no 1531 pp 2321ndash2331 2003

[35] S Laakmann G Gerdts R Erler T Knebelsberger P MartınezArbizu and M J Raupach ldquoComparison of molecular speciesidentification for North Sea calanoid copepods (Crustacea)using proteome fingerprints and DNA sequencesrdquo MolecularEcology Resources vol 13 no 5 pp 862ndash876 2013

[36] R J Machida M U Miya M Nishida and S NishidaldquoMolecular phylogeny and evolution of the pelagic copepodgenusNeocalanus (Crustacea Copepoda)rdquoMarine Biology vol148 no 5 pp 1071ndash1079 2006

[37] M Taniguchi ldquoMolecular phylogeny of Neocalanus copepodsin the subarctic Pacific Ocean with notes on non-geographical

genetic variations for Neocalanus cristatusrdquo Journal of PlanktonResearch vol 26 no 10 pp 1249ndash1255 2004

[38] C E Lee ldquoGlobal phylogeography of a cryptic copepod speciescomplex and reproductive isolation between genetically prox-imate lsquopopulationsrsquordquo Evolution vol 54 no 6 pp 2014ndash20272000

[39] S J Adamowicz S Menu-Marque P D N Hebert and APurvis ldquoMolecular systematics and patterns of morphologicalevolution in the Centropagidae (Copepoda Calanoida) ofArgentinardquo Biological Journal of the Linnean Society vol 90 no2 pp 279ndash292 2007

[40] E Goetze ldquoGlobal population genetic structure and biogeogra-phy of the oceanic copepods Eucalanus hyalinus and E spiniferrdquoEvolution vol 59 no 11 pp 2378ndash2398 2005

[41] R J Machida and A Tsuda ldquoDissimilarity of species and formsof planktonic Neocalanus copepods using mitochondrial COI12S nuclear ITS and 28S gene sequencesrdquo PLoS ONE vol 5no 4 Article ID e10278 2010

[42] G A Wyngaard M Hołynska and J A Schulte ldquoPhylogenyof the freshwater copepodMesocyclops (Crustacea Cyclopidae)based on combined molecular and morphological data withnotes on biogeographyrdquoMolecular Phylogenetics and Evolutionvol 55 no 3 pp 753ndash764 2010

[43] V Alekseev H J Dumont J Pensaert D Baribwegure and J RVanfleteren ldquoA redescription of Eucyclops serrulatus (Fischer1851) (Crustacea Copepoda Cyclopoida) and some relatedtaxa with a phylogeny of the E serrulatus-grouprdquo ZoologicaScripta vol 35 no 2 pp 123ndash147 2006

[44] M R Miracle V Alekseev V Monchenko V Sentandreuand E Vicente ldquoMolecular-genetic-based contribution to thetaxonomy of the Acanthocyclops robustus grouprdquo Journal ofNatural History vol 47 no 5ndash12 pp 863ndash888 2013

[45] M Blaha M Hulak J Sloukova and J Tesitel ldquoMolecular andmorphological patterns acrossAcanthocyclops vernalis-robustusspecies complex (Copepoda Cyclopoida)rdquo Zoologica Scriptavol 39 no 3 pp 259ndash268 2010

[46] T Karanovic and M Krajicek ldquoWhen anthropogenic translo-cation meets cryptic speciation globalized bouillon originatesmolecular variability of the cosmopolitan freshwater cyclopoidMacrocyclops albidus (Crustacea Copepoda)rdquo Annales de Lim-nologie vol 48 no 1 pp 63ndash80 2012

[47] T Karanovic and M Krajicek ldquoFirst molecular data on theWestern Australian Diacyclops (Copepoda Cyclopoida) con-firm morpho-species but question size differentiation andmonophyly of the Alticola-grouprdquo Crustaceana vol 85 no 12-13 pp 1549ndash1569 2012

[48] T Y Mayor N G Sheveleva L V Sukhanova O A Timo-shkin and S V Kirilrsquochik ldquoMolecular-phylogenetic analysis ofcyclopoids (Copepoda Cyclopoida) from Lake Baikal and itswater catchment basinrdquo Russian Journal of Genetics vol 46 no11 pp 1373ndash1380 2010

[49] T Karanovic and S J B Cooper ldquoMolecular andmorphologicalevidence for short range endemism in the Kinnecaris solitariacomplex (Copepoda Parastenocarididae) with descriptions ofseven new speciesrdquo Zootaxa no 3026 pp 1ndash64 2011

[50] F Marrone S L Brutto and M Arculeo ldquoMolecular evidencefor the presence of cryptic evolutionary lineages in the freshwa-ter copepod genus Hemidiaptomus GO Sars 1903 (CalanoidaDiaptomidae)rdquo Hydrobiologia vol 644 no 1 pp 115ndash125 2010

[51] R Scheihing L Cardenas R F Nespolo et al ldquoMorphologicaland molecular analysis of centropagids from the high Andean


plateau (Copepoda Calanoidea)rdquoHydrobiologia vol 637 no 1pp 45ndash52 2010

[52] R A Thum and R G Harrison ldquoDeep genetic divergencesamongmorphologically similar and parapatric Skistodiaptomus(Copepoda Calanoida Diaptomidae) challenge the hypothesisof Pleistocene speciationrdquo Biological Journal of the LinneanSociety vol 96 no 1 pp 150ndash165 2009

[53] H Y Soh S W Kwon W Lee and Y H Yoon ldquoA new Pseu-dodiaptomus (Copepoda Calanoida) from Korea supported bymolecular datardquo Zootaxa no 3368 pp 229ndash244 2012

[54] M A Marszalek S Dayanandan and E J Maly ldquoPhylogeny ofthe genus Hesperodiaptomus (Copepoda) based on nucleotidesequence data of the nuclear ribosomal generdquo Hydrobiologiavol 624 no 1 pp 61ndash69 2009

[55] R A Thum ldquoUsing 18S rDNA to resolve diaptomid copepod(Copepoda Calanoida Diaptomidae) phylogeny an examplewith the North American generardquo Hydrobiologia vol 519 no1ndash3 pp 135ndash141 2004

[56] R Huys J Llewellyn-Hughes P D Olson and K Naga-sawa ldquoSmall subunit rDNA and Bayesian inference revealPectenophilus ornatus (Copepoda incertae sedis) as highlytransformed Mytilicolidae and support assignment of Chon-dracanthidae and Xarifiidae to Lichomolgoidea (Cyclopoida)rdquoBiological Journal of the Linnean Society vol 87 no 3 pp 403ndash425 2006

[57] R Huys J Llewellyn-Hughes S Conroy-Dalton P D Olson JN Spinks and D A Johnston ldquoExtraordinary host switchingin siphonostomatoid copepods and the demise of the Monstril-loida integrating molecular data ontogeny and antennularymorphologyrdquoMolecular Phylogenetics and Evolution vol 43 no2 pp 368ndash378 2007

[58] J Ki K Lee H G Park S Chullasorn H Dahms and J LeeldquoPhylogeography of the copepod Tigriopus japonicus along theNorthwest Pacific rimrdquo Journal of Plankton Research vol 31 no2 pp 209ndash221 2009

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[61] R S Burton R J Byrne and P D Rawson ldquoThree divergentmitochondrial genomes from California populations of thecopepod Tigriopus californicusrdquo Gene vol 403 no 1-2 pp 53ndash59 2007

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[64] D M Hillis and M T Dixon ldquoRibosomal DNA molecularevolution and phylogenetic inferencerdquo Quarterly Review ofBiology vol 66 no 4 pp 411ndash453 1991

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[68] T Sang D J Crawford and T F Stuessy ldquoDocumentationof reticulate evolution in peonies (Paeonia) using internaltranscribed spacer sequences of nuclear ribosomal DNA impli-cations for biogeography and concerted evolutionrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 92 no 15 pp 6813ndash6817 1995

[69] S Giessler and C C Englbrecht ldquoDynamic reticulate evolutionin a Daphnia multispecies complexrdquo Journal of ExperimentalZoology A Ecological Genetics and Physiology vol 311 no 7 pp530ndash548 2009

[70] H Kauserud and T Schumacher ldquoRibosomal DNA variationrecombination and inheritance in the basidiomycete Trichap-tum abietinum implications for reticulate evolutionrdquo Heredityvol 91 no 2 pp 163ndash172 2003

[71] P M W Wyatt C S Pitts and R K Butlin ldquoA molecu-lar approach to detect hybridization between bream Abramisbrama roach Rutlius rutilus and rudd Scardinius erythrophthal-musrdquo Journal of Fish Biology vol 69 pp 52ndash71 2006

[72] J Fuertes Aguilar and G Nieto Feliner ldquoAdditive polymor-phisms and reticulation in an ITS phylogeny of thrifts (ArmeriaPlumbaginaceae)rdquo Molecular Phylogenetics and Evolution vol28 no 3 pp 430ndash447 2003

[73] H Yamaji T Fukuda J Yokoyama et al ldquoReticulate evolutionand phylogeography in Asarumsect Asiasarum (Aristolochi-aceae) documented in internal transcribed spacer sequences(ITS) of nuclear ribosomal DNArdquo Molecular Phylogenetics andEvolution vol 44 no 2 pp 863ndash884 2007

[74] M A Hershkovitz C C Hernandez-Pellicer and M T KArroyo ldquoRibosomal DNA evidence for the diversification ofTropaeolum sect Chilensia (Tropaeolaceae)rdquo Plant Systematicsand Evolution vol 260 no 1 pp 1ndash24 2006

[75] A Hugall J Stanton and C Moritz ldquoReticulate evolution andthe origins of ribosomal internal transcribed spacer diversity inapomictic Meloidogynerdquo Molecular Biology and Evolution vol16 no 2 pp 157ndash164 1999

[76] J F Wendel A Schnabel and T Seelanan ldquoBidirectional inter-locus concerted evolution following allopolyploid speciation incotton (Gossypium)rdquo Proceedings of the National Academy ofSciences of the United States of America vol 92 no 1 pp 280ndash284 1995

[77] K OrsquoDonnell and E Cigelnik ldquoTwo divergent intragenomicrDNA ITS2 types within a monophyletic lineage of the fungusFusarium are nonorthologousrdquo Molecular Phylogenetics andEvolution vol 7 no 1 pp 103ndash116 1997

[78] K OrsquoDonnell E Cigelnik and H I Nirenberg ldquoMolecularsystematics and phylogeography of the Gibberella fujikuroispecies complexrdquoMycologia vol 90 no 3 pp 465ndash493 1998

[79] CM BrasierD E L Cooke and JMDuncan ldquoOrigin of a newPhytophthora pathogen through interspecific hybridizationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 96 no 10 pp 5878ndash5883 1999

[80] K W Hughes and R H Petersen ldquoApparent recombination orgene conversion in the ribosomal ITS region of a Flammulina(Fungi Agaricales) hybridrdquo Molecular Biology and Evolutionvol 18 no 1 pp 94ndash96 2001

[81] G Newcombe B Stirling S McDonald and H D BradshawJr ldquoMelampsora times columbiana a natural hybrid ofM medusaeand M occidentalisrdquo Mycological Research vol 104 no 3 pp261ndash274 2000


[82] K H Chu C P Li and H Y Ho ldquoThe first internal transcribedspacer (ITS-1) of ribosomal DNA as a molecular marker forphylogenetic and population analyses in crustaceardquo MarineBiotechnology vol 3 no 4 pp 355ndash361 2001

[83] L H Rieseberg ldquoHybrid origins of plant speciesrdquo AnnualReview of Ecology and Systematics vol 28 no 1 pp 359ndash3891997

[84] R Cui M Schumer K Kruesi R Walter P Andolfatto andG G Rosenthal ldquoPhylogenomics reveals extensive reticulateevolution in Xiphophorus fishesrdquo Evolution vol 67 no 8 pp2166ndash2179 2013

[85] L Bullini ldquoOrigin and evolution of animal hybrid speciesrdquoTrends in Ecology and Evolution vol 9 no 11 pp 422ndash426 1994

[86] T E Dowling and C L Secor ldquoThe role of hybridization andintrogression in the diversification of animalsrdquo Annual Reviewof Ecology and Systematics vol 28 pp 593ndash619 1997

[87] J Mallet ldquoHybridization as an invasion of the genomerdquo Trendsin Ecology and Evolution vol 20 no 5 pp 229ndash237 2005

[88] U Arnason R Spilliaert A Palsdottir and A ArnasonldquoMolecular identification of hybrids between the two largestwhale species the blue whale (Balaenoptera musculus) and thefin whale (B physalus)rdquo Hereditas vol 115 no 2 pp 183ndash1891991

[89] A V Z Brower ldquoIntrogression of wing pattern alleles andspeciation via homoploid hybridization inHeliconius butterfliesa review of evidence from the genomerdquo Proceedings BiologicalSciences vol 280 no 1752 Article ID 20122302 2013

[90] H L Carson K Y Kaneshiro and F C Val ldquoNatural hybridiza-tion between the sympatric Hawaiian species Drosophila sil-vestris and Drosophila heteroneurardquo Evolution vol 43 no 1 pp190ndash203 1989

[91] S Gieszligler and C C Englbrecht ldquoDynamic reticulate evolutionin a Daphnia multispecies complexrdquo Journal of ExperimentalZoology A Ecological Genetics and Physiology vol 311 no 7 pp531ndash549 2009

[92] S Gieszligler EMader andK Schwenk ldquoMorphological evolutionand genetic differentiation in Daphnia species complexesrdquoJournal of Evolutionary Biology vol 12 no 4 pp 710ndash723 1999

[93] D J Taylor P D N Hebert and J K Colbourne ldquoPhylogeneticsand evolution of the Daphnia longispina group (Crustacea)based on 12S rDNA sequence and allozyme variationrdquo Molec-ular Phylogenetics and Evolution vol 5 no 3 pp 495ndash510 1996

[94] R Vergilino S Markova M Ventura M Manca and FDufresne ldquoReticulate evolution of the Daphnia pulex complexas revealed by nuclear markersrdquoMolecular Ecology vol 20 no6 pp 1191ndash1207 2011

[95] D V Mukha and A P Sidorenko ldquoDetection and analy-sis of Tetrahymena pyriformis 26S ribosomal DNA domainsequences differing in degree of evolutionary conservationrdquoMolekulyarnaya Biologiya vol 29 no 3 pp 529ndash537 1995

[96] D V Mukha and A P Sidorenko ldquoIdentification of highlyconservative domains within the 17s ribosomal dna sequencefrom Tetrahymena pyriformisrdquo Genetika vol 32 no 11 pp1494ndash1497 1996

[97] D VMukha A P Sidorenko I V Lazebnaya BMWiegmannand C Schal ldquoAnalysis of intraspecies polymorphism in theribosomal DNA cluster of the cockroach Blattella germanicardquoInsect Molecular Biology vol 9 no 2 pp 217ndash222 2000

[98] D Mukha B M Wiegmann and C Schal ldquoEvolution andphylogenetic information content of the ribosomal DNA repeatunit in the Blattodea (Insecta)rdquo Insect Biochemistry and Molec-ular Biology vol 32 no 9 pp 951ndash960 2002

[99] F Sanger S Nicklen and A R Coulson ldquoDNA sequencingwith chain-terminating inhibitorsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 74 no12 pp 5463ndash5467 1977

[100] M A Larkin G Blackshields N P Brown et al ldquoClustalW andClustal X version 20rdquo Bioinformatics vol 23 no 21 pp 2947ndash2948 2007

[101] M Goujon H McWilliam W Li et al ldquoA new bioinformaticsanalysis tools framework at EMBL-EBIrdquoNucleic Acids Researchvol 38 no 2 Article ID gkq313 pp W695ndashW699 2010

[102] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[103] X Xia and Z Xie ldquoDAMBE software package for data analysisinmolecular biology and evolutionrdquo Journal of Heredity vol 92no 4 pp 371ndash373 2001

[104] X Xia Data Analysis in Molecular Biology and EvolutionKluwer Academic Publishers Boston Mass USA 2000

[105] X Xia Z Xie M Salemi L Chen and Y Wang ldquoAn index ofsubstitution saturation and its applicationrdquo Molecular Phyloge-netics and Evolution vol 26 no 1 pp 1ndash7 2003

[106] P Lemey M Salemi and A-M Vandamme EdsThe Phyloge-netic Handbook A Practical Approach to Phylogenetic Analysisand Hypothesis Testing Cambridge University Press Cam-bridge UK 2009

[107] W M Fitch ldquoToward defining the course of evolution mini-mum change for a specific tree topologyrdquo Systematic Biologyvol 20 no 4 pp 406ndash416 1971

[108] J Felsenstein ldquoEvolutionary trees from DNA sequences amaximum likelihood approachrdquo Journal ofMolecular Evolutionvol 17 no 6 pp 368ndash376 1981

[109] A Rzhetsky and M Nei ldquoA simple method for estimatingand testing minimum-evolution treesrdquo Molecular Biology andEvolution vol 9 no 5 pp 945ndash967 1992

[110] J P Huelsenbeck F Ronquist R Nielsen and J P BollbackldquoBayesian inference of phylogeny and its impact on evolution-ary biologyrdquo Science vol 294 no 5550 pp 2310ndash2314 2001

[111] J Felsenstein ldquoConfidence limits on phylogenies an approachusing the bootstraprdquo Evolution vol 39 no 4 pp 783ndash791 1985

[112] M Hasegawa H Kishino and T Yano ldquoDating of the human-ape splitting by a molecular clock of mitochondrial DNArdquoJournal of Molecular Evolution vol 22 no 2 pp 160ndash174 1985

[113] K Katoh K Kuma H Toh and T Miyata ldquoMAFFT version5 improvement in accuracy of multiple sequence alignmentrdquoNucleic Acids Research vol 33 no 2 pp 511ndash518 2005

[114] A Dereeper S Audic J Claverie and G Blanc ldquoBLAST-EXPLORER helps you building datasets for phylogenetic anal-ysisrdquo BMC Evolutionary Biology vol 10 no 1 article 8 2010

[115] A Dereeper V Guignon G Blanc et al ldquoPhylogenyfr robustphylogenetic analysis for the non-specialistrdquo Nucleic AcidsResearch vol 36 pp W465ndashW469 2008

[116] J Castresana ldquoSelection of conserved blocks from multiplealignments for their use in phylogenetic analysisrdquo MolecularBiology and Evolution vol 17 no 4 pp 540ndash552 2000

[117] K Tamura ldquoEstimation of the number of nucleotide substitu-tions when there are strong transition-transversion and G+C-content biasesrdquo Molecular Biology and Evolution vol 9 no 4pp 678ndash687 1992


[118] J P Huelsenbeck and F Ronquist ldquoMRBAYES bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[119] F Ronquist and J P Huelsenbeck ldquoMrBayes 3 bayesian phylo-genetic inference under mixed modelsrdquo Bioinformatics vol 19no 12 pp 1572ndash1574 2003

[120] J A A Nylander MrModeltest v2 Program Distributed by theAuthor Evolutionary Biology Centre Uppsala University 2004

[121] P Legendre and V Makarenkov ldquoReconstruction of biogeo-graphic and evolutionary networks using reticulogramsrdquo Sys-tematic Biology vol 51 no 2 pp 199ndash216 2002

[122] V Makarenkov ldquoT-REX reconstructing and visualizing phylo-genetic trees and reticulation networksrdquo Bioinformatics vol 17no 7 pp 664ndash668 2001

[123] V Makarenkov ldquoAn algorithm for the fitting of a tree metricaccording to a weighted least-squares criterionrdquo Journal ofClassification vol 16 no 1 pp 3ndash26 1999

[124] A Rocha-Olivares J W Fleeger and D W Foltz ldquoDecouplingof molecular and morphological evolution in deep lineages ofa meiobenthic harpacticoid copepodrdquo Molecular Biology andEvolution vol 18 no 6 pp 1088ndash1102 2001

[125] BW Frost and A Bucklin ldquoMorphological andmolecular phy-logenetic analysis of evolutionary lineageswithinClausocalanus(Copepoda Calanoida)rdquo Journal of Crustacean Biology vol 29no 1 pp 111ndash120 2009

[126] T A Heath S M Hedtke and D M Hillis ldquoTaxon samplingand the accuracy of phylogenetic analysesrdquo Journal of Systemat-ics and Evolution vol 46 no 3 pp 239ndash257 2008

[127] J Bergsten ldquoA review of long-branch attractionrdquo Cladistics vol21 no 2 pp 163ndash193 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


IGS IGS18S 18S58S

ITS1 ITS228S 28S

DAMS18 DAMS28

Figure 1 Organization of eukaryotic tandemly repeated rDNAclusters 18S 58S and 28S ribosomal RNA genes ITS1 and ITS2internal transcribed spacers IGS intergenic spacer Arrows indicatethe locations of the DAMS18 and DAMS28 primers

taxa furthermore amplification of COI is difficult in somecopepods [59ndash61] However these DNA fragments mightbe informative for analyses of population differentiationor cryptic speciation [9 11ndash15 38ndash40 50 52] Thereforecomparison of nuclear rDNA regions might be informativefor the phylogenetic analysis of copepods

In most eukaryotes rRNA genes are located in a multi-gene family of genomic clusters of repeated sequencesWithin these clusters the 18S 58S and 28S rRNA genes areseparated by internal transcribed spacers (ITS1 and ITS2)and an intergenic spacer [62] (Figure 1) Ribosomal DNA(rDNA) is a reliable and informative phylogenetic marker[63] that contains sequences with different rates of evolution-ary variability In most eukaryotes the most evolutionarilyconserved genes are the rRNA genes comparison of theirsequences allows estimation of the evolutionary distances atintergeneric and higher taxonomic levels Comparison of themore evolutionarily variable spacer sequences enables thestudy of phylogenetic relationships at the species and popula-tion levels [63ndash65]Therefore comparison of different regionsof rDNA enables the phylogenetic analysis of organisms overextended evolutionary distances

Phylogenetic relationships among cyclopoid copepods athigher systematic levels (ordinal familial and generic) havebeen resolved using the 18S and 28S nuclear rRNA genes [2829 55ndash57] and the relationships at the lower taxonomic levels(species and populations) have been resolved using the ITS2of the nuclear rDNA gene cluster [30 40 42 52 58]

Notably analysis of the evolutionary history of livingorganisms based on only one molecular marker can uncoverbifurcating phylogenetic trees revealing branched evolutionHowever it recently becomes evident that evolution is notalways tree-like Comparisons of gene trees based on differentgenetic loci often reveal conflicting tree topologies Thesediscrepancies are not always due to the problems withthe sampling and the gene tree reconstruction methodsReticulation events such as horizontal gene transfer (HGT)and hybridization may be responsible for contradictionsin lineages During an HGT event a DNA segment istransferred from one organism to another which is not itsoffspring whereas hybridization describes the origin of a newspecies through an interspecies mating Both processes yieldgenomes that are mixtures of DNA regions derived fromdifferent species Consequently evolutionary relationshipsbetween species whose past includes reticulation can oftenbe better represented by using phylogenetic networks ratherthan trees [65ndash67]

In view of the above comparing phylogenetic treesbased on different molecular markers may be used for theanalysis of evolutionary events caused by reticulate evolutionPhylogenetic signals from various molecular markers arepotentially divergent during reticulate evolution resultingin phylogenetic trees with alternative positions for the indi-vidual branches [68ndash71] Comparative analysis of the rDNAITS1 and ITS2 sequences is suitable for studying phylogeneticrelationships in terms of branching and reticulate evolution[63 68 70 72ndash82]

Reticulate evolution is primarily driven by hybrid speci-ation which is common among plants [83] but also occursamong animals particularly including fish [84] amphibiansand several invertebrates [85ndash87] In both mammals [88] andarthropods [89 90] a single instance of hybrid speciationhas been well described Interspecies hybridization typicallyresults in complicated relationships within species com-plexes characterized by indistinct species borders Reticulateevolution among crustaceans has been observed only withinspecies complexes of daphnids [91ndash94]

In this study we analyzed the phylogenetic relationshipswithin a small group of cyclopoid copepods representing sev-eral genera of freshwater (CyclopsThermocyclopsDiacyclopsMegacyclops Macrocyclops and Mesocyclops) and marine(Oithona and Paracyclopina) organisms Specific freshwaterspecies were selected for analysis because these species areimportant for the maintenance of food chains in Russianfreshwater ecosystems The aim of this study was to analyzethe sequence characteristics of the rDNA 28S gene ITS1 andITS2 regions as phylogenetic markers for the selected groupof organisms

2 Materials and Methods

21 Samples Collection Nine freshwater species of theCyclopidae family were collected near the Borok settle-ment in the Yaroslavskaya region of Russia Mesocyclopsleuckarti (Claus 1857) Cyclops strenuus (Fischer 1851)and Cyclops insignis (Claus 1857) (population no 1) fromthe Barskiy Pond (58∘31015840593510158401015840N 38∘151015840101610158401015840E) Thermo-cyclops oithonoides (Sars 1863) from the Sunoga pond(58∘21015840346610158401015840N 38∘141015840412910158401015840E) Thermocyclops crassus (Fis-cher 1853) Macrocyclops distinctus (Richard 1887) Macro-cyclops albidus (Jurine 1820) Diacyclops bicuspidatus (Claus1857) and Megacyclops viridis (Jurine 1820) (populationno 1) from the Ikhteologichesky Canal (58∘31015840556210158401015840N38∘151015840210510158401015840E) and Megacyclops viridis (Jurine 1820) (pop-ulation no 2) from a pond in the flood zone of the RybinskReservoir (58∘4101584047010158401015840N 38∘151015840398810158401015840E)

Cyclops kolensis (Lilljeborg 1901) and Cyclops insignis(Claus 1857) (population no 2) were collected from theAndreevsky small pond in Vorobrsquoevy Gory Moscow Russia(55∘421015840354010158401015840N 37∘34101584066110158401015840E) Two marine species Oncaeasp (Claus) and Oithona similis (Claus 1866) were collectedfrom the Norwegian Sea (68∘521015840366710158401015840N 3∘81015840219110158401015840E)

Individuals of each species were collected for furtheranalysis at the specified locations over a 05- to 1-hour period

























Oncaea sp


Oithona similis

Oithona nana

Oithona simplex

Paracyclopina nana

Cyclops kolensis

Cyclops strenuus











100100

100100

100100

100100

7976

9999

9999

8189 9394

57628684

5467




Cyclops kolensis

Cyclops strenuus









9998

99100

99100

96918291

9699








Table2Pairw

iseun

correctedgenetic

distance

amon

g18

sequ

enceso

fCyclopo

idab

ased

oncomparis

onof

28SrRNAgene

Speciesn

ame

12

34

56

78

910

1112

1314

1516

171

Oncaeasp

2Pa

racyclo

pina

nana

0229

3Oith

onasim

ilis

0230

0148

4Oith

onahelgo

land

ica0227

0143

0011

5Oith

onanana

0233

0181

0155

0146

6Oith

onasim

plex

0244

0152

0175

0166

0177

7Cy

clops

kolen

sis0209

0223

0197

0195

0244

0212

8Cy

clops

strenuu

s0207

0220

0197

0195

0242

0207

0005

9Cy

clops

insig

nisM

oscow

0216

0223

0204

0203

0245

0201

0037

0032

10C

insig

nisB

orok

0209

0216

0198

0197

0241

0197

0027

0023

0014

11Diacyclo

psbicuspidatus

0186

0200

0183

0180

0224

0194

0090

0085

0098

0087

12Th

ermocyclops

oithonoides

0220

0203

0191

0186

0216

0184

0128

0123

0120

0116

0098

13Th

ermocyclops

crassus

0218

0203

0181

0178

0220

0204

0127

0125

0127

0125

0093

0084

14Mesocyclops

leuckarti

0198

0183

0174

0171

0215

0181

0133

0128

0123

0122

0090

0087

0079

15Macrocyclo

psalbidu

s0221

0198

0197

0191

0215

0178

0145

0143

0137

0134

0099

0131

0114

0105

16Macrocyclo

psdistinctus

0230

0216

0192

0192

0218

0191

0152

0148

0143

0142

0113

0127

0143

0131

0123

17Megacyclops

virid

isBo

rok1

0213

0207

0195

0192

0230

0191

0137

0134

0134

0123

0105

0116

0117

0114

0130

0139

18MviridisB

orok2

0207

0210

0206

0203

0236

0198

0123

0122

0127

0119

0085

0114

0110

0105

0125

0130

0050

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop


sitions


Table3Pairw

iseun

correctedgenetic

distance

amon

g12

Cyclo

pidaes

equences

basedon

comparis

onof

concatenated

ITS1IT

S2rRNAsequ

ences

Speciesn

ame

12

34

56

78

910

111

Cyclo

pskolen

sis2

Cyclo

psstrenuu

s000

63

Cyclo

psinsig

nisM

oscow

0054

004

84

Cinsig

nisB

orok

004

8004

2000

65

Diacyclo

psbicuspidatus

0250

0244

0232

0232

6Th

ermocyclops

oithonoides

0333

0330

0330

0330

0313

7Th

ermocyclops

crassus

0301

0298

0295

0295

0262

0188

8Mesocyclops

leuckarti

0324

0321

0310

0310

0286

0280

0241

9Macrocyclo

psdistinctuslowast

0414

0411

040

8040

50393

0429

040

80369lowast

10Macrocyclo

psalbidu

s0393

0390

0381

0378

0357

0369

0366

0336

0321

11Megacyclops

virid

isBo

rok1lowast

0381

0384

0396

0390

0387

0387

0387

0390lowast

0455

0438

12MviridisB

orok2lowast

0375

0378

0384

0378

0363

0366

0357

0381lowast

0438

0429

0149

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop

tion)F

inaldatasets

336po

sitions

forc

oncatenatedITS1IT

S2Th

enam

esof

thethreetaxa

mostevolutio

narilydista

ntfro

mMesocyclops

andthev

alueso

fthe

correspo

ndinggenetic

dista

nces

ares

hownwith

asteris

k


Cyclops kolensis








13

14

15

16

17

18

19

20

21

22







001

100

100100

100

100

100100

100

100100

100

65

999

999

802

998

512 97

779

703

703743

676

882

98

M viridis (Borok2)

M viridis (Borok1)

M distinctus

M albidus


T oithonoides

D bicuspidatus


C strenuus

C kolensis









Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899



(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)




4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Oncaea sp


Oithona similis

Oithona nana

Oithona simplex

Paracyclopina nana

Cyclops kolensis

Cyclops strenuus











100100

100100

100100

100100

7976

9999

9999

8189 9394

57628684

5467




Cyclops kolensis

Cyclops strenuus









9998

99100

99100

96918291

9699








Table2Pairw

iseun

correctedgenetic

distance

amon

g18

sequ

enceso

fCyclopo

idab

ased

oncomparis

onof

28SrRNAgene

Speciesn

ame

12

34

56

78

910

1112

1314

1516

171

Oncaeasp

2Pa

racyclo

pina

nana

0229

3Oith

onasim

ilis

0230

0148

4Oith

onahelgo

land

ica0227

0143

0011

5Oith

onanana

0233

0181

0155

0146

6Oith

onasim

plex

0244

0152

0175

0166

0177

7Cy

clops

kolen

sis0209

0223

0197

0195

0244

0212

8Cy

clops

strenuu

s0207

0220

0197

0195

0242

0207

0005

9Cy

clops

insig

nisM

oscow

0216

0223

0204

0203

0245

0201

0037

0032

10C

insig

nisB

orok

0209

0216

0198

0197

0241

0197

0027

0023

0014

11Diacyclo

psbicuspidatus

0186

0200

0183

0180

0224

0194

0090

0085

0098

0087

12Th

ermocyclops

oithonoides

0220

0203

0191

0186

0216

0184

0128

0123

0120

0116

0098

13Th

ermocyclops

crassus

0218

0203

0181

0178

0220

0204

0127

0125

0127

0125

0093

0084

14Mesocyclops

leuckarti

0198

0183

0174

0171

0215

0181

0133

0128

0123

0122

0090

0087

0079

15Macrocyclo

psalbidu

s0221

0198

0197

0191

0215

0178

0145

0143

0137

0134

0099

0131

0114

0105

16Macrocyclo

psdistinctus

0230

0216

0192

0192

0218

0191

0152

0148

0143

0142

0113

0127

0143

0131

0123

17Megacyclops

virid

isBo

rok1

0213

0207

0195

0192

0230

0191

0137

0134

0134

0123

0105

0116

0117

0114

0130

0139

18MviridisB

orok2

0207

0210

0206

0203

0236

0198

0123

0122

0127

0119

0085

0114

0110

0105

0125

0130

0050

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop


sitions


Table3Pairw

iseun

correctedgenetic

distance

amon

g12

Cyclo

pidaes

equences

basedon

comparis

onof

concatenated

ITS1IT

S2rRNAsequ

ences

Speciesn

ame

12

34

56

78

910

111

Cyclo

pskolen

sis2

Cyclo

psstrenuu

s000

63

Cyclo

psinsig

nisM

oscow

0054

004

84

Cinsig

nisB

orok

004

8004

2000

65

Diacyclo

psbicuspidatus

0250

0244

0232

0232

6Th

ermocyclops

oithonoides

0333

0330

0330

0330

0313

7Th

ermocyclops

crassus

0301

0298

0295

0295

0262

0188

8Mesocyclops

leuckarti

0324

0321

0310

0310

0286

0280

0241

9Macrocyclo

psdistinctuslowast

0414

0411

040

8040

50393

0429

040

80369lowast

10Macrocyclo

psalbidu

s0393

0390

0381

0378

0357

0369

0366

0336

0321

11Megacyclops

virid

isBo

rok1lowast

0381

0384

0396

0390

0387

0387

0387

0390lowast

0455

0438

12MviridisB

orok2lowast

0375

0378

0384

0378

0363

0366

0357

0381lowast

0438

0429

0149

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop

tion)F

inaldatasets

336po

sitions

forc

oncatenatedITS1IT

S2Th

enam

esof

thethreetaxa

mostevolutio

narilydista

ntfro

mMesocyclops

andthev

alueso

fthe

correspo

ndinggenetic

dista

nces

ares

hownwith

asteris

k


Cyclops kolensis








13

14

15

16

17

18

19

20

21

22







001

100

100100

100

100

100100

100

100100

100

65

999

999

802

998

512 97

779

703

703743

676

882

98

M viridis (Borok2)

M viridis (Borok1)

M distinctus

M albidus


T oithonoides

D bicuspidatus


C strenuus

C kolensis









Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899



(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)




4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Pairw

iseun

correctedgenetic

distance

amon

g18

sequ

enceso

fCyclopo

idab

ased

oncomparis

onof

28SrRNAgene

Speciesn

ame

12

34

56

78

910

1112

1314

1516

171

Oncaeasp

2Pa

racyclo

pina

nana

0229

3Oith

onasim

ilis

0230

0148

4Oith

onahelgo

land

ica0227

0143

0011

5Oith

onanana

0233

0181

0155

0146

6Oith

onasim

plex

0244

0152

0175

0166

0177

7Cy

clops

kolen

sis0209

0223

0197

0195

0244

0212

8Cy

clops

strenuu

s0207

0220

0197

0195

0242

0207

0005

9Cy

clops

insig

nisM

oscow

0216

0223

0204

0203

0245

0201

0037

0032

10C

insig

nisB

orok

0209

0216

0198

0197

0241

0197

0027

0023

0014

11Diacyclo

psbicuspidatus

0186

0200

0183

0180

0224

0194

0090

0085

0098

0087

12Th

ermocyclops

oithonoides

0220

0203

0191

0186

0216

0184

0128

0123

0120

0116

0098

13Th

ermocyclops

crassus

0218

0203

0181

0178

0220

0204

0127

0125

0127

0125

0093

0084

14Mesocyclops

leuckarti

0198

0183

0174

0171

0215

0181

0133

0128

0123

0122

0090

0087

0079

15Macrocyclo

psalbidu

s0221

0198

0197

0191

0215

0178

0145

0143

0137

0134

0099

0131

0114

0105

16Macrocyclo

psdistinctus

0230

0216

0192

0192

0218

0191

0152

0148

0143

0142

0113

0127

0143

0131

0123

17Megacyclops

virid

isBo

rok1

0213

0207

0195

0192

0230

0191

0137

0134

0134

0123

0105

0116

0117

0114

0130

0139

18MviridisB

orok2

0207

0210

0206

0203

0236

0198

0123

0122

0127

0119

0085

0114

0110

0105

0125

0130

0050

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop


sitions


Table3Pairw

iseun

correctedgenetic

distance

amon

g12

Cyclo

pidaes

equences

basedon

comparis

onof

concatenated

ITS1IT

S2rRNAsequ

ences

Speciesn

ame

12

34

56

78

910

111

Cyclo

pskolen

sis2

Cyclo

psstrenuu

s000

63

Cyclo

psinsig

nisM

oscow

0054

004

84

Cinsig

nisB

orok

004

8004

2000

65

Diacyclo

psbicuspidatus

0250

0244

0232

0232

6Th

ermocyclops

oithonoides

0333

0330

0330

0330

0313

7Th

ermocyclops

crassus

0301

0298

0295

0295

0262

0188

8Mesocyclops

leuckarti

0324

0321

0310

0310

0286

0280

0241

9Macrocyclo

psdistinctuslowast

0414

0411

040

8040

50393

0429

040

80369lowast

10Macrocyclo

psalbidu

s0393

0390

0381

0378

0357

0369

0366

0336

0321

11Megacyclops

virid

isBo

rok1lowast

0381

0384

0396

0390

0387

0387

0387

0390lowast

0455

0438

12MviridisB

orok2lowast

0375

0378

0384

0378

0363

0366

0357

0381lowast

0438

0429

0149

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop

tion)F

inaldatasets

336po

sitions

forc

oncatenatedITS1IT

S2Th

enam

esof

thethreetaxa

mostevolutio

narilydista

ntfro

mMesocyclops

andthev

alueso

fthe

correspo

ndinggenetic

dista

nces

ares

hownwith

asteris

k


Cyclops kolensis








13

14

15

16

17

18

19

20

21

22







001

100

100100

100

100

100100

100

100100

100

65

999

999

802

998

512 97

779

703

703743

676

882

98

M viridis (Borok2)

M viridis (Borok1)

M distinctus

M albidus


T oithonoides

D bicuspidatus


C strenuus

C kolensis









Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899



(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)




4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table3Pairw

iseun

correctedgenetic

distance

amon

g12

Cyclo

pidaes

equences

basedon

comparis

onof

concatenated

ITS1IT

S2rRNAsequ

ences

Speciesn

ame

12

34

56

78

910

111

Cyclo

pskolen

sis2

Cyclo

psstrenuu

s000

63

Cyclo

psinsig

nisM

oscow

0054

004

84

Cinsig

nisB

orok

004

8004

2000

65

Diacyclo

psbicuspidatus

0250

0244

0232

0232

6Th

ermocyclops

oithonoides

0333

0330

0330

0330

0313

7Th

ermocyclops

crassus

0301

0298

0295

0295

0262

0188

8Mesocyclops

leuckarti

0324

0321

0310

0310

0286

0280

0241

9Macrocyclo

psdistinctuslowast

0414

0411

040

8040

50393

0429

040

80369lowast

10Macrocyclo

psalbidu

s0393

0390

0381

0378

0357

0369

0366

0336

0321

11Megacyclops

virid

isBo

rok1lowast

0381

0384

0396

0390

0387

0387

0387

0390lowast

0455

0438

12MviridisB

orok2lowast

0375

0378

0384

0378

0363

0366

0357

0381lowast

0438

0429

0149

Allpo

sitions

containing

gaps

andmiss

ingdataweree

liminated

(com

pleted

eletionop

tion)F

inaldatasets

336po

sitions

forc

oncatenatedITS1IT

S2Th

enam

esof

thethreetaxa

mostevolutio

narilydista

ntfro

mMesocyclops

andthev

alueso

fthe

correspo

ndinggenetic

dista

nces

ares

hownwith

asteris

k


Cyclops kolensis








13

14

15

16

17

18

19

20

21

22







001

100

100100

100

100

100100

100

100100

100

65

999

999

802

998

512 97

779

703

703743

676

882

98

M viridis (Borok2)

M viridis (Borok1)

M distinctus

M albidus


T oithonoides

D bicuspidatus


C strenuus

C kolensis









Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899



(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)




4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cyclops kolensis








13

14

15

16

17

18

19

20

21

22







001

100

100100

100

100

100100

100

100100

100

65

999

999

802

998

512 97

779

703

703743

676

882

98

M viridis (Borok2)

M viridis (Borok1)

M distinctus

M albidus


T oithonoides

D bicuspidatus


C strenuus

C kolensis









Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899



(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)




4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


001

100

100100

100

100

100100

100

100100

100

65

999

999

802

998

512 97

779

703

703743

676

882

98

M viridis (Borok2)

M viridis (Borok1)

M distinctus

M albidus


T oithonoides

D bicuspidatus


C strenuus

C kolensis









Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899



(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)




4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Cyclops kolensis

Cyclops strenuus









9999

5475

7692

9594

8697

9899



(a)

Cyclops kolensis

Cyclops strenuus









100

100

100

100

100

100

094

086

073



(b)

Cyclops kolensis

Cyclops strenuus









8572

82677281

7281

7772

61849998



99mdash

(c)

Cyclops kolensis

Cyclops strenuus









061

100

100

100

100

100

096

095

070



(d)

Cyclops kolensis

Cyclops strenuus








9897

5159 7554

66968477

100mdash

(e)

Cyclops kolensis

Cyclops strenuus








8165

7772

9794

97979798

10099

(f)




4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Conclusion




Acknowledgments


References














































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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