Research Article Physical Mapping of the 5S and...

Research ArticlePhysical Mapping of the 5S and 18S rDNA inTen Species of Hypostomus Laceacutepegravede 1803 (SiluriformesLoricariidae) Evolutionary Tendencies in the Genus

Vanessa Bueno1 Paulo Ceacutesar Venere2 Jocicleacuteia Thums Konerat3

Claacuteudio Henrique Zawadzki4 Marcelo Ricardo Vicari5 and Vladimir Pavan Margarido3

1 Universidade Tecnologica Federal do Parana Prolongamento da Rua Cerejeira sn Sao Luiz 85892-00l Santa Helena PR Brazil2 Departamento de Biologia e Zoologia Universidade Federal de Mato Grosso Instituto de BiocienciasAvenida Fernando Correa da Costa sn Coxipo 78060-900 Cuiaba MT Brazil

3 Universidade Estadual do Oeste do Parana Centro de Ciencias Biologicas e da Saude Rua Universitaria 206985801-110 Cascavel PR Brazil

4Departamento de BiologiaNupelia Universidade Estadual de Maringa Avenida Colombo 5790 87020-900 Maringa PR Brazil5 Departamento de Biologia Estrutural Molecular e Genetica Universidade Estadual de Ponta GrossaAvenida Carlos Cavalcanti 4748 84030-900 Ponta Grossa PR Brazil

Correspondence should be addressed to Vladimir Pavan Margarido vladimirmargaridounioestebr

Received 30 July 2014 Accepted 24 September 2014 Published 27 October 2014

Academic Editor Adayabalam S Balajee

Copyright copy 2014 Vanessa Bueno et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Hypostomus is a diverse group with unclear aspects regarding its biology including the mechanisms that led to chromosomediversification within the group Fluorescence in situ hybridization (FISH) with 5S and 18S rDNA probes was performed onten Hypostomini species Hypostomus faveolus H cochliodon H albopunctatus H aff paulinus and H topavae had only onechromosomepairwith 18S rDNA sites whileH ancistroidesH commersoniH hermanniH regani andH strigaticepshadmultiple18S rDNA sites Regarding the 5S rDNA genes H ancistroides H regani H albopunctatus H aff paulinus and H topavae had5S rDNA sites on only one chromosome pair and H faveolus H cochliodon H commersoni H hermanni and H strigaticepshad multiple 5S rDNA sites Most species had 18S rDNA sites in the telomeric region of the chromosomes All species but Hcochliodon had 5S rDNA in the centromericpericentromeric region of one metacentric pair Obtained results are discussed basedon existent phylogenies for the genus with comments on possible dispersionmechanisms to justify the variability of the rDNA sitesin Hypostomus

1 Introduction

Loricariidae is a species-rich and diverse family distributedthrough Central and South America [1 2] It is composed ofseven subfamilies Delturinae Hypoptomatinae Hypostom-inae Lithogeneinae Loricariinae Neoplecostominae andOtothyrinae [1 3ndash5] The Hypostominae contain a greatnumber of nominal species with unclear status and thesystematics of the subfamily is not well resolved [3] Arm-bruster [1] proposed the division of the subfamily in fivetribes namely Corymbophanini Rhinelepini HypostominiPterygoplichthini and AncistriniThe only genus recognized

for Hypostomini would beHypostomus with AphanotorulusCochliodon Isorineloricaria Squaliforma and Watwata assynonyms However molecular studies based on mitochon-drial rRNA gene sequences D-loop and ITS sequences indi-cated relevant distinctions among Squaliforma Isorinelori-caria Aphanotorulus and Hypostomus therefore not sup-porting the synonymization of these genera withHypostomus[6 7] Further analyses have not been performed on theHypostomini to verify which genera should be recognizedand there seems to be no consensus about which phylogenyshould be adopted forHypostomus with a number of further

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 943825 8 pageshttpdxdoiorg1011552014943825

2 The Scientific World Journal

studies about other aspects of the genera considering eachhypothesis as valid

Karyotypic studies of Hypostomus initiated with theanalysis of Hypostomus plecostomus (Linnaeus 1758) [8] Thediploid number found 2119899 = 54 chromosomes is the lowerdiploid number observed for the genus Further studiesshowed diploid numbers ranging from 2119899 = 64 to 2119899 = 84chromosomes [9 10] indicating that the specimens analyzedby Muramoto et al [8] might have been misidentified [9]The number of species cytogenetically analyzed is increasingbut is still far from representing a significant portion ofthe genus [11] Also most studies discuss only the diploidnumber and location of the AgNORs ignoring importantmarkers as heterochromatin distribution and mapping ofrDNAs sites

Few species ofHypostomus had their rDNA sites mappedThe lack of information on the number and location ofthe 5S and 18S rDNA sites in Hypostomus hinders a broadcomparative analysis for the genus Since the relationshipsamong species of Hypostomus are still unclear the presentstudy aims to perform the mapping of 5S and 18S rDNAof ten species of Hypostomus comparing the results withavailable data for this group to verify the existence ofpossible evolutionary trends on the genus regarding thistrait

2 Material and Methods

The specimens were captured from three distinct Brazilianlocalities Piquiri River (municipality of Nova LaranjeirasParana state Upper Parana River basin) Iguacu River(municipality of Foz do Iguacu Parana state Middle ParanaRiver basin) and Taquaralzinho River (municipality of Barrado Garcas Mato Grosso state Upper Araguaia River basins)The collection sites number ofmales and females and catalognumbers of voucher specimens which were deposited in theColecao Ictiologica do Nucleo de Pesquisas em LimnologiaIctiologia e Aquicultura-Nupelia-Universidade Estadual deMaringa Brazil are summarized in Table 1 The specimenswere anesthetized and sacrificed through clove-oil over-doses [13] Metaphasic cells were obtained from the kidneythrough the air-drying technique [14] Fluorescence in situhybridization was performed according to Pinkel et al [15]with modifications suggested by Margarido and Moreira-Filho [16] The 18S rDNA probes were obtained accordingto Hatanaka and Galetti [17] and 5S rDNA probes wereobtained according toMartins and Galetti Jr [18]The probeswere marked through nick translation with biotin-16-dUTP(18S rDNA) and digoxigenin-11-dUTP (5S rDNA) (Roche)Detection and amplification of the hybridization signals wereperformed with avidin-FITC and antiavidin biotin (Sigma-Aldrich) for the 18S rDNA probes and antidigoxigeninrhodamine (Roche Applied Science) for the 5S rDNA probesThe slides were counterstained with DAPI and analyzedon the epifluorescence microscope Olympus BX 61 Thirtymetaphases per individual were analyzed Chromosomeswere classified according to Levan et al [19]

3 ResultsHypostomus faveolus Zawadzki Birindelli and Lima 2008H cochliodon Kner 1854 H albopunctatus (Regan 1908) Haff paulinus (Ihering 1905) and H topavae (Godoy 1969)had only one chromosome pair with 18S rDNA sites whileH ancistroides (Ihering 1911) H commersoni Valenciennes1836 H hermanni (Ihering 1905) H regani (Ihering 1905)andH strigaticeps (Regan 1908) hadmultiple 18S rDNA sitesAll species had 18S rDNA sites located in the telomeric regionof the chromosomes As for the 5S rDNA H ancistroidesH regani H albopunctatus H aff paulinus and H topavaehad 5S rDNA sites on only one chromosome pair and Hfaveolus H cochliodon H commersoni H hermanni andH strigaticeps had multiple 5S rDNA sites (Figures 1 2 and3) The number and position of both 5S and 18S rDNAdiffered between the populations for H commersoni andboth populations showed syntenic sites The chromosomesize difference in H commersoni (pair 31) is due to a hete-rochromatic block that is amplified in just one homologousof the chromosome pair Results are summarized in Table 2along with the available data for localization of rDNA inHypostomus

4 Discussion

A single pair bearing 5S and 18S rDNA sites has beenconsidered plesiomorphic for Loricariidae given that thischaracteristic was observed in the outgroup Trichomycteri-dae and some other Loricariidae genera (NeoplecostomusKronichthys Isbrueckerichthys and Parotocinclus) consideredphylogenetically basal through morphological analysis [127] Artoni and Bertollo [28] also consider singleNORs as theancestral phenotype for Loricariidae These characteristicsobserved onbasal genera for Loricariidae besides other tribesof Hypostominae such as Pterygoplichthini and Ancistrini[23 29] support the hypothesis that the presence of one siteof 5S and 18S rDNAs is basal for Hypostomus

It is known that the diploid numbers of Hypostomus arelikely correlated with their phylogeny and distribution [9]The following discussion relies on this correlation and onexistent phylogenies for the genus therefore the discussionwill be organized according to diploid number ranges Thecorrelation among diploid numbers data obtained from thepresent paper and phylogenetic relationships of Hyposto-mus are simplified in Figure 4 Hypostomus faveolus and Hcochliodon are the species with the lower diploid numbersin Hypostomus that had the 5S and 18S rDNA locationmapped Both species had a single pair bearing 18S rDNAalthough on different positions on the chromosome foreach species and multiple chromosomes bearing 5S rDNAConsidering the diploid number of 54 chromosomes basalfor the family Loricariidae lower diploid numbers would beconsidered basal for Hypostomus [30] Also a phylogeneticanalysis performed by Martinez [12] with the sequences ofmitochondrial cytochrome b and partial sequences of the 16SrRNA and nuclear F-reticulon 4 genes consideredH faveolusthe most basal species for Hypostomus The status of Hfaveolus as a basal species is compatible with our results since

The Scientific World Journal 3

Table 1 Species collection sites and number of analyzed specimens and catalog numbers (NUP) of the specimens deposited in theichthyological collection of the Nucleo de Pesquisas em Limnologia Ictiologia e Aquicultura

Species Males Females Locality NUP

Hypostomus albopunctatus 3 3 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 13532

Hypostomus ancistroides 4 11 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 3902

Hypostomus cochliodon 4 1 Iguacu River Foz do Iguacu25∘3810158405310158401015840 S 54∘2710158402810158401015840W 13541

Hypostomus commersoni 0 1 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 13540

1 1 Iguacu River Foz do Iguacu25∘3810158405310158401015840 S 54∘2710158402810158401015840W

Hypostomus faveolus 7 2 Taquaralzinho River Barra do Garcas15∘4010158404210158401015840 S 52∘1710158405210158401015840W 13539

Hypostomus hermanni 5 4 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 4927

Hypostomus regani 5 6 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 13534

Hypostomus aff paulinus 6 7 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 13535

Hypostomus strigaticeps 8 7 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 13536

Hypostomus topavae 9 6 Piquiri River Nova Laranjeiras24∘5610158405410158401015840 S 52∘3510158404910158401015840W 11430

Table 2 Available data regarding 5S18S rDNA distribution in Hypostomus

Species 2119899 Karyotypic formula 5S 18S Locality ReferenceH cochliodon 64 12119898 + 16119904119898 + 16119904119905 + 20119886 Multiple Simple Parana River basin Present paperH faveolus 64 18119898 + 8119904119898 + 22119904119905 + 16119886 Multiple Simple Araguaia River basin Present paperH affinis 66 14119898 + 14119904119898 + 12119904119905 + 26119886 Multiple Multiple Paraıba do Sul River basin [20 21]H tapijara 66 14119898 + 24119904119898 + 14119904119905 + 14119886 Multiple Multiple Ribeira do Iguape basin [22]H prope plecostomus 68 12119898 + 16119904119898 + 12119904119905 + 28119886 mdash Simple Orinoco River basin [23]H ancistroides 68 10119898 + 26119904119898 + 32119886 mdash Multiple Paranapanema River basin [24]H ancistroides 68 14119898 + 14119904119898 + 8119904119905 + 32119886 Simple Multiple Parana River basin Present paperH ancistroides 68 14119898 + 16119904119898 + 22119904119905 + 16119886 Multiple Multiple Paranapanema River basin [22]H commersoni 68 12119898 + 14119904119898 + 14119904119905 + 28119886 Multiple Multiple Parana River basin (Iguacu River) Present paperH commersoni 68 12119898 + 14119904119898 + 14119904119905 + 28119886 Multiple Multiple Parana River basin (Piquiri River) Present paperH hermanni 72 10119898 + 8119904119898 + 32119904119905 + 22119886 Multiple Multiple Parana River basin Present paperH regani 72 8119898 + 16119904119898 + 20119904119905 + 28119886 Multiple Simple Sao Francisco River basin [25]H regani 72 10119898 + 18119904119898 + 44119904119905 minus 119886 mdash Multiple Paranapanema River basin [24]H regani 72 12119898 + 8119904119898 + 10119904119905 + 42119886 Simple Multiple Parana River basin Present paperH strigaticeps 72 10119898 + 16119904119898 + 46119904119905 minus 119886 mdash Multiple Paranapanema River basin [24]H strigaticeps 72 12119898 + 12119904119898 + 18119904119905 + 30119886 Multiple Multiple Parana River basin Present paperH albopunctatus 74 8119898 + 14119904119898 + 16119904119905 + 36119886 Simple Simple Parana River basin Present paperH paulinus 74 10119898 + 12119904119898 + 20119904119905 + 32119886 Simple Simple Parana River basin Present paperH paulinus 76 6119898 + 16119904119898 + 54119904119905 minus 119886 mdash Simple Paranapanema River basin [24]H nigromaculatus 76 12119898 + 22119904119898 + 30119904119905 + 12119886 Simple Simple Paranapanema River basin [22]H iheringii 80 8119898 + 16119904119898 + 28119904119905 + 28119886 Simple Multiple Paranapanema River basin [26]H topavae 80 14119898 + 10119904119898 + 26119904119905 + 30119886 Simple Simple Parana River basin Present paper


1 2 3 4 5 6 7 8 9

10 11 12 13

14 15 16 17 18 19 20 21 22 23

24

25 26 27 28 29 30 31 32

m

a

sm

st

(a)

1 2 3 4 5 6

7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22

23 24 25 26 27 28 29 30 31 32

m

a

sm

st

(b)

1 2 3 4 5 6

7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

31 32 33 34

m

a

sm

st

(c)

1 2 3 4 5 6

7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

31 32 33 34

m

a

sm

st

(d)

Figure 1 Double FISH karyotypes with 5S rDNA probes (rhodamine red) and 18S rDNA probes (FITC green) of (a) Hypostomus faveolus(b) Hypostomus cochliodon (c) Hypostomus commersoni from the Piquiri River and (d) Hypostomus commersoni from the Iguacu River Thebar represents 5 120583m

this species presented one 18S rDNA site which corroboratesthe hypothesis that the presence of one 18S rDNA site is basalfor Hypostomus As for the 5S rDNA it is not possible todetermine whether multiple pairs bearing 5S rDNA sites arean ancestral condition for the genus or an apomorphy for theanalyzed species

The species with 2119899 = 68 and 2119899 = 72 chromosomes(H ancistroides H commersoni H hermanni H regani andH strigaticeps) hadmultiple chromosomes bearing 18S rDNAsites Most species also presented multiple chromosomesbearing 5S rDNA sites with only H ancistroides and Hreganiwith 5S rDNA on a single chromosome pairThere wasvariation on the location of some sites among the analyzedpopulations of H commersoni Only a small number ofspecimens of H commersoni were collected therefore it wasnot possible to verify how the rDNA is distributed withinthe populations There is also a description of H reganifrom Piumhi River (Sao Francisco River basin state of SaoPaulo) with multiple pairs bearing 5S rDNA and a single pairbearing 18S rDNA [25] It differs from that observed for thepopulation from thePiquiri River (Parana River basin Paranastate) that showed a single pair bearing 5S rDNA andmultiplepairs bearing 18S rDNA Analyses of other populations ofH ancistroides (Parana River basin Sao Paulo state) [22]

and H strigaticeps (Parana River basin between Parana andSao Paulo states) [24] also showed different results fromthat observed on the present study with multiple 5S rDNAsites for H ancistroides and two pairs bearing 18S rDNAsites for H strigaticeps Considering the small number ofspecies analyzed through in situ hybridization there seemsto be a relevant occurrence of variation on the number andposition of the rDNA sites Although this group includesspecies with similar diploid numbers (2119899 = 68 to 2119899 = 72)and number of 5S and 18S sites these characteristics are notsynapomorphic [1 7 12] The variation on the number ofrDNA sites even among populations of the same species andthe existence of phylogenetic hypotheses that separate specieswith similar number and location of rDNA sites indicatethat this characteristic might not be reliable to establishrelationships withinHypostomus but seems to have potentialas a population marker

The variation on the number and location of 18S rDNAsites which occurs even among individuals of the samespecies suggests the existence of dispersion mechanisms onthese sites The particular chromosome evolution of thisgenus through centric fissions and pericentric inversions [1130] may be partly responsible for this diversity in Hyposto-mus However it is known that some retrotransposons are


1 2 3 4 5 6 7

8 9 10 11 12 13 14

15 16 17 18

19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9

10 11 12 13 14 15 16 17 18 19

20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(b)

1 2 3 4 5 6

7 8 9 10

11 12 13 14 15

16 17 18 19 20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(c)

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36

m

a

sm

st

(d)

Figure 2Double FISHkaryotypeswith 5S rDNAprobes (rhodamine red) and 18S rDNAprobes (FITC green) of (a)Hypostomus ancistroides(b) Hypostomus hermanni (c) Hypostomus regani (d) Hypostomus strigaticeps The bar represents 5120583m

specific for rDNA sequences and that these sites are favorablefor the invasion of mobile elements [31ndash33] Many typesof transposable elements (TEs) are described for teleosteanfishes [34] A possible role of TEs as a source of rRNAgenes movement which could generate the spreading ofribosomal clusterscopies was visualized in fishes [35ndash37]Hence the organization of rRNA gene clusters seems toreflect their intense and particular evolutionary pathwayand not the evolutionary history of the associated taxa insome fish group [36 38] Similar mechanisms might beresponsible for the variation on the number and positionof 18S rDNA sites observed in Hypostomus The presence ofadjacent heterochromatin on these sites may also facilitategenetic exchanges among non-homologous chromosomescausing the dispersion of these sequences on the genome

The species with 2119899 = 74 and 2119899 = 80 chromosomes(H albopunctatus H aff paulinus and H topavae) showedsingle chromosome pairs bearing 5S and 18S rDNA sitesHowever H iheringii (Regan 1908) analyzed by Traldi et al[26] showed a single pair bearing 5S rDNA andmultiple pairsbearing 18S rDNA Also some species within this diploidnumber group have been reported with multiple NORs such

as 119867 aff agna (2119899 = 74) [39] H nigromaculatus (Schubart1964) (2119899 = 76) [40] and Hypostomus sp E (2119899 = 80) [40]therefore multiple 18S rDNA sites In fact H nigromaculatusfrom the Lapa stream Sao Paulo state was reported to have 5Sand 18S rDNA each on only one chromosome pair [22] whilepopulations from Mogi-Guacu and Tibagi Rivers showedmultiple AgNORs pointing to the existence of multiple 18rDNA sites on such populations [40] These results indicatethat it is probable that this group of species also showsvariations on the number and locations of rDNA sites amongthem If it really is so the whole group of species with 2119899 =68 to 2119899 = 84 would be very heterogeneous regarding thenumber and location of the 18S rDNA sites indicating thatthis character changed recurrently during the chromosomaldiversification of the genus All species in this diploid numberrange (2119899 = 74 to 2119899 = 84) analyzed until the presentmomentpresent a single pair bearing 5S rDNA sites Further analysesare necessary to determinewhether the increase of the diploidnumber is associated with the loss of 5S rDNA sites

The presence of a centromericpericentromeric 5S rDNAsite on the short arm of a metacentricsubmetacentric pairis a particular feature of the 5S rDNA observed on most


1 2 3 4

5 6 7 8 9 10 11

12 13 14 15 16 17 18 19

20 21 22 23 24 25 26 27 28 29

30 31 32 33 34 35

35

36 37

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36 37

m

a

sm

st

(b)

1 2 3 4 5 6 7

8 9 10 11 12

13 14 15 16 17 18 19 20 21 22

23 24 25

26 27 28 29 30 31 32 33 34 35

36 37 38 39 40

m

sm

st

a

(c)

Figure 3 Double FISH karyotypes with 5S rDNA probes (rhodamine red) and 18S rDNA probes (FITC green) of (a) Hypostomusalbopunctatus with a variation of the position of the 18S rDNA sites evidenced on the box (b) Hypostomus aff paulinus and (c) Hypostomustopavae The bar represents 5120583m

H albopunctatus (2n = 74)

H nigromaculatus (2n = 76)

H regani (2n = 72)

H commersoni (2n = 68)

H affinis (2n = 66)

H ancistroides (2n = 68)

H plecostomus (2n = 68)

(2n = 64)H cochliodonlowast

(2n = 64)H faveoluslowast

Simple Simple D4

Simple SimpleD3

Various Various

Multiple Multiple

Multiple Multiple

Various MultipleD2

Simple

Multiple Simple D1

Multiple Simple D4

5S 18S Clade

ndash

Figure 4 Data on rDNA sites number plotted against the phylogenetic relationship between speciesThe cladogram is only representative andis based on the phylogeny proposed byMontoya-Burgos (2003) with addition of species marked with an asterisk ldquoCladesrdquo refer to the cladesproposed by the same author Hypostomus cochliodon is added considering information from Armbruster [1] and H faveolus consideringinformation fromMartinez [12]


Hypostomus species analyzed on the present paper and alsoon other previously analyzed populationsspecies [20ndash2224 25] All species of Hypostomus analyzed so far but Hcochliodon have this 5S rDNA site on this particular locationTraldi et al [22] consider the frequent occurrence of thissite in this particular location a possible primitive conditionfor the genus Possibly the location of this site favoredits permanence on the karyotype of most species whiletelomeric sites seem to vary more easily Further analyses arenecessary to identify whether the lack of this particular 5SrDNA site occurs in other species of the H cochliodon groupor only inH cochliodon or even on the studied population ofthis species

Syntenic 5S and 18S rDNAs were observed in H com-mersoni The occurrence of syntenic 5S and 18S rDNAs isconsidered primitive for Loricariidae with Trichomycteridaeas outgroup [27] This characteristic was already describedfor species of the subfamilies Neoplecostominae and Hypop-topomatinae [27] and in Hypostominae for the tribesAncistrini [29] and Hypostomini [22] For Hypostomini twospecies presented syntenic 5S and 18S rDNAsH ancistroidesand H tapijara Oyakawa Akama and Zanata 2005 [22]On the present study we presented one more species ofHypostomini (H commersoni) that presented syntenic 5Sand 18S rDNAs All Hypostominae tribes that were analyzedthrough in situ hybridization with 5S and 18S rDNA probesuntil nowpresent specieswith syntenic rDNAsAlthough thiscondition may also be the primitive condition for Hypos-tominae given its occurrence on different tribes furtherstudies are necessary to determine if it was maintained inHypostomus If H faveolus really is a basal species for thegenus the possibility that the syntenic condition of the rDNAclasses was lost early in the genus and then it reappeared duethe apparent mobility of the rDNA on the genome must beconsidered as well

The analysis here presented still includes a rather smallnumber of species considering the whole genus Howeverwe could observe a great variability on the number andlocation of the rDNA sites reinforcing the hypothesis pro-posed by Artoni and Bertollo [41] that Hypostomus has adivergent chromosomal evolution when compared to otherHypostominae that tend to maintain more ancestral charac-teristics such lower diploid numbers and one chromosomepair bearing AgNORs therefore 18S rDNA Further studiesincluding more species especially from northern basins andanalyses on the rDNAs and adjacent sequences would greatlycontribute to clarify the dispersion mechanisms of thesesequences within the genome of Hypostomus

Conflict of Interests

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

Acknowledgments

The authors are grateful to Instituto Chico Mendes deConservacao da Biodiversidade (ICMBio) for authorizing

the capture of fishes (License SISBIO 10522-1) The authorsthank Unioeste and the Nucleo de Pesquisas em LimnologiaIctiologia e Aquicultura (Nupelia) for logistical supportThis study was financed by Fundacao Araucaria (FundacaoAraucaria de Apoio ao Desenvolvimento Cientıfico e Tec-nologico do Estado do Parana) CAPES (Coordenadoria deAperfeicoamento de Ensino Superior) and CNPq (ConselhoNacional de Desenvolvimento Cientıfico e Tecnologico)

References

[1] J W Armbruster ldquoPhylogenetic relationships of the sucker-mouth armoured catfishes (Loricariidae) with emphasis on theHypostominae and the Ancistrinaerdquo Zoological Journal of theLinnean Society vol 141 no 1 pp 1ndash80 2004

[2] C J Ferraris Jr ldquoChecklist of catfishes recent and fossil (Oste-ichthyes Siluriformes) and catalogue of siluriform primarytypesrdquo Zootaxa vol 1418 pp 1ndash628 2007

[3] CWeber ldquoHypostominaerdquo inCheck List of the Freshwater Fishesof South and Central America R E Reis S O Kullander and CJ Ferraris Jr Eds pp 351ndash372 Edipucrs Porto Alegre Brazil2003

[4] R E Reis E H L Pereira and J W Armbruster ldquoDelturinaea new loricariid catfish subfamily (Teleostei Siluriformes) withrevisions ofDelturus andHemipsilichthysrdquo Zoological Journal ofthe Linnean Society vol 147 no 2 pp 277ndash299 2006

[5] M C Chiachio C Oliveira and J I Montoya-Burgos ldquoMolec-ular systematic and historical biogeography of the armoredNeotropical catfishes Hypoptopomatinae and Neoplecostomi-nae (Siluriformes Loricariidae)rdquo Molecular Phylogenetics andEvolution vol 49 no 2 pp 606ndash617 2008

[6] J I Montoya-Burgos S Muller C Weber and J PawlowskildquoPhylogenetic relationships of the Loricariidae (Siluriformes)based on mitochondrial rRNA gene sequencesrdquo in Phylogenyand Classification of Neotropical Fishes L R Malabarba R EReis R P Vari Z M S Lucena and C A S Lucena Eds pp363ndash374 Edipucrs Porto Alegre Brazil 1998

[7] J I Montoya-Burgos ldquoHistorical biogeography of the catfishgenus Hypostomus (Siluriformes Loricariidae) with impli-cations on the diversification of Neotropical ichthyofaunardquoMolecular Ecology vol 12 no 7 pp 1855ndash1867 2003

[8] J Muramoto S Ohno and N B Atkin ldquoOn the diploid stateof the fish order Ostariophysirdquo Chromosoma vol 24 no 1 pp59ndash66 1968

[9] V Bueno P C Venere C H Zawadzki and V P MargaridoldquoKaryotypic diversification inHypostomus Lacepede 1803 (Silu-riformes Loricariidae) biogeographical and phylogenetic per-spectivesrdquo Reviews in Fish Biology and Fisheries vol 23 no 1pp 103ndash112 2013

[10] S S Cereali E Pomini R Rosa C H Zawadzki O Froehlichand L Giuliano-Caetano ldquoKaryotype description of two speciesof Hypostomus (Siluriformes Loricariidae) of the Planalto daBodoquena Brazilrdquo Genetics and Molecular Research vol 7 no3 pp 583ndash591 2008

[11] V Bueno C H Zawadzki and V P Margarido ldquoTrends inchromosome evolution in the genus Hypostomus Lacepede1803 (Osteichthyes Loricariidae) a new perspective about thecorrelation between diploid number and chromosomes typesrdquoReviews in Fish Biology and Fisheries vol 22 no 1 pp 241ndash2502012


[12] E R M Martinez Estudo da evolucao do genero Hypostomus(Teleostei Siluriformes Loricariidae) com base em caracterescromossomicos e sequencias deDNA [PhD thesis] UniversidadeEstadual Paulista Sao Paulo Brazil 2009

[13] S P Griffiths ldquoThe use of clove oil as an anaesthetic andmethodfor sampling intertidal rockpool fishesrdquo The Journal of FishBiology vol 57 no 6 pp 1453ndash1464 2000

[14] L A C Bertollo C S Takahashi and O Moreira FilholdquoCytotaxonomic considerations on Hoplias lacerdae (PiscesErythrinidae)rdquo Brazilian Journal of Genetics vol 1 no 2 pp103ndash120 1978

[15] D Pinkel T Straume and J W Gray ldquoCytogenetic analysisusing quantitative high-sensitivity fluorescence hybridizationrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 83 no 9 pp 2934ndash2938 1986

[16] V P Margarido and O Moreira-Filho ldquoKaryotypic differen-tiation through chromosome fusion and number reductionin Imparfinis hollandi (Ostariophysi Heptapteridae)rdquo Geneticsand Molecular Biology vol 31 no 1 pp 235ndash238 2008

[17] T Hatanaka and P M Galetti ldquoMapping of the 18S and 5Sribosomal RNA genes in the fish Prochilodus argenteusAgassiz1829 (Characiformes Prochilodontidae)rdquoGenetica vol 122 no3 pp 239ndash244 2004

[18] C Martins and P M Galetti Jr ldquoChromosomal localizationof 5S rDNA genes in Leporinus fish (Anostomidae Characi-formes)rdquo Chromosome Research vol 7 no 5 pp 363ndash367 1999

[19] A Levan K Fredga and A A Sandberg ldquoNomenclature forcentromeric position on chromosomesrdquo Hereditas vol 52 no2 pp 201ndash220 1964

[20] K F Kavalco R Pazza L A C Bertollo and O Moreira-FilholdquoGene mapping of 5s rDNA sites in eight fish species fromthe Paraba do Sul river basin Brazilrdquo Cytogenetic and GenomeResearch vol 106 no 1 pp 107ndash110 2004

[21] K F Kavalco R Pazza L A C Bertollo and O Moreira-FilholdquoKaryotypic diversity and evolution of Loricariidae (PiscesSiluriformes)rdquo Heredity vol 94 no 2 pp 180ndash186 2005

[22] J B Traldi D R Blanco M R Vicari et al ldquoChromoso-mal diversity in Hypostomus (Siluriformes Loricariidae) withemphasis on physical mapping of 18S and 5S rDNA sitesrdquoGenetics andMolecular Research vol 12 no 1 pp 463ndash471 2013

[23] A L Alves R S de Borba A P B Pozzobon et al ldquoLocalizationof 18S ribosomal genes in suckermouth armoured catfishesLoricariidae (Teleostei Siluriformes) with discussion on theAg-NOR evolutionrdquo Comparative Cytogenetics vol 6 no 3 pp315ndash321 2012

[24] M Rubert R Da Rosa F C Jerep L A C Bertollo andL Giuliano-Caetano ldquoCytogenetic characterization of fourspecies of the genus Hypostomus Lacepede 1803 (SiluriformesLoricariidae) with comments on its chromosomal diversityrdquoComparative Cytogenetics vol 5 no 5 pp 397ndash410 2011

[25] E de Oliveira Mendes-Neto M R Vicari R F Artoni andO Moreira-Filho ldquoDescription of karyotype in Hypostomusregani (Ihering 1905) (Teleostei Loricariidae) from the Piumhiriver in Brazil with comments on karyotype variation found inHypostomusrdquo Comparative Cytogenetics vol 5 no 2 pp 133ndash142 2011

[26] J B Traldi M R Vicari D R Blanco J D F Martinez R FArtoni and O Moreira-Filho ldquoFirst karyotype description ofHypostomus iheringii (Regan 1908) a case of heterochromaticpolymorphismrdquoComparative Cytogenetics vol 6 no 2 pp 115ndash125 2012

[27] K Ziemniczak A V Barros K O Rosa et al ldquoCompara-tive cytogenetics of Loricariidae (Actinopterygii Siluriformes)emphasis in Neoplecostominae and Hypoptopomatinaerdquo Ital-ian Journal of Zoology vol 79 no 4 pp 492ndash501 2012

[28] R F Artoni and L A C Bertollo ldquoCytogenetic studies onHypostominae (Pisces Siluriformes Loricariidae) Considera-tions on karyotype evolution in the genus Hypostomusrdquo Cary-ologia vol 49 no 1 pp 81ndash90 1996

[29] S Mariotto L Centofante M R Vicari R F Artoni andO Moreira-Filho ldquoChromosomal diversification in ribosomalDNA sites in Ancistrus Kner 1854 (Loricariidae Ancistrini)from three hydrographic basins of Mato Grosso Brazilrdquo Com-parative Cytogenetics vol 5 no 4 pp 31ndash42 2011

[30] R F Artoni and L A C Bertollo ldquoTrends in the karyotypeevolution of Loricariidae fish (Siluriformes)rdquoHereditas vol 134no 3 pp 201ndash210 2001

[31] Y Xiong W D Burke J L Jakubczak and T H EickbushldquoRibosomal DNA insertion elements R1Bm and R2Bm cantranspose in a sequence specific manner to locations outsidethe 28S genesrdquoNucleic Acids Research vol 16 no 22 pp 10561ndash10573 1988

[32] K K Kojima and H Fujiwara ldquoEvolution of target specificityin R1 clade non-LTR retrotransposonsrdquo Molecular Biology andEvolution vol 20 no 3 pp 351ndash361 2003

[33] K K Kojima and H Fujiwara ldquoCross-genome screening ofnovel sequence-specific non-LTR retrotransposons variousmulticopy RNA genes and microsatellites are selected as tar-getsrdquoMolecular Biology and Evolution vol 21 no 2 pp 207ndash2172004

[34] J N Volff ldquoGenome evolution and biodiversity in teleost fishrdquoHeredity vol 94 no 3 pp 280ndash294 2005

[35] M da Silva D A Matoso M R Vicari M C de Almeida VP Margarido and R F Artoni ldquoPhysical mapping of 5S rDNAin two species of knifefishes Gymnotus pantanal and Gym-notus paraguensis (Gymnotiformes)rdquo Cytogenetic and GenomeResearch vol 134 no 4 pp 303ndash307 2011

[36] S S R Milhomem P C Scacchetti J C Pieczarka et al ldquoAreNORs always located on homeologous chromosomes A fishinvestigation with rDNA and whole chromosome probes inGymnotus fishes (gymnotiformes)rdquo PLoS ONE vol 8 no 2Article ID e55608 2013

[37] D Piscor D B Ribacinko-Piscor C A Fernandes and P PParise-Maltempi ldquoCytogenetic analysis in three Bryconameri-cus species (Characiformes Characidae) first description of the5S rDNA-bearing chromosome pairs in the genusrdquo MolecularCytogenetics vol 6 no 1 article 13 2013

[38] R T Nakajima D C Cabral-de-Mello G T Valente P CVenere and C Martins ldquoEvolutionary dynamics of rRNA geneclusters in cichlid fishrdquo BMC Evolutionary Biology vol 12 no 1article 198 2012

[39] E R M Martinez C H Zawadzki F Foresti and C OliveiraldquoCytogenetic analysis of five Hypostomus species (SiluriformesLoricariidae)rdquoGenetics andMolecular Biology vol 34 no 4 pp562ndash568 2011

[40] M Rubert C H Zawadzki and L Giuliano-Caetano ldquoCyto-genetic characterization of Hypostomus nigromaculatus (Siluri-formes Loricariidae)rdquo Neotropical Ichthyology vol 6 no 1 pp93ndash100 2008

[41] R F Artoni and L A C Bertollo ldquoNature and distributionof constitutive heterochromatin in fishes genus Hypostomus(Loricariidae)rdquo Genetica vol 106 no 3 pp 209ndash214 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

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Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Zoology


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GenomicsInternational Journal of


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BioinformaticsAdvances in

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Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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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


studies about other aspects of the genera considering eachhypothesis as valid

Karyotypic studies of Hypostomus initiated with theanalysis of Hypostomus plecostomus (Linnaeus 1758) [8] Thediploid number found 2119899 = 54 chromosomes is the lowerdiploid number observed for the genus Further studiesshowed diploid numbers ranging from 2119899 = 64 to 2119899 = 84chromosomes [9 10] indicating that the specimens analyzedby Muramoto et al [8] might have been misidentified [9]The number of species cytogenetically analyzed is increasingbut is still far from representing a significant portion ofthe genus [11] Also most studies discuss only the diploidnumber and location of the AgNORs ignoring importantmarkers as heterochromatin distribution and mapping ofrDNAs sites

Few species ofHypostomus had their rDNA sites mappedThe lack of information on the number and location ofthe 5S and 18S rDNA sites in Hypostomus hinders a broadcomparative analysis for the genus Since the relationshipsamong species of Hypostomus are still unclear the presentstudy aims to perform the mapping of 5S and 18S rDNAof ten species of Hypostomus comparing the results withavailable data for this group to verify the existence ofpossible evolutionary trends on the genus regarding thistrait

2 Material and Methods

The specimens were captured from three distinct Brazilianlocalities Piquiri River (municipality of Nova LaranjeirasParana state Upper Parana River basin) Iguacu River(municipality of Foz do Iguacu Parana state Middle ParanaRiver basin) and Taquaralzinho River (municipality of Barrado Garcas Mato Grosso state Upper Araguaia River basins)The collection sites number ofmales and females and catalognumbers of voucher specimens which were deposited in theColecao Ictiologica do Nucleo de Pesquisas em LimnologiaIctiologia e Aquicultura-Nupelia-Universidade Estadual deMaringa Brazil are summarized in Table 1 The specimenswere anesthetized and sacrificed through clove-oil over-doses [13] Metaphasic cells were obtained from the kidneythrough the air-drying technique [14] Fluorescence in situhybridization was performed according to Pinkel et al [15]with modifications suggested by Margarido and Moreira-Filho [16] The 18S rDNA probes were obtained accordingto Hatanaka and Galetti [17] and 5S rDNA probes wereobtained according toMartins and Galetti Jr [18]The probeswere marked through nick translation with biotin-16-dUTP(18S rDNA) and digoxigenin-11-dUTP (5S rDNA) (Roche)Detection and amplification of the hybridization signals wereperformed with avidin-FITC and antiavidin biotin (Sigma-Aldrich) for the 18S rDNA probes and antidigoxigeninrhodamine (Roche Applied Science) for the 5S rDNA probesThe slides were counterstained with DAPI and analyzedon the epifluorescence microscope Olympus BX 61 Thirtymetaphases per individual were analyzed Chromosomeswere classified according to Levan et al [19]

3 ResultsHypostomus faveolus Zawadzki Birindelli and Lima 2008H cochliodon Kner 1854 H albopunctatus (Regan 1908) Haff paulinus (Ihering 1905) and H topavae (Godoy 1969)had only one chromosome pair with 18S rDNA sites whileH ancistroides (Ihering 1911) H commersoni Valenciennes1836 H hermanni (Ihering 1905) H regani (Ihering 1905)andH strigaticeps (Regan 1908) hadmultiple 18S rDNA sitesAll species had 18S rDNA sites located in the telomeric regionof the chromosomes As for the 5S rDNA H ancistroidesH regani H albopunctatus H aff paulinus and H topavaehad 5S rDNA sites on only one chromosome pair and Hfaveolus H cochliodon H commersoni H hermanni andH strigaticeps had multiple 5S rDNA sites (Figures 1 2 and3) The number and position of both 5S and 18S rDNAdiffered between the populations for H commersoni andboth populations showed syntenic sites The chromosomesize difference in H commersoni (pair 31) is due to a hete-rochromatic block that is amplified in just one homologousof the chromosome pair Results are summarized in Table 2along with the available data for localization of rDNA inHypostomus

4 Discussion

A single pair bearing 5S and 18S rDNA sites has beenconsidered plesiomorphic for Loricariidae given that thischaracteristic was observed in the outgroup Trichomycteri-dae and some other Loricariidae genera (NeoplecostomusKronichthys Isbrueckerichthys and Parotocinclus) consideredphylogenetically basal through morphological analysis [127] Artoni and Bertollo [28] also consider singleNORs as theancestral phenotype for Loricariidae These characteristicsobserved onbasal genera for Loricariidae besides other tribesof Hypostominae such as Pterygoplichthini and Ancistrini[23 29] support the hypothesis that the presence of one siteof 5S and 18S rDNAs is basal for Hypostomus

It is known that the diploid numbers of Hypostomus arelikely correlated with their phylogeny and distribution [9]The following discussion relies on this correlation and onexistent phylogenies for the genus therefore the discussionwill be organized according to diploid number ranges Thecorrelation among diploid numbers data obtained from thepresent paper and phylogenetic relationships of Hyposto-mus are simplified in Figure 4 Hypostomus faveolus and Hcochliodon are the species with the lower diploid numbersin Hypostomus that had the 5S and 18S rDNA locationmapped Both species had a single pair bearing 18S rDNAalthough on different positions on the chromosome foreach species and multiple chromosomes bearing 5S rDNAConsidering the diploid number of 54 chromosomes basalfor the family Loricariidae lower diploid numbers would beconsidered basal for Hypostomus [30] Also a phylogeneticanalysis performed by Martinez [12] with the sequences ofmitochondrial cytochrome b and partial sequences of the 16SrRNA and nuclear F-reticulon 4 genes consideredH faveolusthe most basal species for Hypostomus The status of Hfaveolus as a basal species is compatible with our results since


















1 2 3 4 5 6 7 8 9

10 11 12 13

14 15 16 17 18 19 20 21 22 23

24

25 26 27 28 29 30 31 32

m

a

sm

st

(a)

1 2 3 4 5 6

7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22

23 24 25 26 27 28 29 30 31 32

m

a

sm

st

(b)

1 2 3 4 5 6

7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

31 32 33 34

m

a

sm

st

(c)

1 2 3 4 5 6

7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

31 32 33 34

m

a

sm

st

(d)







1 2 3 4 5 6 7

8 9 10 11 12 13 14

15 16 17 18

19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9

10 11 12 13 14 15 16 17 18 19

20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(b)

1 2 3 4 5 6

7 8 9 10

11 12 13 14 15

16 17 18 19 20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(c)

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36

m

a

sm

st

(d)







1 2 3 4

5 6 7 8 9 10 11

12 13 14 15 16 17 18 19

20 21 22 23 24 25 26 27 28 29

30 31 32 33 34 35

35

36 37

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36 37

m

a

sm

st

(b)

1 2 3 4 5 6 7

8 9 10 11 12

13 14 15 16 17 18 19 20 21 22

23 24 25

26 27 28 29 30 31 32 33 34 35

36 37 38 39 40

m

sm

st

a

(c)




H regani (2n = 72)


H affinis (2n = 66)





Simple Simple D4

Simple SimpleD3

Various Various

Multiple Multiple

Multiple Multiple

Various MultipleD2

Simple

Multiple Simple D1

Multiple Simple D4

5S 18S Clade

ndash








Acknowledgments



References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















1 2 3 4 5 6 7 8 9

10 11 12 13

14 15 16 17 18 19 20 21 22 23

24

25 26 27 28 29 30 31 32

m

a

sm

st

(a)

1 2 3 4 5 6

7 8 9 10 11 12 13 14

15 16 17 18 19 20 21 22

23 24 25 26 27 28 29 30 31 32

m

a

sm

st

(b)

1 2 3 4 5 6

7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

31 32 33 34

m

a

sm

st

(c)

1 2 3 4 5 6

7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

31 32 33 34

m

a

sm

st

(d)







1 2 3 4 5 6 7

8 9 10 11 12 13 14

15 16 17 18

19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9

10 11 12 13 14 15 16 17 18 19

20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(b)

1 2 3 4 5 6

7 8 9 10

11 12 13 14 15

16 17 18 19 20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(c)

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36

m

a

sm

st

(d)







1 2 3 4

5 6 7 8 9 10 11

12 13 14 15 16 17 18 19

20 21 22 23 24 25 26 27 28 29

30 31 32 33 34 35

35

36 37

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36 37

m

a

sm

st

(b)

1 2 3 4 5 6 7

8 9 10 11 12

13 14 15 16 17 18 19 20 21 22

23 24 25

26 27 28 29 30 31 32 33 34 35

36 37 38 39 40

m

sm

st

a

(c)




H regani (2n = 72)


H affinis (2n = 66)





Simple Simple D4

Simple SimpleD3

Various Various

Multiple Multiple

Multiple Multiple

Various MultipleD2

Simple

Multiple Simple D1

Multiple Simple D4

5S 18S Clade

ndash








Acknowledgments



References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7

8 9 10 11 12 13 14

15 16 17 18

19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9

10 11 12 13 14 15 16 17 18 19

20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(b)

1 2 3 4 5 6

7 8 9 10

11 12 13 14 15

16 17 18 19 20 21 22 23 24 25

26 27 28 29 30 31 32 33 34 35

36

m

a

sm

st

(c)

1 2 3 4 5 6

7 8 9 10 11 12

13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36

m

a

sm

st

(d)







1 2 3 4

5 6 7 8 9 10 11

12 13 14 15 16 17 18 19

20 21 22 23 24 25 26 27 28 29

30 31 32 33 34 35

35

36 37

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36 37

m

a

sm

st

(b)

1 2 3 4 5 6 7

8 9 10 11 12

13 14 15 16 17 18 19 20 21 22

23 24 25

26 27 28 29 30 31 32 33 34 35

36 37 38 39 40

m

sm

st

a

(c)




H regani (2n = 72)


H affinis (2n = 66)





Simple Simple D4

Simple SimpleD3

Various Various

Multiple Multiple

Multiple Multiple

Various MultipleD2

Simple

Multiple Simple D1

Multiple Simple D4

5S 18S Clade

ndash








Acknowledgments



References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4

5 6 7 8 9 10 11

12 13 14 15 16 17 18 19

20 21 22 23 24 25 26 27 28 29

30 31 32 33 34 35

35

36 37

m

a

sm

st

(a)

1 2 3 4 5

6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 21

22 23 24 25 26 27 28 29 30 31

32 33 34 35 36 37

m

a

sm

st

(b)

1 2 3 4 5 6 7

8 9 10 11 12

13 14 15 16 17 18 19 20 21 22

23 24 25

26 27 28 29 30 31 32 33 34 35

36 37 38 39 40

m

sm

st

a

(c)




H regani (2n = 72)


H affinis (2n = 66)





Simple Simple D4

Simple SimpleD3

Various Various

Multiple Multiple

Multiple Multiple

Various MultipleD2

Simple

Multiple Simple D1

Multiple Simple D4

5S 18S Clade

ndash








Acknowledgments



References


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







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

Research Article Physical Mapping of the 5S and...

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Transcript of Research Article Physical Mapping of the 5S and...