Cytogenetics of anew speciesof Paratelmatobius cardosoi group (Anura: Leptodactylidae), with thedescription of an apparent caseof pericentricin version

LucianaB. Lourenço 1,PauloC.A. Garcia 2,ShirleiM. Recco-Pimentel 3

1;3Departamento deBiologia Celular, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), 13084-971,Campinas, SP ,Brasil correspondingauthor: e-mail: [email protected] e-mail: [email protected] 2 Departamento deZoologiae Cursode Pó s-Graduaç ão, Institutode Biociê ncias, Universidade Estadual Paulista (UNESP),13506-900, Rio Claro, SP ,Brasil e-mail: [email protected]

Abstract. Thekaryotype of a new species of Paratelmatobius from the P. cardosoi groupis described. As with other Paratelmatobius and Scythrophrys karyotypes, Paratelmatobius sp. (aff. cardosoi)showsa diploid numberof 24chromosomes, in addition to other similarities withthe former karyotypes.The Paratelmatobius sp. (aff. cardosoi)karyotypediffers from that of P. cardosoi inthe morphology of pair 4, the NOR location andthe C-bands in pairs 3 and8 (exclusiveto Paratelmatobius sp.)and those of pairs 7 and9 (exclusiveto P. cardosoi).Bothkaryotypes also differ in the amount of heterochromatinin pair 1. Thepresence ofinterstitial heterochromatinin the long arm ofpair1 andthe interstitial C-bands in both arms ofchromosome5 are apparently synapomorphiccharacters of P. cardosoi and Paratelmatobius sp. (aff. cardosoi),since theyare absentin the other Paratelmatobius and Scythrophrys karyotypes.In Paratelmatobius sp. (aff. cardosoi),thenucleolus organizer regionis onthe short arm ofa small metacentric chromosome(pair 9), an arrangement similar tothe NOR- bearingchromosome pair in the karyotype of P.poecilogaster andin karyotype II of Scythrophrys .Aconspicuous heteromorphismunrelated to the sex determiningmechanism was alsoobserved and probably arose froma pericentricinversion.

Introduction The genus Paratelmatobius Lutzand Carvalho comprises Ž vespecies placed in two groups(Pombal Jr .andHaddad, 1999). The P. cardosoi groupincludes P. cardosoi and P.mantiqueira while the P. lutzii groupcontains P. lutzii, P.poecilogaster ,andprobably P. gaigeae. All Paratelmatobius speciesare endemic to Braziland P.mantiqueira , P. lutzii and P. gaigeae were consideredas missingspecies by thoseauthors.

© KoninklijkeBrill NV ,Leiden,2003 Amphibia-Reptilia24: 47-55 Alsoavailable online - www.brill.nl 48 LucianaB. Lourenço, PauloC.A. Garcia, ShirleiM. Recco-Pimentel

Comparativecytogenetic analyses of livingspecies of Paratelmatobius (Lourenço etal., 2000), P. lutzii (De Luccaet al., 1974) and genus Scythrophrys (Lourenço etal.,in press) haveshown several similarities between these genera and have allowed the evolutionary studyof somechromosomal characters in this group. Inthe present paper, we describethe karyotype of anewspecies of Paratelmatobius in the P. cardosoi groupand reassert the evolutionary hypothesis proposed in our previous studies of Paratelmatobius and Scythrophrys (Lourenço etal., 2000, in press). An inter- estingcase of an autossomal inversion is also described. This new species, here called Paratelmatobius sp. (aff. cardosoi),was foundin theSerra doMarin the State of Paraná, Brazil,and differs from P. cardosoi byitsadvertisement call.

Materialsand methods

Sixmales andtwo females of Paratelmatobius sp. (aff. cardosoi)collectedfrom Piraquara, Paraná , Brazil, in October1999 were studiedcytogenetically. Chromosome preparations were obtainedfrom intestinal and testes cell suspensions,as describedby Schmid (1978) and Schmid et al.(1979). Conventional staining with a 10% Giemsa solution,the C-banding technique (King, 1980) and the Ag-NOR method(Howell and Black, 1980) were done.Chromosomes were classiŽed according to Green andSessions (1991). All the specimens were deposited inCélio F.B. Haddad collection (CFBH), Departamento deZoologia,Universidade Estadual Paulista, Rio Claro, SP, Brasil.

Results

Thediploid complement of Paratelmatobius sp. (aff. cardosoi)consistsof 24 chromo- somes,with six pairs of metacentricchromosomes and sixof submetacentricchromosomes (Žgs 1-3,5; table1). In all the specimens analyzed, a conspicuoussecondary constriction was observedin the short arm ofhomologue9 inGiemsa-stainedmetaphases (Ž g. 1)and was identiŽed asan NOR sitein silver-stainedpreparations (Ž g. 2).Additionally, a smaller secondaryconstriction was observedinterstitially in the long arm ofchromosome 5 in moredecondensed metaphases (Ž g. 1a). All the centromeric regions of the Paratelmato- bius sp. (aff. cardosoi)karyotypeshowed a smallamount of constitutiveheterochromatin (Žg. 3).Non-centromeric heterochromatic bands were easilyvisualized in pairs1, 3,5,and adjacentto the NOR inpair9 (Žg. 3).In some metaphases, a smalltelomeric C-band was observedin theshort arm ofpair8 (Žg. 3).Considering the small size of thisband, it was probablynot detected in allthe metaphases because of technicalproblems in performing C-banding. Aninteresting dimorphism was notedfor pair1 inoneof the females. In this specimen, pair1 was deeplyheteromorphic in all metaphases analyzed (Ž gs 1-3). While one homologue(1a) was asubmetacentricchromosome with an arm ratioof 1.72, the other homologue(1b) could almost be considered a subtelocentricchromosome since its arm ratiowas 2.88and, according to theclassiŽ cation system of Greenand Sessions (1991), the Cytogeneticsof anew species of Paratelmatobiuscardosoi 49

Figure 1. Paratelmatobius sp. (aff. cardosoi)karyotype.In a,aspecimen witha homomorphicpair 1. Theinset showsa secondaryconstriction in pair 5 (arrow).In b,aspecimen witha heteromorphicpair 1. The inset shows anotherpair 1 ofthesame specimen. Notethe variable size ofmorph1b relative to1a. The secondary constriction oftheNOR inpair 9 is alsoshown in a and b (arrow). arm ratioof asubtelocentricchromosome is 3.01-7.00.These morphs of pair1 alsodiffered intheir C-banding pattern. Thus, while morph 1a showed interstitial heterochromatin inits long arm andnearly all its short arm was heterochromatic,in morph 1b, the longarm was heterochromaticand no heterochromatin was detectedin the short arm (Žg. 3). Intercellular variation was observedin the size of chromosome1b relative to its homologouschromosome 1a. Whilein some metaphases morphs 1a and1b showedalmost thesame size, in others, chromosome 1b was clearlysmaller than 1a (Žg. 1b). Themeiotic analysis of four males showed that, in 92% of 119 diakineses, the homomorphicpair 1 hada clearrod-like conŽ guration, probably the result of aterminal associationof thelong arms (Žg. 4). Such an association was particularlyidentiŽ able in 50 LucianaB. Lourenço, PauloC.A. Garcia, ShirleiM. Recco-Pimentel

Figure 2. Silver-stainedkaryotype. Note the active NOR inpair 9. The inset shows the heteromorphic pair 1 in onefemale specimen.

Figure 3. C-bandedkaryotype. The insets show a homomorphicpair 1, anda pair8 witha clear telomeric C-band intheshort arm. Thesmall interstitialbands of pair5 are indicatedby arrows.

Table 1. Morphometricdata basedon measurements from39 metaphases of Paratelmatobius sp. (aff. cardosoi).

Chromosomes 11b2 34567891011 12 r.l.14.44 11.96 11.47 10.66 10.42 9.40 8.69 6.98 5.72 5.54 5.32 4.94 4.77 a.r.1.72 2.88 1.58 2.30 1.96 1.25 1.15 1.88 1.91 1.08 1.26 1.71 1.32 c.c. sm sm m sm sm m m sm sm m m sm m r.l.:relative length(%); a.r.: arm ratio;c.c.: centromeric classiŽcation; m: metacentric; sm: submetacentric; st:subtelocentric.

C-bandeddiakinesis in whichthe heterochromatic markers of chromosome1 (almostthe wholearm andan interstitialband in thelong arm) couldbe seen(Ž g. 4b). Cytogeneticsof anew species of Paratelmatobiuscardosoi 51

Figure 4. Giemsa-stained ( a)andC-banded ( b)diakinesesfrom a specimen withhomomorphic pair 1. Note the rod-likeconŽ guration of this pair and the position of the short arms. Theterminal associationof the long arms is indicated(arrow).

Discussion InterspeciŽc analysis Thediploid number of Paratelmatobius sp. (aff. cardosoi)isthe same as in P. cardosoi, P.poecilogaster (Lourenço etal., 2000), and P. lutzii (De Luccaet al., 1974). The morphologyof severalchromosome pairs is also very similar among these species (Ž g. 5). The Paratelmatobius sp. (aff. cardosoi)karyotypediffers from thoseof P.poecilogaster and P. lutzii especiallyin pair 6, which is metacentric in Paratelmatobius sp. (aff. cardosoi)andsubtelocentric in P.poecilogaster and P. lutzii.Onthe other hand, pair 4 of Paratelmatobius sp. (aff. cardosoi)differs morphologicallyfrom thatpair 4 of P. cardosoi. Thelocation of the NOR alsodistinguishes Paratelmatobius sp. (aff. cardosoi) from P. cardosoi, since in P. cardosoi pair7 isthe NOR-bearing chromosome pair. Apart from theheterochromatic bands of pair 4 of P. cardosoi,whichare characteristic of this species,the non-centromeric bands of pairs 7 and9 of P. cardosoi arealso not found in the Paratelmatobius sp. (aff. cardosoi)karyotype.The interstitial band in pair 3 and thetelomeric band in pair 8 thatare present in Paratelmatobius sp. (aff. cardosoi) do not occur in P. cardosoi.Additionally,while in P. cardosoi theshort arm ofchromosome1 has aninterstitial C-band, nearly all the short arm ofchromosome1a in Paratelmatobius sp. (aff. cardosoi)isheterochromatic. All of these differences provide a clearkaryological distinctionbetween the specimens studied here and P. cardosoi,andcorroborate that Paratelmatobius sp. (aff. cardosoi)isan undescribed species. Bothkaryotypes have interstitial heterochromatin in the long arm ofpair1 (morph1a in Paratelmatobius sp.),although the amount of this heterochromatin is greater in Paratel- matobius sp. (aff. cardosoi) than in P. cardosoi.Inaddition, pair 5 of Paratelmatobius sp. (aff. cardosoi)stronglyresembles pair 5 (especiallymorph 5b) of P. cardosoi. The 52 LucianaB. Lourenço, PauloC.A. Garcia, ShirleiM. Recco-Pimentel

Figure 5. Idiogramsof the karyotypes of Paratelmatobius sp. (aff. cardosoi) (a), P. cardosoi (b; based on Lourenço et al.,2000), P.poecilogaster (c;basedon Lourenço et al.,2000), group I andII of Scythrophrys (d and e;basedon Lourenço et al.,2001, and manuscript in preparation). Solid blocks: dark C-bands. Gray blocks:faint C-bands.Open regions: secondary constrictions. Checkered circles: NORs. similarmorphology and C-banding pattern in both arms ofthese chromosomes is a fur- therevidence of their homeology. The long arm ofboth these chromosomes also has a secondaryconstriction. Interstitial heterochromatin in thelong arm ofchromosome1 and theC-banding pattern of chromosome5 mentionedabove were notfound in Paratelmato- biuspoecilogaster (Lourenço etal.,2000). Also, such characteristics were notseen in the genus Scythrophrys (Lourenço etal., in press;Ž g.5),which is closelyrelated to Paratel- matobius (Heyer,1975; Garcia, 1996; Lourenç o etal., in press).Although one specimen of Scythrophrys showeda faintC-band in the long arm ofonehomologue of pair1, itdif- feredfrom theinterstitial heterochromatin in the long arm ofpair 1 of P. cardosoi and Paratelmatobius sp. (aff. cardosoi)alsobecause it was associatedwith a NOR inthat Scythrophrys .So,we concludethat those characteristics of pairs 1 and5 of P. cardosoi and Paratelmatobius sp. (aff. cardosoi)canbe synapomorphic characters that assemble thesespecies. Cytogeneticsof anew species of Paratelmatobiuscardosoi 53

TheNOR-bearing chromosome pair of Paratelmatobius sp. (aff. cardosoi)isprobably homeologousto pair10 of P.poecilogaster andto pair 10 ofkaryotypeII of Scythrophrys , bothof which carry a NOR (Lourenço etal., 2000, in press). The classiŽ cation of this pairas 9inthe Paratelmatobius sp. (aff. cardosoi)karyotypeand not as pair 10 (as inthe otherkaryotypes) is probablybecause of aconspicuousC-band between the NOR andthe telomereof theshortarm ofthischromosome, which is absentin pair 10 of P.poecilogaster andof karyotypeII in Scythrophrys .Thisheterochromatic band is apparently responsible for thegreater size of the short arm ofthat chromosome in Paratelmatobius sp. (aff. cardosoi). Theintrachromosomal location of theNOR inpair 10 ofkaryotypeII of Scythrophrys differs slightlyfrom thatin pair 10 of P.poecilogaster and pair 9 of Paratelmatobius sp. (aff. cardosoi). In Scythrophrys ,theNOR ismoreterminal, while in Paratelmatobius sp. (aff. cardosoi) and in P.poecilogaster itis proximal to the centromere. This variation mayhave been generated by a hypotheticalparacentric inversion. Anyway, the location ofa NOR inasmallmetacentric/ submetacentricchromosome is anancestral characteristic relativeto the other NOR sitepresent in Paratelmatobius (inpairs 7 and8; Lourenço etal., 2000) and Scythrophrys (inpairs 1 and5),as proposedelsewhere (Lourenç o etal.,in press). Thus,the locationof theNOR inpair 10 of P.poecilogaster andin thehomeologous pair 9 of Paratelmatobius sp. (aff. cardosoi)cannotbe used to group these species separately from P. cardosoi,whichdoes not have this NOR. Thecomparison between the Paratelmatobius sp. (aff. cardosoi) and Scythrophrys karyotypesalso showed that, as in Paratelmatobius sp. (aff. cardosoi),karyotypeI of Scythrophrys hasa telomericC-band in the short arm ofpair8, whereasin karyotype II theentire arm ofthischromosome is heterochromatic.If thepresence of heterochromatin inthis region is considered a homeologouscharacteristic, then the common ancestor of Paratelmatobius and Scythrophrys musthave this heterochromatin before being lost in P.poecilogaster and P. cardosoi.Analternative hypothesis is that the telomeric C- bandin pair 8 of Paratelmatobius sp. (aff. cardosoi)isnot homeologous to the non- centromericheterochromatin of the Scythrophrys karyotypesso that these heterochromatin aroseindependently in these genera. Overall,these cytogenetic data agree with the two groups of Paratelmatobius proposed byPombal Jr. andHaddad (1999), and allow the inclusion of Paratelmatobius sp. (aff. cardosoi) in the P. cardosoi group.

IntraspeciŽc variationin chromosome pair 1

Of theeight specimens of Paratelmatobius sp. (aff. cardosoi)examined,one female showedheteromorphism in pair 1 thatwas easilydetected in all preparations. While chromosome1a of Paratelmatobius sp. (aff. cardosoi)was similarin morphology to chromosome1 ofthe other Paratelmatobius and Scythrophrys species(see above),the unusualmorph 1b ofthekaryotype described here was observedonly in Paratelmatobius 54 LucianaB. Lourenço, PauloC.A. Garcia, ShirleiM. Recco-Pimentel sp. (aff. cardosoi).Onepossible mechanism to explainthe appearance of morph1b could bea pericentricinversion in morph 1a. This hypothesis is supported by the variation in thearm ratiosof themorphs and by theirdistinct patterns of heterochromatindistribution. However,this mechanism cannot fully explain the decrease in theamount of euchromatin inmorph 1b compared to 1a, particularly since this decrease may have arisen from a deletionor heterochromatinization. Thein uence of thisheteromorphism on themeiotic pairing/ crossingof pair1 was not evaluatedbecause this phenomenon was seenin only one female. However, the meiotic analysisof homomorphicpairs of chromosome1 suggestedthat the large amount of het- erochromatinpresent impaired correct pairing/ crossingand prevented the ring conŽ gura- tion.Such an in uence of heterochromatin has been reported for severalorganisms (see reviewby John,1988). Theheteromorphism in pair 1 of Paratelmatobius sp. (aff. cardosoi)was notrelated tothe sex determining mechanism since in another female examined the morphs were homomorphic,as was thecase for males.

Acknowledgements. Thiswork was supportedby the Brazilian agencyFAPESP (Fundaç ão deAmparo à Pesquisado Estado de Sã o Paulo,grants n ± 97/00459-9and n ± 98/06087-9).

References

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Received: April2, 2002.Accepted: July 12,2002.