Penil morphology in three species of Brzilin Tuco-tucos, Ctenomys torqutus, C. minutus, nd C. flmrioni (Rodenti: Ctenomyide) Roch-Bros, O. *, Bernrdo, JSL.,, Loguercio, MFC., Freits, TRO. c, Sntos-Mllet, JR. d nd Bidu, CJ. Lortório de Zoologi de Verterdos (Tetrpod) LAZOVERTE, Deprtmento de Zoologi, Instituto de Biologi Roerto Alcântr Gomes, Universidde do Estdo do Rio de Jneiro UERJ, Rio de Jneiro, RJ, Brzil Lortório de Biologi e Prsitologi de Mmíferos Silvestres Reservtórios, Instituto Oswldo Cruz FIOCRUZ, Pvilhão Arthur Neiv, sl 14, Rio de Jneiro, RJ, Brzil c Deprtmento de Genétic, Instituto de Biociêncis, Universidde Federl do Rio Grnde do Sul UFRGS, Porto Alegre, RS, Brzil d Lortório de Trnsmissores de Leishmnioses, Setor de Entomologi Médic e Forense SEMEF, Instituto Oswldo Cruz FIOCRUZ, Pvilhão Crlos Chgs, 5º ndr, sl 16, Rio de Jneiro, RJ, Brzil *e-mil: or-ros@hotmil.com Received Jnury 31, 2012 Accepted My 30, 2012 Distriuted Ferury 28, 2013 (With 5 figures) Astrct The present study nlyses the glns penis nd culum morphology of three Brzilin tuco-tucos, Ctenomys torqutus Lichtenstein, 1830, Ctenomys minutus Nehring, 1887 nd Ctenomys flmrioni Trvi, 1981, in order to identify possile vritions nd understnd some more out this txonomiclly complex group. We used fixed penis from 15 previously listed dult specimens. For more detiled culum nlysis, the penis underwent dissection nd diphnistion, wheres to nlyse the glns penis surfce we used Scnning Electron Microscopy (SEM). Results showed striking differences in culum morphology mong the three species. While C. minutus hve prticulr V-shped proximl culum tip, C. flmrioni culum is thin throughout the shft with rounded proximl nd distl tips. Ctenomys torqutus hve shorter nd lrger culum, similr to wht hs previously een descried for the species. Glns penis surfce microstructure nlyses lso reveled inter-specific differences, with penil spines vrying in shpe, size nd, especilly density. Although C. torqutus hs reltively smll penis, it hs the lrgest penil spine density, which suggests more complex penil ornmenttion in this species. Keywords: Ctenomys, Glns penis, morphology, Bculum, scnning electron microscopy. Morfologi penin de três espécies de tuco-tuco do Brsil Ctenomys torqutus, C. minutus e C. flmrioni (Rodenti: Ctenomyide) Resumo O presente estudo nlis morfologi do glns penis e do culum de três tuco-tucos do Brzil Ctenomys torqutus Lichtenstein, 1830, Ctenomys minutus Nehring, 1887, e Ctenomys flmrioni Trvi, 1981 com finlidde de identificr possíveis vrições e elucidr mis sore grupo txonômico complexo. Form usdos pênis fixdos de 15 indivíduos dultos ds espécies listds nteriormente. Pr um nálise mis detlhd do culum, o pênis foi sumetido dissecções e difnizção, enqunto que, pr nálise d superfície do glns penis, foi usd técnic de microscopi eletrônic de vrredur (MEV). Os resultdos exiirm significtivs diferençs n morfologi do culum entre s três espécies. Enqunto C. minutus present um culum com um pont em form de V crcterístic, o culum de C. flmrioni é fino o longo do osso, com s ponts proximl e distl rredondds. Ctenomys torqutus present um culum mis curto e lrgo, similr o descrito nteriormente pr espécie. Análises d microestrutur n superfície do glns penis revelrm diferençs interespecífics, com os espinhos peninos vrindo em form, tmnho e, especilmente, densidde. Apesr de C. torqutus ter um pênis reltivmente pequeno, present mior densidde de espinhos peninos, o que sugere um complex ornmentção penin nest espécie. Plvrs-chve: Ctenomys, Glns penis, morfologi, Bculum, microscopi eletrônic de vrredur. 201
Roch-Bros, O. et l. 1. Introduction Structures such s penis nd culum re n importnt prt of the mmmlin reproductive system nd they hve een suject of severl scientific studies (Ptterson nd Theler Junior, 1982; Brdley nd Schmidly, 1987; Simson et l., 1995). Penis morphology hs lrgely een used in txonomicl nd systemtic studies (Brdley nd Schmidly, 1987; Brdley et l., 1989), s in other studies focused on etter understnding copultion nd sexul selection systems (Altun nd Less, 1985; Simson et l., 1995; Prg et l., 2006). The culum or os penis is one locted within the mle penis nd it is the most diverse of ones (Romer, 1962; Ptterson nd Theler, 1982). It ws first descried y Thoms (1915) nd it is found in the mmmlin orders Rodenti, Chiropter, Insectivor, Crnivor nd Primtes (Hmilton Junior, 1949). If the culum hs n dptive function is still in dete, ut three min hypotheses for its function hve een proposed so fr: firstly, to fcilitte intromission through extr rigidity nd support to overcome vginl friction; secondly, to llow prolonged intromission to ssist sperm trnsport; nd lst, to provide extr rigidity to stimulte femle ovultion (Lrivière nd Ferguson, 2002). Among the rodents Octodontide, the genus Ctenomys comprises fossoril herivorous rodents (Nowk, 1999). This genus hs huge criotipic diversity, including species vrying from 2n = 10 to 2n = 70, which reflects in complex evolutionry history, with still conflicting phylogenetic reltions mong species (Vitullo et l., 1988; Less nd Cook, 1998; Contrers nd Bidu, 1999; Mscheretti et l., 2000; Giménez et l., 2002; Gvs nd Freits, 2003, 2004; Bidu, 2006). Although, some evidence suggests tht increse in penis ornmenttion nd decrese in culum size re relted to solitry ehviour (Prg et l., 2006), studies in Ctenomys penis indicte intr- nd inter-specific vritions such s in the intromitent sc nd even in the culum (Altun nd Less, 1985; Less nd Cook, 1989; Contrers et l., 1993). Herein, we im to descrie culum nd glns penis morphology in three species of Ctenomys genus found in the southern Brzilin territory: Ctenomys torqutus Lichtenstein, 1830, Ctenomys minutus Nehring, 1887 nd Ctenomys flmrioni Trvi, 1981, in order to elucidte some more out the morphology of these inconspicuous species. 2. Mteril nd Methods Overll, fifteen penises of dult specimens were exmined. They were seprted from the ody t the se, nd then preserved in 10% formlin nd stored in the refrigertor. All specimens were collected in the stte of Rio Grnde do Sul nd re deposited in the mmml collection of the Universidde Federl do Rio Grnde do Sul UFRGS, Genetics Deprtment. Numers of collection nd loclities re s follows: C. minutus: TRO022 - Lgo Emo, TRO391 - Cpivri, TRO410, TRO416 nd TRO418 - Lgo dos Brros, TRO435 - Osório nd TRO442 - Pitngueir; C. torqutus: TRO086 - Estção Ecológic do Tim, TRO373 - Brro Duro nd SN - no dt; C. flmrioni: TRO029 nd TRO031 - Pri d Cidreir, TRO051 - Pri de Cpão de novo, NC190 - no dt nd TRO049 - Lgo dos Brros. Four C. minutus specimens nd two C. flmrioni were nlysed using the diphnistion technique. We used the Dingerkus nd Uhler (1977) protocol with the following modifictions: the enzimtic tretment consisted of immersing the mteril in pncretin solution, insted of in trypsin solution; t the lcoholicl series, the mteril ws treted pssing from 30% glicerine solution to 100% glicerine solution (rupt osmotic shock). One penis of ech species ws hydrted nd then dissected ventrlly to remove the culum. Bcul were photogrphed using Sony DSC S75 digitl cmer. To study glns penis surfce microstructure, two specimens of ech species were used. The mteril ws fixed in 2.5% glutrldehyde in sodium ccodylte uffer 0.1 M, ph 7.2 for 1 hour t room temperture. Soon fter tht, cut ws done on the glns penis sis to seprte the glns from the penis ody. Lter, it ws wshed twice in uffer nd post fixed in 1% osmium tetroxide in sodium ccodylte uffer 0.1 M, ph 7.2 for 1 hour t room temperture in the drk. The mteril ws then dehydrted in n incresing series of cetone (7.5%, 15%, 30%, 50%, 70%, 90% nd 100%) nd sujected to the dry method y superdry CO 2 criticl point in Blzers devices. Then, the smples were put on metl rckets, using doule-side tpes nd were covered y gold in n evportion system clled sputtering, in which gold is removed from mssive electrode y ion omrdment in high vcuum, using Blzers devices. Finlly, the smples were oserved in scnning electron microscope Jeol JSM 6390LV (Akishim, Tokyo, Jpn), t the Pltform de Microspcopi Eletrônic do Instituto Oswldo Cruz, t 15 Kv. All imges were cptured directly in computer using the softwre Sem Aforce, which ws lso used to mesure dorsl nd ventrl glns penis spines length. 3. Results In ll species, culum ws thin nd flt structure, locted in the glns penis dorsl region s shown y diphnistion nd y dissections (Figures 1 nd 2). But ech species showed unique morphology. The C. minutus culum hs reltively V-shped proximl tip. Its midlength is slightly nrrower thn oth tips. The distl tip is round with sutle concvity in the ventrl surfce. Length ws 9.34 mm (Figure 2, ). Menwhile, C. flmrioni culum is thin throughout the shft. Both proximl nd distl tips re round nd its midlength is slightly constricted. It lso hs slightly concve ventrl surfce. Length ws 10.40 mm (Figure 2c, d). And C. torqutus culum is reltively shorter nd lrger thn in the other species. It hs round proximl nd distl tips. Its midlength is similr in width to oth tips. There ws no concvity in the ventrl region. Length is 6.41 m (Figure 2e, f). 202
Penil Morphology of Tuco-tucos Figure 1. Diphnised penis of Ctenomys minutus, on lterl () nd dorsl () views, showing the culum position (drk grey). Both scles represent 0.5 cm. The glns penis surfce microstructure nlyses through Scnning Electron Microscopy (SEM) reveled inter-specific differences. Smll spines vried in shpe, size nd density mong the three species, s well s in ventrl nd dorsl regions. All specimens hd proximl-directed spines, with their ses lrger thn their pices. In most cses, spines were shrp nd internlly curved. In C. minutus, glns tips re recovered y spines, excluding the edge of the urethr (Figure 3). In the dorsl surfce, spines re very shrp; most of them with single tip, ut some doule-tipped (Figure 3-d). In the ventrl surfce, ner the glns, spines hd fltter shpe, very close to the surfce nd with two or three tips (Figure 3e f). In the medin region (ventrl view), ner the glns se, spines hd shrper shpe, like the dorsl spines (Figure 3g-h). Density ws out 12 spines/mm2. Men spine length ws: 245.14 ± 49.81 µm (n = 41). The spines oserved in C. flmrioni specimens hd different shpe. In the dorsl region, they hve shrp shpe nd sometimes utton shpe, nd reltively smll density (Figure 4-d), while in the ventrl region, spines hd round shpe, flt nd with two or three tips (Figure 4e-h). Density ws out 9.5 spines/mm2. Men length of the spines ws 226.26 ± 32.42 µm (n = 41). Ctenomys torqutus spines cover the entire glns tip, except t the urethr entry edges (Figure 5). Dorsl surfce hs shrp spines, with convex shpes (Figure 5 d), wheres ventrl surfce hs different shped spines: shorter, rounded nd concve (Figure 5e-h). Density ws out 17.5 spines/mm2. Men length of the spines ws 189 ± 23.9 µm (n = 41). 4. Discussion Our results confirmed the diverse penil morphology previously descried in tuco-tucos y Less nd Cook (1989) nd Blontin et l. (1996). The three species hve single-tipped cul differing from the triloed ones descried for Ctenomys rionegrensis Lngguth nd Aell, 1970 in Uruguy nd Argentin (Reig et l., 1966; Altun nd Less, 1985). Although ll cul re locted in the glns penis dorsl region s commonly found in others rodents (Simson et l., 1995), mny striking differences re evident when compring the three studied species. The C. minutus pddle-shped culum hs similr morphology to the one descried for the Uruguyn species Ctenomys personi Less nd Lngguth, 1983 (Altun nd Less, 1985), even though the former hs more pronounced V-shped proximl tip. We lso oserved sutle concvity in the ventrl region of the culum s previously descried y Contrers et l. (1993) to Octodontide rodents. 203
Roch-Bros, O. et l. c d e f Figure 2. Bcul of C. minutus (, ), C. flmrioni (c, d) nd C. torqutus (e, f) in dorsl nd ventrl views, respectively. The scles represent 0.5 cm. Nevertheless, Reig et l. (1966) descried clerly different culum for C. minutus from Snto Antonio d Ptrulh, south Brzil. The Snto Antonio de Ptrulh smple hs more rounded proximl tip nd nrrower midlength nd distl pex, wheres our smple hs very pronounced pddle-shped proximl tip nd slight constriction in the midlength. According to Freits (2001), t this Brzilin region it is lso found Ctenomys lmi Freits, 2001, recently descried species similr to C. minutus. Proly 204 Reig et l. (1966) nlysed C. lmi culum insted of C. minutus culum, ut further investigtions re needed to corroorte this ffirmtion. As expected C. torqutus culum morphology ws very similr to the pddle-shped cul descried for C. torqutus from Médnos in Argentin (Reig et l., 1966). Both re very lrge throughout the shft compred to the ones from the other Ctenomys species. Bculum from C. flmrioni is first descried in the present study
Penil Morphology of Tuco-tucos c d e f g h Figure 3. Surfce of the glns penis of Ctenomys minutus showing smll spines. Oserve tht the spines re proximl directed. Dorsl view, tip of the glns, n increse of 33 (). Dorsl view, medin region, n increse of 180 (). Dorsl view, medin region, n increse of 33 (c). Dorsl view, medin region, n increse of 80 (d). Ventrl view, tip of glns, n increse of 30. The rrow indicte doule-tipped spine (e). Ventrl view, tip of glns, n increse of 270. You cn oserve triple-tipped spine in detil (f). Ventrl view, medin region, n increse of 90 (g). Ventrl view, medin region, n increse of 180 (h). 205
Roch-Bros, O. et l. c d e f g h Figure 4. Surfce of the glns penis of C. flmrioni showing spines. Oserve tht the spines re proximl directed. Dorsl view, tip of the glns, n increse of 30 (). Dorsl view, medin region, n increse of 120 (). Dorsl view, medin region, n increse of 120 (c). Dorsl view, medin region, n increse of 90 (d). Ventrl view, tip of glns, n increse of 30 (e). Ventrl view, medin region, n increse of 85 (f). Ventrl view, medin region, n increse of 140. Ventrl view, medin region, n increse of 250 (h). 206
Penil Morphology of Tuco-tucos c d e f g h Figure 5. Surfce of the glns penis of C. torqutus showing spines. Dorsl view, tip of the glns, n increse of 30 (). Dorsl view, medin region, n increse of 30 (). Dorsl view, medin region, n increse of 190 (c). Dorsl view, medin region, n increse of 95 (d). Ventrl view, tip of glns, n increse of 30 (e). Ventrl view, medin region, n increse of 75 (f). Ventrl view, medin region, n increse of 230 (g). Ventrl view, medin region, n increse of 230 (h). 207
Roch-Bros, O. et l. nd it clerly differs from ll previous morpho-types. It hs oth proximl nd distl tips quite round nd its midlength slightly constricted. Similrly to C. minutus, it is lso sutly concve t its ventrl region. Bcul length lso vried mong the three species. While C. minutus nd C. flmrioni hd reltively similr lengths, C. torqutus hd much smller culum. Bculr length is usully considered to hve llometric trends lthough it my vry independently from hed nd ody length (Less nd Cook, 1989). In contrst, in Ctenomys, the lrger species (C. torqutus) hs the smller culum. According to Long nd Frnk (1968), these morphologicl vritions coupled with genetics my e closely relted to reproductive isoltion nd specition phenomen. Our results reveled tht in the three nlysed species, glns penis surfce is covered y smll spines, whose shpe nd density seem to e species-specific. These smll spines re very similr to penile spines found in others rodent groups (Brdley nd Schmidly, 1987; Prg et l., 2006). The penile spines of the three species nlysed hve lrger ses thn tips. Some of them re shrp nd in ll three species they re fcing the penile se (proximl directed) s seen in other rodent fmilies: Bthyergide, which re lso fossoril rodents like Ctenomys nd Peromyscus from the Cricetide fmily (Brdley nd Schmidly, 1987; Brdley et l., 1989; Prg et l., 2006). Nevertheless, the numer of spines per re vried in ech species. Ctenomys torqutus despite hving reltively smller penis thn the others, showed the lrgest density, while C. flmrioni nd C. minutus hd nerly the sme numer of spines per re. According to Prg et l. (2006), the complexity of rodent penis morphology, specificlly the surfce structures, is closely relted to the level of sociility. The more socil the niml is the less is the complexity of spines. Tht is, more solitry nimls hve higher level of complexity of penile structures, s is the cse of Ctenomys. According to Altun nd Less (1985), these microstructures would hve greter dptive vlue precisely in nimls with solitry lifestyle. Perhps this suggests more solitry socil hit in C. torqutus thn in C. minutus nd C. flmrioni; nonetheless, we must tke into ccount tht interctions etween socil nd reproductive tuco-tucos re much more diverse nd complex thn expected for common solitry nd territoril species (Zenuto et l., 2002). Spines nd other penis ornmenttions cn hve vrious functions such s, for exmple, induce ovultion or ct s encourgement to mle cceptnce through vginl stimultion, or cn even ensure genitl lock, holding the wlls of the vgin during intercourse (Altun nd Less, 1985). In species with high sperm competition, these spines my lso hve the function of removing the semen deposited in the vgin of the femle from previous mtings (Prg et l., 2006). 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