Reproductive biology of thesouthern Brazilianpitviper Bothrops neuwiedi pubescens (Serpentes, Viperidae)

Marília T. Hartmann 1,OtavioA.V .Marques 2,SelmaM. Almeida-Santos 2

1 Departamento deZoologia,Instituto de Biociê ncias, Caixa Postal199, UNESP ,13506-900Rio Claro, Sã o Paulo,Brasil e-mail: [email protected] 2 Laboratório de Herpetologia, Instituto Butantan, A v.Vital Brazil 1500,05535-900, Sã o Paulo,SP ,Brasil e-mail: [email protected] e-mail: [email protected]

Abstract. Dissectionof 286 specimens ofthe Bothropsneuwiedi pubescens ,combinedwith data oncaptive individuals,provided information on the reproductive biology of this viperid snake from southern Brazil. Females attainedlarger bodysizes thanmales, andreproduction was seasonal withmating taking place in autumnwhen males were more frequentlyencountered. Vitellogenesis occurred from summer tospring (January- September),sperm storageduring autumn and winter (May-September), ovulation and fertilization in early spring (September),embryonic development during middle spring and summer (October-March),and parturition in the summer (January-March).Embryonic development was estimated tolast fromthree toŽ vemonths, a shortertime thanwas previouslyreported. The number of offspringof Bothropsneuwiedi pubescens bornin one litter can varyfrom four to 25 ( x 11/.Fecundityis correlated withmaternal bodysize. Neonates measure 17-25cm SVL.Inferred growth rate ofjuveniles was low(10 mm/ monthin Ž rst year),with males attainingsexual maturity at about16 months, but maturity is delayedin females forat least twoadditional years.

Introduction Thepitvipers of thegenus Bothrops arewidely distributed in Central and South America (Campbelland Lamar, 1989). Reproductive data are available for onlya few ofthe morethan 20 species occurring in Brazil, such as Bothropsjararaca (Sazima,1992; Janeiro-Cinquiniet al., 1993; Almeida-Santos and Orsi, 2002), B. moojeni (Leloup,1975), B. atrox (Hogeand Federsoni Jr., 1976) and B. neuwiedi (Alveset al., 1998, 2000; Valdujoet al.,2002). Bothropsneuwiedi pubescens inhabitsdeciduous forests of southern Braziland Uruguay (Almeida, 1999). This species is a conspicuouscomponent of the southernBrazilian snake fauna (Almeida, 1999; Cechin, 1999), and is responsible for many humansnakebites in the region (Santos-Costa, 1999). Apart from scatteredand anecdotal

© KoninklijkeBrill NV ,Leiden,2004 Amphibia-Reptilia25: 77-85 Alsoavailable online - www.brill.nl 78 M.T.Hartmann, O.A.V .Marques,S.M. Almeida-Santos reports(Melgarejo, 1978), scant information has beenpublishedregarding the reproductive ecologyof this species. W eprovidedata on body size, reproductive timing, clutch size, neonatesize, and infer about the growth and age at maturity of thistaxon.

Materialsand methods

Thepresent study is based on the dissection of 286 specimens of Bothropsneuwiedi pubescens from collectionsof theMuseu de Ciências da Pontifí cia Universidade Cató lica doRio Grande do Sul (MCP), Universidade Federal de Santa Maria (ZUFSM) inRio Grandedo Sul,and of theInstituto Butantan (IB) inSã o Paulo.The specimens examined originatedfrom RioGrande do Sulstate (27 to 32 S, 50 to 56 W), southernBrazil. This areais covered mainly by pampas grassland, deciduous forests, disturbed and cultivated areas(Lindman and Ferri, 1974;Vieira, 1984). The climate of thearea is highly seasonal, withhigher temperatures from springto theonsetof autumnand lower temperatures in the winterand most of the autumn. Rainfall is well distributed over most of theyear (1600 mm) (Vieira,1984; Melhem-Adas, 1996) (Ž g. 1). Thefollowing data were takenfrom eachspecimen: (1) snout-ventlength (SVL); (2) sex; (3) reproductivematurity (females were consideredmature if theyhad embryosin oviducts orvitellogenic follicles; males were consideredmature if they had enlarged testes or opaquedeferent ducts indicating the presence of sperm,see Shine, 1980); (4) diameterof thelargest ovarian follicle and the number and length (SVL) ofembryos.Degree of sexual sizedimorphism (SSD) was 1—(meanadult SL Vofthelarger sex/ meanadult SL Vof thesmaller sex) (see Shine,1994). W einferredgrowth rates from seasonaldistributions of bodysizes, a methodthat relies on identiŽcation of ageclasses based on bodysizes (Shine, 1978a).Data on captive snakes were obtainedfrom registersof MCP .Seasonalactivity

Figure 1. Annualdistribution of mean monthly( C)andrainfall (mm) inRio Grande do Sul State from1961 to1990. Lines represent mean monthlytemperature andbars denoterainfall. Data fromInstituto Nacional de Metereologia,Brazil. Reproductivebiology of Bothropsneuwiedi pubescens 79 isbased on collection dates of MCP,ZUFSM andIB specimens(see similarmethods in Marqueset al.,2001).

Results

Bodysize. Bothrops neuwiedi pubescens isa mediumsized snake with adult SVL averaging64.6 cm ( s 14:2, n 188).Mature females averaged 76.2 cm SVL ( s 10:9, n 84,range 62-111.5 cm), andmature males averaged 54.9 cm SVL ( s 8:6, n 104, range38-83). The difference in bodysize between sexes was highlysigniŽ cant ( t 14:9, df 186, P < 0:001/.Thedegree of sexualsex dimorphism (SSD) was 0.39.

Reproductivecycles. Enlargedfollicles were presentthroughout the year (Ž g. 2).Ovula- tionoccurred in earlyspring, and oviductal embryos were foundfrom Octoberto March (Žg. 2).From Octoberto December the embryos were inan early stage of development, andfrom Januaryto Marchwere fully-developed(Ž g. 2).Thus, we calculatedthat the ges- tationperiod extended from threeto Ž vemonths.The data from 11captivegravid females arein agreement with those from dissectedspecimens, since parturition was recordedin January (n 3),February ( n 3)andMarch ( n 5).One captive snake collected in Aprilof 1996and maintained alone gave birth on 16January of 1998(data from MCP), providingclear evidence of sperm retention.

Mating. Courtshipof B. n. pubescens was observedin theŽ eldon dayMarch 1997. The female(ZUFSM 1293)measured 850 mm SVL andthe male (ZUFSM 1294)measured 595mm SVL. Bothindividuals were mature,but the female had no vitellogenicfollicles. Courtshipand matingin captivitywas recordedin July and August(G. Pontes,pers. com.).

Figure 2. Seasonalvariation in diameters ofthe largest ovarianfollicles in adult female Bothropsneuwiedi pubescens. OE oviductalembryos. Triangles represent early embryos(SVL < 4cm) andsquares represent fully-developmentembryos (SVL > 12 cm). 80 M.T.Hartmann, O.A.V .Marques,S.M. Almeida-Santos

Figure 3. Seasonalabundance of male andfemales Bothropsneuwiedi pubescens ,as showby collectiondata for museum specimens. Shadedbars represent adultmales ( n 99/,openbars are mature females ( n 83/, and blackbars are immatures ( n 66/.

Figure 4. Relationshipbetween female snout-ventlength and litter size in Bothropsneuwiedi pubescens , from 2 southernBrazil ( F1;11 25:3, r 0:669, P < 0:001/.

Seasonalactivity. Adultmales and females were collectedyear round, except for July (Žg. 3).Both males and females were moreoften collected during the summer and onset of autumn(from Januaryto April) (  2 105:09, n 182 df 11; P < 0:01).The number ofmales collected outnumbered females in March and April (Ž g. 3). The frequency of juvenileswas higherthan expected from Januaryto March and September to November, andlower than expected from Aprilto August and in December (  2 16:02, n 66 df 11; P < 0:14; Ž g. 3).

Littersize. Thelitter size averaged 11 ( s 6:2,range4-25, n 13)and was positively correlatedwith female body size (Ž g. 4).Neonates born in captivity averaged 21 cmSVL (s 2:1cm,range 17-25 cm, n 35)and individual body mass averaged10.12 g (s 4:65g,range3.87-16.80 g, n 6). Reproductivebiology of Bothropsneuwiedi pubescens 81

Figure 5. Seasonaldistribution of body size of Bothropsneuwiedi pubescens ,received inthe PontiŽ cia UniversidadeCató lica fromRio Grande do Sul and Universidade Federal deSanta Maria from1990 to 1997. Soliddots show juveniles, circles showmature females, crosses showmature males.

Growthrates. Seasonaldistributions of SVL suggestperiod of recruitmentand growth patternsof juvenile B. n. pubescens (Žg. 5).Newborn snakes (SVL 20cm) appearedto occurin latesummer and probably grew toalengthof about30 cmSVL bytheend of the calendaryear of theirbirth. Apparently, males attain maturity at an ageof approximately 16months, whereas maturity in females is delayed for atleast two additional years.

Discussion Femalesattain a muchlarger body size than males in Bothropsneuwiedi pubescens , similarto other species of thegenus (Soló rzano and Cerdas, 1989; Sazima, 1992; Sazima andManzani, 1998; Nogueira, 2001). This is themost common condition among snakes andis generally correlated with absence of male-male combat behavior (Shine 1978b; Shine,1994). Combat behavior in the genus Bothrops maybe restricted to the B. atrox group,which includes B. atrox, B. leucurus and B. moojeni (seeAlmeida-Santos and Salomão, 2002), since our experience throughout many years at the Instituto Butantan (where thousandsof specimens of Bothrops, including B. neuwiedi havebeen housed) male-malecombat was onlyobserved in three species of the B. atrox group.Moreover, B. n.pubescens havehigher sexual dimorphism for size(SSD 0.39)than members of 82 M.T.Hartmann, O.A.V .Marques,S.M. Almeida-Santos the B. atrox groupin which male-male combat had been reported (0.15 for B. atrox and 0.10 for B. moojeni;Shine,1994, and data obtained from Nogueira,2001), suggesting that male-malecombat behavior is absentin B. n.pubescens . Reproductivecycles of this species seem to be seasonal and associated with warm periodsof theyear. Field data conŽ rm fullmating with a peakof maleactivity in March andApril indicating mate-searching (Duvall et al., 1992). Embryonic development occurs inthe spring and/ orthesummer when higher and more constant body temperatures are easierto maintain. High temperatures increase the rate of embryonic development and mayreduce the incidence of anomalies(V inegar,1974). Recruitment of juvenilesduring warmer monthsis apparently widespread in the genus Bothrops (Sazima,1992; Martins andOliveira, 1999; Almeida-Santos and Salomã o, 2002; Marques and Sazima, 2003). Mostbirths occur when prey availability for juvenilesis apparently high, since most frogsand lizards are abundant during the warm seasonin southern Brazil (Almeida, 1999; Cechin,1999). Environmental factors such as humidity and temperature may be themain reasonsfor thepresence of both young snakes and prey in the warmer months.Length ofgestation in B. neuwiedi was estimatedto befrom 205to 320 days in captivity(Alves etal., 1998), whereas we calculatedit to be between90 and150 days. Studies of captive animalshave often overestimated gestation length (e.g., Peñ aranda, 1995; Cruz et al., 1989) becauseof spermstorage. Similar to data obtained here for onecaptivefemale, Alves et al. (1998)reported parturition to be625days after mating in anothercaptive female of B. n. pubescens.Thesefemales apparently stored sperm for approximatelysixteen months when theirbody mass startedto increase, indicating the beginning of secondary vitellogenesis (Almeida-Santosand Salomã o, 2002 — seeSaint-Girons, 1975, 1982; Schuett, 1982, 1992;Almeida-Santos and Salomã o, 1997;2002 for discussionabout storage of sperm). Mating in B.n.pubescens occursin earlyautumn, with females storing sperm until spring whenfertilization can occurs. Bothropsneuwiedi showsthe presence of uterinemuscular twisting(UMT) innon-vitellogenic and vitellogenic stages (Almeida-Santos and Salomã o, 2002).The female observed in courtship in theŽ elddid not have vitellogenic follicles and probablydid not give birth the following year .Thedata from animalsin captivityconŽ rm thatfemales of speciesfrom thegenus Bothrops havethe ability to storesperm for more thanone season by meansof uterinemuscular twisting (UMT) (see Almeida-Santosand Salomão, 2002). Theseasonal activity pattern recorded in B.n.pubescens seemsto be related to the climaticvariation in the region, with few activeindividuals during colder months. The few specimenscollected during the winter were foundon relatively warmer days(Almeida, 1999).In some temperate species, peaks of activityin spring and late autumn are associated tomigration among different hibernation and foraging sites (Gibbons and Semlitsch, 1987).This does not seem to be the case in B.n.pubescens ,asthis species presents a unimodalseasonal activity pattern (with a peakfrom Januaryto April). As reportedfor otherspecies (Secor, 1994), it ispossible that pregnant females of B.n.pubescens become moresedentary and probably inhabit secretive microhabitats, thus diminishing encounter Reproductivebiology of Bothropsneuwiedi pubescens 83 probability.A smallnumber of pregnantfemales in thetotal sample could also be dueto thissedentary behavior . Encounterrate of maleswas higherin March and April, which could be explainedby anincrease in activityrelated to the mating period (cf. Secor,1994). In juveniles, different selectivepressures in uence activity. Possibly, much of the time is devoted to foraging activitiesin order to assimilateenergy for growth,and thus, activities may be lessseasonal (Whitinget al., 1996). Thesmall body size is characteristic of the B. neuwiedi groupand may be responsible for lowsize of neonates and low fecundity presented for B.n.pubescens , as well as B.n.pauloensis (Valdujoet al., 2002; see data for other Bothrops speciesin Leloup, 1975;Soló rzano and Cerdas, 1989; Sazima, 1992). The inferred growth rate from seasonal distributionof juvenile B. n. pubescens isapproximately 10 mm SVL/month.This value iswithin the range record for juvenileand adult of B. jararaca studiedin the Ž eld(5- 18mm/ month,see Sazima, 1992). The growth rate in B. n. pubescens isrelatively low, similarto thatveriŽ ed for otherviperids, which is probablydue to thestout body of this groupof snakes (see Shine,1980). The long delay in sexual maturation recorded here istypical of other neotropical pitvipers (Sazima, 1992; MacCoy and Censky, 1992) and canbe related to high survivorship of this group of venomous snakes (see Shine,1980; Williams,1996).

Acknowledgements. We are gratefulto A.S. Abe, G. Gollmann,L. Luiselli,C.F.D. Rocha, and two anonymous referees whocritically read themanuscript. The English text was improvedby A.d’Heursel Baldisseri.We thank M.Martins,S.Z. Cechin, P .A.Hartmann,J. Melchiors,L.O.M. Giasson, A. Malmann,and V .J.Germano for theirvarious forms ofassistance. S.Z.Cechin and M. Di-Bernardofor allowing us toexamine specimens under theircare. S.Z.Cechin, Setor de Zoologia,Universidade Federal deSantaMaria forlogistical support and many facilities alongthe study. This study is partof the master’ s thesisof the Ž rst authorat thePrograma de Pó s Graduaçã oem Biologia,Á rea deZoologia,of theUniversidade Estadual Paulista, UNESP ,Campusde RioClaro. FAPESP,CAPESand CNPq for Ž nancialsupport.

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Received: November 20,2002. Accepted: April9, 2003.