developmentofsecondarysexualcharactersandtheir ...hooks in males, were examined under a...

Journal of Fish Biology (2018) 92, 131–149

doi:10.1111/jfb.13499, available online at wileyonlinelibrary.com

Development of secondary sexual characters and theirrelationship to ontogeny and seasonal reproductive periodin Hyphessobrycon igneus (Ostariophysi: Characiformes)

L. Schönhofen Longoni, J. Giora* and C. Bernhardt Fialho

Universidade Federal do Rio Grande do Sul, Departamento de Zoologia, Laboratório deIctiologia, Avenida Bento Gonçalves, 9500, Agronomia, CEP: 91509900, Porto Alegre, Rio

Grande do Sul, Brazil

(Received 9 March 2017, Accepted 2 October 2017)

Sexual dimorphism in size, anal-fin shape and coloration of Hyphessobrycon igneus, Characidae, wereexamined. Males were more frequent at larger body sizes, confirming body size as a sexually dimorphictrait. Anal-fin shape and the colour of all fins were the same for females and juveniles, differing only inadult males. Likewise, only adult males had bony hooks on fin rays; larger and more sexually maturemales had the most numerous and developed hooks and hooks were most developed in degree and num-ber during peak reproductive periods. Fin hooks regressed in number and developmental degree afterthe reproductive period, but restarted development with the beginning of the new reproductive periodwithout completely disappearing. Results show that bony hooks have a development and regressioncycle related to reproductive seasonality.

© 2017 The Fisheries Society of the British Isles

Key words: bony hooks; Characidae; fin morphology; gonad histology; reproduction.

INTRODUCTION

The order Characiformes is a highly diverse group of fishes with a wide array of formsand behaviours (Vazzoller & Menezes, 1992) and includes approximately 37% of allNeotropical freshwater fish species (Reis et al., 2003). Characidae, with 1108 validspecies, is the most species rich family within Characiformes (Eschmeyer & Fong,2016). The characid species Hyphessobrycon igneus Miquelarema, Menni, López &Casciotta, 1980 occurs in the lower Río Paraná in Argentina and in the Laguna dosPatos and Rio Tramandaí systems and coastal lagoons in the southern Brazilian Stateof Rio Grande do Sul. According to Carvalho (2011), until recently, specimens of H.igneus collected from the drainages of Rio Grande do Sul were erroneously identifiedas Hyphessobrycon bifasciatus Ellis 1911.

There are many documented cases of sexual dimorphism among fishes, which areparticularly informative in this respect (Py Daniel & Cox-Fernandes, 2005). Bodysize is the most frequently sexually dimorphic trait in fishes (Nikolskii, 1963), espe-cially in Characiformes and Siluriformes. Females are often larger than males in such

*Author to whom correspondence should be addressed. Tel.: +55 51 33087722; email: [email protected]

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© 2017 The Fisheries Society of the British Isles

132 L . S C HÖ N H O F E N L O N G O N I E T A L.

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Fig. 1. Development of fin-ray hooks in males of Hyphessobrycon igneus: (a) weakly developed, (b) developedand (c) greatly developed. Scale bar= 0·2 mm.

orders, with this difference considered as a reproductive strategy of these groups toincrease fecundity (Lowe-McConnel, 1987; Wootton, 1990; Agostinho & Júlio, 1999;Gomiero & Braga, 2007). Sexual dimorphism in size also arises as a consequence ofsexual selection, operating through competition among members of one sex for mem-bers of the opposite sex and for spawning grounds (Forsgren et al., 2002). Fishes canalso show sexual dimorphism in the shape of caudal, pelvic, pectoral and anal fins(Py Daniel & Cox-Fernandes, 2005). Sex differences in fin shape, size and colorationhave been reported for many species of Characidae (Miquelarena & Aquino, 1995;Canan & Gurgel, 1997; Miquelarena & Aquino, 1999; Bertaco & Lucinda, 2005; Car-valho, 2006; Carvalho & Bertaco, 2006; Matheus, 2006; Miquelarena & López, 2006;Oyakawa et al., 2006; Bertaco et al., 2007; Alcaraz et al., 2009; Menezes & Weitz-man, 2009; Zanata & Camelier, 2009; Carvalho et al., 2010; Lucena & Malabarba,2010; Miquelarena & López, 2010; Zanata & Camelier, 2010; Jerep & Malabarba,2011; Camelier & Zanata, 2014). The development of bony hooks on fin rays (Fig. 1)is a male secondary sexual character often used in phylogenetic analyses of manyCharacidae taxa (Von Ihering & Azevedo, 1936; Andrade et al., 1984; Garutti, 1990;Azevedo, 2000; Oliveira et al., 2002; Lima & Moreira, 2003; Malabarba & Weitz-man, 2003; Silvano et al., 2003; Azevedo, 2004; Lampert et al., 2004; Bertaco & Car-valho, 2005; Bertaco & Malabarba, 2005; Gonçalves et al., 2005; Andrade et al., 2006;Carvalho, 2006; Miquelarena & López, 2006; Sato et al., 2006; Bertaco et al., 2007;Lampert et al., 2007; Garcia-Alzate & Román-Valencia, 2008; Garcia-Alzate et al.,2010; Miquelarena & López, 2010; Carvalho, 2011; Dala-Corte & Fialho, 2013; Came-lier & Zanata, 2014). These hooks are often interpreted to be contact organs duringspawning (Wiley & Collette, 1970), but their real function is still poorly understood.In many fishes, hooks arise before the reproductive period and regress after reproduc-tion is complete (Collette, 1977). In characins, however, the question of whether thesehooks are maintained or lost after reproduction remains controversial (Von Ihering &Azevedo, 1936; Andrade et al., 1984; Garutti, 1990; Azevedo, 2000; Oliveira et al.,2002; Azevedo, 2004; Lampert et al., 2004; Gonçalves et al., 2005; Andrade et al.,2006; Sato et al., 2006; Lampert et al., 2007; Vieira et al., 2016).

This paper aims to answer the following questions: does H. igneus, like othercharacins, show sexual dimorphism in body size, fin colour and shape and the

© 2017 The Fisheries Society of the British Isles, Journal of Fish Biology 2018, 92, 131–149

D I M O R P H I S M , O N T O G E N Y A N D R E P RO D U C T I O N I N H Y P H E S S O B R Y C O N I G N E U S 133

presence or absence of hooks on fin rays; if the presence–absence of fin hooks issexually dimorphic, are the number of hooks and degree of hook development relatedto gonadal maturation and reproductive period; does H. igneus maintain or lose its finhooks after reproduction?

MATERIALS AND METHODS

Field work and sampling were carried out under the scientific collection Permit Number873510 issued by the Sistema de Autorização e Informação em Biodiversidade of the InstitutoChico Mendes de Conservação da Biodiversidade (Sisbio/ICMBio). This study was conductedaccording to ethical and methodological standards for the use of fishes, approved by the EthicsCommittee on Animal Use of the Universidade Federal do Rio Grande do Sul (Permit Number24434).

Hyphessobrycon igneus individuals were collected at Lagoa Bacopari (30∘ 32′ 00′′ S; 50∘ 25′

34′′ W) in the municipality of Mostardas, on the central coast of Rio Grande do Sul, southernBrazil. This lagoon has a surface area of 9·2 km2 and maximum and mean depths of 11·7 and4·51 m, respectively (Schäfer et al., 2009).

Fish were collected monthly with a trawl from April 2010 to March 2011. In the field, individ-uals were euthanized with clove oil, fixed in 10% formalin and then preserved in 70% ethanol. Inthe laboratory, specimens were measured (standard length, LS), weighted (total mass, MT) anddissected for sex determination and gonad analyses. The gonado-somatic index (IG) was calcu-lated using the formula adapted from Santos (1978): IG = 100MGMT, where MG is gonad mass.

Gonads were visually inspected to determine the macroscopic maturity phase of males andfemales. The gonads of some males and females were also microscopically examined to betterdetermine maturation phase. These gonads were dehydrated in ethanol series, embedded in gly-col methacrylate resin and sectioned into 3 μm thick slices in a Leica RM2245 microtome (www.leicabiosystems.com). The sections were stained with haematoxylin and eosin or toluidin blue.

Based on the gonad micro and macroscopic characteristics, the following phases of maturationfor males and females were determined (Brown-Peterson et al., 2011): immature (IMMAT),developing (DEV), spawning capable (SC), regressing (REGRES) and regenerating (REGEN).The Spawning capable phase was subdivided into spawning capable 1 (SC1), spawning capable2 (SC2) and spawning capable 3 (SC3) for males and spawning capable 1 (SC1), spawningcapable 2 (SC2) and actively spawning (AS) for females. Reproductive period was establishedfrom the monthly variation in both the mean IG and the relative frequencies of the gonadalmaturity phases in males and females.

Anal-fin shape and size of specimens, in addition to coloration and presence–absence ofhooks in males, were examined under a stereomicroscope for possible ontogenetic or sexualvariation. Through qualitative analysis of size and shape, the male anal-fin hooks were classi-fied according to their degree of development as: weakly developed (WD), developed (D) andgreatly developed (GD). Number of rays with hooks was also recorded. Anal-fin morphologyand the coloration of all fins were grouped according to sex, reproductive phase and LS.Individuals were divided into eight LS classes: 17·32–20·52 mm (LS1); 20·53–23·73 mm (LS2);23·74–26·94 mm (LS3); 26·95–30·15 mm (LS4); 30·16–33·36 mm (LS5); 33·37–36·37 mm(LS6); 36·38–39·78 mm (LS7); 39·79–42·99 mm (LS8). A Fisher exact test with residual adjust-ment was performed to investigate possible relationships among these variables. Pearson′scorrelation coefficient (𝛼 = 0·01) was applied to evaluate whether there was a relationshipbetween the number of anal-fin hooks and standard length (LS). One-way analysis of variance(ANOVA) combined with a Tukey honestly significantly different (HSD) for multiple compar-ison (𝛼 = 0·05) was performed to verify whether there was an association between the numberof rays with hooks and the maturity stages. One-way ANOVA combined with a Tukey HSDfor multiple comparison (𝛼 = 0·05) was employed to verify whether there was an associationbetween the degree of anal-fin hook development and LS. A 𝜒2-test, followed by a Fisher exacttest with residual adjustment (𝛼 = 0·05), was applied to verify whether there was an associationbetween maturity stage and the degree of anal-fin hook development. A one-way ANOVAcombined with a Tukey HSD for multiple comparisons (𝛼 = 0·05) was used to evaluate whether


http://www.leicabiosystems.com

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Fig. 2. Standard length (LS)-relative frequency distribution of male ( ) and female ( ) Hyphessobryconigneus: LS1, 17·32–20·52 mm; LS2, 20·53–23·73 mm; LS3, 23·74–26·94 mm; LS4, 26·95–30·15 mm; LS5,30·16–33·36 mm; LS6, 33·37–36·37 mm; LS7, 36·38–39·78 mm; LS8, 39·79–42·99 mm.

there is a relationship between the number of rays with hooks and the collection month. A𝜒2-test, followed by a Fisher exact test with residual adjustment (𝛼 = 0·05), was used to verifywhether there is an association between the degree of development of anal-fin hooks andcollection month. All statistical analyses were performed using SPSS 18 software (IBM; www.01.ibm.com).

Voucher specimens were deposited in the fish collection of the Departamento de Zoologia,Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil (UFRGS 17266 and UFRGS17267).

RESULTS

A total of 785 H. igneus specimens were analysed: 444 females with LS rangingfrom 17·32 to 42·45 mm and 341 males ranging from 17·56 to 41·89 mm. The pro-portion of adult males and females differed from 1:1 in standard length classes LS5(𝜒2 = 11·17, higher number of females), LS6 (𝜒2 = 4·86, higher number of females)and LS7 (𝜒2 = 6·89, higher number of males); showing that females were more fre-quent in intermediate size classes, whereas males were more frequent in higher sizeclasses (Fig. 2).

Descriptions of the male and female maturation phases, based on both the macroand microscopic characteristics of gonads, are provided in Table I and Figs 3–5.The mean monthly values of IG (Fig. 6) and the monthly relative frequencies ofgonadal maturation phases (Fig. 7) indicated that the reproductive period occursfrom August to March, with peaks in September to October and February. Never-theless, a large proportion of males capable of releasing sperm were always presentyear-round.

Colour of all fins and anal-fin shape differed between adult males and females andbetween juvenile and adult males. In females and juvenile males the distal margin of theanal fin was slightly concave and all their fins were red; conversely, the anal fin of adultmales was more developed with the outer margin straight and all their fins were yellow


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Table I. Macroscopic and microscopic descriptions of Hyphessobrycon igneus maleand female gonad developmental stages through the reproductive cycle adapted from

Brown-Peterson et al. (2011)

Developmental stage Female Male

ImmatureNever spawned Small and translucent; only

oogonia and primarygrowth oocytes presenttidily arranged, withoutspace between oocytes orovigerous lamellae[Fig. 2(a)]

Small and translucent; onlyspermatogonia present[Fig. 3(a)]

DevelopingGonad developing for thefirst time or restarting thedevelopment

Enlarging ovaries, startingto become opaque due toonset of vitellogenicprocess; oogonia,primary growth oocyte,pre-vitellogenic oocyteand few vitellogenicoocyte are present[Fig. 2(b)]

Whitish testes;spermatogonia, primaryand secondaryspermatocytes, fewspermatids and scarcespermatozoa are present[Fig. 3(b)]

Spawning capableFish are able to spawn inthis cycle

Large and yellowish toorange ovaries; individualoocytes visiblemacroscopically.

Spawning capable 1:primary growth and fewpre-vitellogenic andmature oocyte arepresent, with vitellogenicoocyte as the maingerminal cell in thegonad [Fig. 2(c)].

Spawning capable 2:primary growth and fewpre-vitellogenic orvitellogenic oocytes arepresent; mature oocyteare the main germinal cellin the gonad [Fig. 2(d)]

Large, firm and opaquetestes.

Spawning capable 1: allstages of spermatogenesis(spermatogonia,spermatocyte, spermatid,spermatozoa) are present.Great amount ofsecondary spermatocyte,spermatids andspermatozoa [Fig. 3(c)].

Spawning capable 2: allstages of spermatogenesisare present; secondaryspermatocytes stillabundant, butspermatozoa are the maingerminal cells in thegonad [Fig. 3(d)].

Spawning capable 3: testesfull of spermatozoa, witha number ofSpermatogonia,spermatocytes andspermatids present, butless abundant [Fig. 3(e)]



Table I. cotinued

Developmental stage Female Male

Actively spawningOvulation and spawning in

progress; large number ofmature oocytes; atreticfollicle and postovulatoryfollicle complex arepresent [Fig. 2(e)]

RegressingCessation of spawning Flacid ovaries, blood vessels

prominent and ovigerouslamellae distended;oogonial proliferation;great amount of primarygrowth oocytes, atreticfollicles andpostovulatory folliclecomplex are present;some vitellogenic ormature residual oocytespresent [Fig. 2(f)]

Small and flaccid testes;little to no activespermatogenesis;spermatogonialproliferation; greatamount ofspermatogonia, fewprimary or secondaryspermatocytes; residualspermatozoa are present[Fig. 3(f)]

RegeneratingSexually mature,reproductively inactive

Small ovaries, blood vesselsreduced but present; onlyoogonia and primarygrowth oocytes present;few atretic follicle or oldpostovulatory folliclecomplex may be present[Fig. 2(g)]

Small and translucenttestes; proliferation ofspermatogoniathroughout testes; smallamount of residualspermatozoa occasionallypresent in the gonad or insperm duct [Fig. 3(g)]

(Fig. 8). The fins of females and juvenile males were thus considered as nondimorphic,whereas those of females and adult males as dimorphic. Seven males in the classes ofstandard length LS1 and LS2 had a red-to-yellow intermediate coloration, were consid-ered as transitioning from nondimorphic to dimorphic states and were excluded fromthe statistical analysis.

Male anal-fin shape was significantly related to standard length (P< 0·001) and matu-rity phase (P< 0·001). Smaller body size classes (LS1, LS2 and LS3) were highly relatedto have a non-dimorphic fin, whereas larger classes (LS5, LS6 and LS7) were related adimorphic fin [Fig. 9(a) and Table S1, Supporting information]. The IMMAT and DEVmaturation phases were highly related to having a non-dimorphic anal fin, whereas theSC2 and SC3 phases were related to having a dimorphic fin [Fig. 9(b) and Table S2,Supporting information].

Male fin colour was also significantly associated with standard length (P< 0·001)and with maturation phases (P< 0·001). Smaller body size males (LS1, LS2 and LS3)were much more likely to have red-coloured fins, whereas larger body sizes (LS6, LS7



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Fig. 3. Histological section of Hyphessobrycon igneus ovary illustrating maturation phases: (a) immature, (b)developing, (c) spawning capable 1, (d) spawning capable 2. pg., primary growth oocyte; pv, previtellogenicoocyte; vt, vitellogenic oocyte; m, mature oocyte. Scale bar= 0·5 mm.

e LS8) were more likely to have yellow-coloured fins [Fig. 9(c) and Table S3, Sup-porting information]. The IMMAT and DEV maturation phases were highly related tored-coloured fins, whereas the SC2 and SC3 phases to yellow-coloured fins [Fig. 9(d)and Table S4, Supporting information]. The shape of the anal-fin and the colour of allfins of males were strongly associated (P< 0·001).

Only males had hooks on pelvic and anal fins. The number of rays with hooks wassignificantly correlated with LS (r2 = 0·85; P< 0·01) and associated with maturationphases (F21,6 = 78·83, P< 0·01). Males in phases SC2 and SC3 had a significantlyhigher mean number of rays with hooks than those in IMMAT, DEV and SC1 phases.The degree of development of hooks (Fig. 1) was also associated with an increasein standard length (F2,7 = 242·45, P< 0·01). The development of hooks was alsoassociated with maturation phases (𝜒2 = 232.160a, P< 0·01; Table S5, Supporting



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Fig. 4. Histological section of Hyphessobrycon igneus ovary illustrating maturation phases: (a) actively spawn-ing, (b) regressing and (c) regenerating. pg., primary growth oocyte; m, mature oocyte; at, atretic follicle; *,postovulatory follicle. Scale bar= 0·5 mm.

information). The absence of hooks was positively associated with IMMAT and withDEV phases, the developed hooks (D) with SC2 and with SC3 phases and the greatlygeveloped hooks (GD) only with SC2 phase. The relationships between the numberand development of hooks and LS, and maturation phases are shown in Fig. 10. Hooksstarted their development at LS3 size class and DEV maturation phase, increasingin both number and size as individuals became larger and entered more advancedmaturation phases. Males in SC2 and SC3 phases had the highest values of IG andthe greatest capability of releasing sperm and had the most numerous and developedfin-ray hooks. The REGRES phase was not well defined in relation to the numberand developmental degree of hooks due to low sample size; nevertheless, a tendencytoward hook regression was noted.

The number (F21,2 = 7·924; P< 0·01) and developmental degree (𝜒2 = 125·584;P< 0·01) of hooks significantly differed among months (Fig. 11). Males with alarge number of hooks were significantly more frequent in September, October andNovember than in April, May, June and March. These last months were those whenmales showed the fewest hooks (Fig. 12). There was an association of April and Marchwith the absence of hooks, of April and May with the presence of WD hooks, of Julyand August with D hooks and of September and October with GD hooks (Table S6,Supporting information).

DISCUSSION

Most Neotropical fish species exhibit seasonal reproduction (Vazzoller & Menezes,1992). According to Azevedo (2004), this kind of reproduction can be seen in about80% of the derived Characidae species. Among small characins, just over 15% ofspecies restrict their reproduction to 3 months at most, 50% take 4 to 6 months toreproduce, >20% extend their reproductive period beyond 6 months, whereas about13% present a continuous reproductive period, lasting from 10 to 12 months a year(Azevedo, 2004). Hyphessobrycon igneus has a reproductive period of over 6 months,showing two spawning peaks a year. This relatively long reproductive period, togetherwith an all-year-round spermatozoid production, probably explains the low number of



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Fig. 5. Histological section of Hyphessobrycon igneus testis illustrating maturation phases: (a), immature, (b)developing, (c) spawning capable 1, (d) spawning capable 2, (e) spawning capable 3, (f) regressing and (g)regenerating. *, spermatogonia; sc1, primary spermatocyte; sc2, secondary spermatocyte; st, spermatid; sz,spermatozoa. Scale ba r= 0·1 mm.

specimens in the regressing phase. As observed, the rapid reorganization of the malegonads enable the fertilization of oocytes spawned during the two reproductive peaksof the species.

Hyphessobrycon igneus is sexually dimorphic in size, with males longer thanfemales. This difference is probably due to a tendency for larger males to matemore successfully and defend their territory from rivals (Forsgren et al., 2002; Bragaet al., 2006). In Hyphessobrycon Durbin 1908, there are territorial species, such asHyphessobrycon eques (Steindachner 1882) (Matheus, 2006) and Hyphessobryconpanamensis Durbin 1908 (Kramer, 1978), wherein males are larger than females. Incontrast, the genus also includes species in which females are larger than males [as



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in Hyphessobrycon meridionalis Ringuelet, Miquelarena & Menni 1978 (Menni &Almirón, 1994) and Hyphessobrycon togoi Miquelarena & López, 2006 (Miquelarena& López, 2006)], or the sexes are similar in size [as in Hyphessobrycon boulengeri(Eigenmann 1907) and Hyphessobrycon sp. (Carvalho, 2006)]. More studies onpopulation structure and the reproductive biology of Hyphessobrycon spp. are neededto assess whether males and females have different growth rates and if these rates arerelated to intraspecific agonistic behaviours.

Additional male behaviours and morphological characters, such as courtship dis-plays and fin colour and shape, also appear to play a role in mate choice in manyfishes (Forsgren et al., 2002), with fin variation being commonly observed in variousfish groups (Py Daniel & Cox-Fernandes, 2005). The differences in male and femaleanal-fin shape observed herein is often used as a character in the study of Characidaesystematics (Miquelarena & Aquino, 1995; Canan & Gurgel, 1997; Miquelarena &Aquino, 1999; Bertaco & Lucinda, 2005; Carvalho, 2006; Carvalho & Bertaco, 2006;Matheus, 2006; Bertaco et al., 2007; Carvalho et al., 2010; Lucena & Malabarba, 2010;Carvalho, 2011; Jerep & Malabarba, 2011). Nevertheless, there can be intragenericvariation in this pattern; for example, anal-fin shape is similar in males and females ofH. togoi (Oyakawa et al., 2006) and Hyphessobrycon nicolasi Miquelarena & López,2010 (Miquelarena & López, 2010).



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Adult male and female fin colour is another form of sexual dimorphism in H. igneus.In Hyphessobrycon more generally, fin colour variation has been observed in thespecies H. bifasciatus, wherein both caudal and anal-fins are red in young, but arelemon-yellow in adults (Oyakawa et al., 2006); in Hyphessobrycon brumado Zanata& Camelier, 2010 and Hyphessobrycon negodagua Lima & Gerhard 2001, males andfemales differ in the colour of the caudal region (Zanata & Camelier, 2010). Sexual

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Fig. 9. Relative frequency distribution by (a) standard length (LS) classes and (b) gonad maturation stage for maleHyphessobrycon igneus with dimorphic ( ) or nondimorphic ( ) anal-fin morphology. Similarly, relativefrequency distribution by (c) LS classes and (d) gonad maturation stage for male H. igneus but with red ( ) oryellow fin coloration. Length classes: LS1, 17·32–20·52 mm; LS2, 20·53–23·73 mm; LS3, 23·74–26·94 mm;LS4, 26·95–30·15 mm; LS5, 30·16–33·36 mm; LS6, 33·37–36·37 mm; LS7, 36·38–39·78 mm; LS8,39·79–42·99 mm. Maturity stages: IMMAT, immature; DEV, developing; SC1, spawning capable 1; SC2,spawning capable 2; SC3, spawning capable 3; REGRES, regressing; REGEN, regenerating.

differences in colour pattern have been reported for other Characidae species. The finsare more intensely coloured in sexually mature males than females in Glandulocaudamelapleurus (Ellis 1911) and in Glandulocauda caerulea Menezes & Weitzman,2009, whereas in Mimagoniates lateralis (Nichols 1913), Mimagoniates microlepis(Steindachner 1877), Mimagoniates rheocharis Menezes & Weitzman 1990 andMimagoniates sylvicola Menezes & Weitzman 1990, sexually mature males have thewhole body more intensely coloured than females (Menezes & Weitzman, 2009). Inmales of Hemigrammus tocantinsi Carvalho et al., 2010 the body and fins are red ororange, whereas females exhibit yellow coloration (Carvalho et al., 2010). Astyanaxvermilion Zanata & Camelier, 2009 also present sexually dimorphic colour patternswith fin margins darker in males than females (Zanata & Camelier, 2009).

It should be noted that young males of H. igneus have the same anal-fin shape andcoloration of all fins as females. The differentiation of males from females beginsat c. 26 mm LS, when they are able to release spermatozoids; i.e. they are sexuallymature. Thus, the characters of fin shape and colour are related to sexual matura-tion in males and, once established, remain throughout adulthood rather than beinglost or undergoing regression. In some Hyphessobrycon spp., including H. igneus,males have hooks on the pelvic and anal fins (Lima & Moreira, 2003; Carvalho, 2006;Garcia-Alzate & Román-Valencia, 2008; Garcia-Alzate et al., 2010). In other Characi-dae species, males lack hooks on their fins (Bertaco & Carvalho, 2005; Garcia-Alzate& Román-Valencia, 2008), have hooks only on their anal fin (Bertaco et al., 2007;Garcia-Alzate & Román-Valencia, 2008; Carvalho, 2011), have hooks on three or more



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40

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Fig. 11. Relative frequency of (a) the number of anal-fin rays having hooks ( , 0–4; , 5–9; , 10–14; , 15–19;, >20) and (b) the degree of hook development ( , absent; , weakly developed; , developed; , greatly

developed) in male Hyphessobrycon igneus by month.

distinct fins (Bertaco & Malabarba, 2005; Garcia-Alzate & Román-Valencia, 2008;Miquelarena & López, 2010), or have hooks on all their fins (Miquelarena & López,2006; Bertaco et al., 2007; Camelier & Zanata, 2014). The latter condition is the mostunusual in the family (Miquelarena & López, 2006; Bertaco et al., 2007). Fin-ray hooksare often associated with reproductive behaviour, although their specific function is yetto be demonstrated. In H. igneus, the number and development of hooks were associ-ated with both LS and stage of maturity. Hook development started when males wereabout 30 mm LS and began to show a large number of spermatozoids in the testis(spawning capable stage). Furthermore, hooks were most numerous and fully devel-oped in the largest specimens (c. 40 mm LS) that were able to release sperm and werecollected during the species reproductive period. The months when male IG values werehighest (indicating greater gonadal development) were also those when the proportionof specimens with well-developed hooks was highest, decreasing thereon. Thus, theresults suggest that in H. igneus fin rays hooks regress after the reproductive period.

Several authors have discussed the presence of hooks on fins in Characidae, buttheir maintenance or loss after the reproductive period is still debated. In characins,some studies have concluded that hooks develop until the fish reaches sexual maturityand show no sign of regression after reproduction (Andrade et al., 1984; Azevedo,2000; Oliveira et al., 2002; Azevedo, 2004; Lampert et al., 2004; Gonçalves et al.,2005; Lampert et al., 2007). According to Azevedo (2004), the great number of maleswith weakly developed, or lacking, hooks present after the reproductive period can be



APR0

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Month

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0·6

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Fig. 12. Monthly variation in number (N) of Hyphessobrycon igneus males with >14 anal-fin rays having hooks( ) and mean gonado-somatic index (IG; ).

associated to the recruitment of young males in the population and not to regression ofhooks in mature males (Azevedo, 2004). In contrast, there are reports for many otherspecies in which hooks develop before reproduction and regress afterwards (Collette,1977) as in Astyanax bimaculatus (l. 1758) (Von Ihering & Azevedo, 1936; Garutti,1990; Sato et al., 2006) and Salminus hilarii Valenciennes 1850 (Andrade et al., 2006).Recently, Vieira et al. (2016) described and compared bony hooks on the anal andpelvic fins of six characid species; however, seasonal variation in hook occurrence wasnot examined.

Most H. igneus specimens collected after the breeding period showed weaklydeveloped hooks, although large adult males lacking hooks were also recorded. Thissuggests that in most males the fin hooks regress after reproduction but do not entirelydisappear, remaining present though less developed. The long reproductive periodwith a short interval before the subsequent period may explain the maintenance ofweakly developed hooks throughout the year. These structures begin to regress afterreproduction, but with the beginning of a new reproductive period they resume theirdevelopment before disappearing completely, increasing in number and size onceagain. These results suggest that long reproductive periods might make it less likelyto observe the development and regression of bony hooks in anal and pelvic-fin raysof many small characin species.

We are grateful to T. Schmidt Dias for conducting the collect expeditions in the Lagoa Baco-pari. This project was funded by Programa Nacional de Pós Doutorado – PNPD-CAPES (pro-cess 2282/09) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq(process 476821/2003-7; 478002/2006-8).

Supporting Information

Supporting Information may be found in the online version of this paper:Table S1. Association between the morphology of the anal fin and the standard lengthclasses (LS) in males of Hyphessobrycon igneus by Fisher’s exact test (𝛼 = 0·05).



Table S2. Association between the morphology of the anal fin and the maturationstage in males of Hyphessobrycon igneus by Fisher’s exact test (𝛼 = 0·05).

Table S3. Association between the colour of the fins and the standard length (LS)classes in males of Hyphessobrycon igneus by Fisher’s exact test (𝛼 = 0·05).

Table S4. Association between the colour of the fins and the maturation phase inmales of Hyphessobrycon igneus, by Fisher’s exact test (𝛼 = 0·05).

Table S5. Association between maturation stage and the development of the hooksof males of Hyphessobrycon igneus, by Fisher’s exact test (𝛼 = 0·05).

Table S6. Association between month and degree of development of hooks in malesof Hyphessobrycon igneus, by Fisher’s exact test (𝛼 = 0·05).

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