Development of Secondary Sexual Characters and Their Relationship to Ontogeny and Seasonal Reproductive Period in Hyphessobrycon Igneus (Ostariophysi: Characiformes)

Journal of Fish Biology (2018) 92, 131–149 doi:10.1111/jfb.13499, available online at wileyonlinelibrary.com

Development of secondary sexual characters and their relationship to ontogeny and seasonal reproductive period in Hyphessobrycon igneus (Ostariophysi: Characiformes)

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

Universidade Federal do Rio Grande do Sul, Departamento de Zoologia, Laboratório de Ictiologia, 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, were examined. Males were more frequent at larger body sizes, confirming body size as a sexually dimorphic trait. Anal-fin shape and the colour of all fins were the same for females and juveniles, differing onlyin adult males. Likewise, only adult males had bony hooks on fin rays; larger and more sexually mature males had the most numerous and developed hooks and hooks were most developed in degree and number during peak reproductive periods. Fin hooks regressed in number and developmental degree after the reproductive period, but restarted development with the beginning of the new reproductive period without completely disappearing. Results show that bony hooks have a development and regression cycle 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 forms and behaviours (Vazzoller & Menezes, 1992) and includes approximately 37% of all Neotropical freshwater fish species (Reis et al., 2003). Characidae, with 1108 valid species, 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 dos Patos and Rio Tramandaí systems and coastal lagoons in the southern Brazilian State of 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 identified as Hyphessobrycon bifasciatus Ellis 1911. There are many documented cases of sexual dimorphism among fishes, which are particularly informative in this respect (Py Daniel & Cox-Fernandes, 2005). Body size 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] 131

(a) (b) (c)

Fig. 1. Development of fin-ray hooks in males of Hyphessobrycon igneus: (a) weakly developed, (b) developed and (c) greatly developed. Scale bar = 0·2 mm. orders, with this difference considered as a reproductive strategy of these groups to increase fecundity (Lowe-McConnel, 1987; Wootton, 1990; Agostinho & Júlio, 1999; Gomiero & Braga, 2007). Sexual dimorphism in size also arises as a consequence of sexual selection, operating through competition among members of one sex for members of the opposite sex and for spawning grounds (Forsgren et al., 2002). Fishes can also 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 coloration have 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 many Characidae 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 during spawning (Wiley & Collette, 1970), but their real function is still poorly understood. In many fishes, hooks arise before the reproductive period and regress after reproduction is complete (Collette, 1977). In characins, however, the question of whether these hooks 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 other characins, 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 DIMORPHISM, ONTOGENY AND REPRODUCTION IN HYPHESSOBRYCON IGNEUS 133 presence or absence of hooks on fin rays; if the presence–absence of fin hooks is sexually dimorphic, are the number of hooks and degree of hook development related to gonadal maturation and reproductive period; does H. igneus maintain or lose its fin hooks after reproduction?

MATERIALS AND METHODS Field work and sampling were carried out under the scientific collection Permit Number 873510 issued by the Sistema de Autorização e Informação em Biodiversidade of the Instituto Chico Mendes de Conservação da Biodiversidade (Sisbio/ICMBio). This study was conducted according to ethical and methodological standards for the use of fishes, approved by the Ethics Committee on Animal Use of the Universidade Federal do Rio Grande do Sul (Permit Number 24434). 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, southern Brazil. This lagoon has a surface area of 9·2km2 and maximum and mean depths of 11·7and 4·51 m, respectively (Schäfer et al., 2009). Fish were collected monthly with a trawl from April 2010 to March 2011. In the field, individuals were euthanized with clove oil, fixed in 10% formalin and then preserved in 70% ethanol. In the laboratory, specimens were measured (standard length, LS), weighted (total mass, MT)and dissected for sex determination and gonad analyses. The gonado-somatic index (IG) was calcu- lated using the formula adapted from Santos (1978): IG = 100MGMT,whereMG is gonad mass. Gonads were visually inspected to determine the macroscopic maturity phase of males and females. The gonads of some males and females were also microscopically examined to better determine 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 maturation for 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 capable 2 (SC2) and spawning capable 3 (SC3) for males and spawning capable 1 (SC1), spawning capable 2 (SC2) and actively spawning (AS) for females. Reproductive period was established from the monthly variation in both the mean IG and the relative frequencies of the gonadal maturity phases in males and females. Anal-fin shape and size of specimens, in addition to coloration and presence–absence of hooks in males, were examined under a stereomicroscope for possible ontogenetic or sexual variation. 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)and greatly developed (GD). Number of rays with hooks was also recorded. Anal-fin morphology and 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 adjustment was performed to investigate possible relationships among these variables. Pearson′s correlation coefficient𝛼 ( = 0·01) was applied to evaluate whether there was a relationship between 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 comparison (𝛼 = 0·05) was performed to verify whether there was an association between the number of rays with hooks and the maturity stages. One-way ANOVA combined with a Tukey HSD for multiple comparison (𝛼 = 0·05) was employed to verify whether there was an association 𝜒2 between the degree of anal-fin hook development and LS.A -test, followed by a Fisher exact test with residual adjustment (𝛼 = 0·05), was applied to verify whether there was an association between maturity stage and the degree of anal-fin hook development. A one-way ANOVA combined with a Tukey HSD for multiple comparisons (𝛼 = 0·05) was used to evaluate whether

(%) 20 y enc u 15 e freq v 10 Relati 5

LS1 LS2 LS3 LS4 LS5 LS6 LS7 LS8

LS class

Fig. 2. Standard length (LS)-relative frequency distribution of male ( ) and female ( ) Hyphessobrycon igneus: 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. A 𝜒2-test, followed by a Fisher exact test with residual adjustment (𝛼 = 0·05), was used to verify whether there is an association between the degree of development of anal-fin hooks and collection 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 UFRGS 17267).

RESULTS

A total of 785 H. igneus specimens were analysed: 444 females with LS ranging from 17·32 to 42·45 mm and 341 males ranging from 17·56 to 41·89 mm. The proportion of adult males and females differed from 1:1 in standard length classes LS5 𝜒2 𝜒2 ( = 11·17, higher number of females), LS6 ( = 4·86, higher number of females) 𝜒2 and LS7 ( = 6·89, higher number of males); showing that females were more frequent in intermediate size classes, whereas males were more frequent in higher size classes (Fig. 2). Descriptions of the male and female maturation phases, based on both the macro and 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 of gonadal maturation phases (Fig. 7) indicated that the reproductive period occurs from August to March, with peaks in September to October and February. Never- theless, a large proportion of males capable of releasing sperm were always present year-round. Colour of all fins and anal-fin shape differed between adult males and females and between juvenile and adult males. In females and juvenile males the distal margin of the anal fin was slightly concave and all their fins were red; conversely, the anal fin ofadult males was more developed with the outer margin straight and all their fins were yellow

Table I. Macroscopic and microscopic descriptions of Hyphessobrycon igneus male and female gonad developmental stages through the reproductive cycle adapted from Brown-Peterson et al. (2011)

Developmental stage Female Male Immature Never spawned Small and translucent; only Small and translucent; only oogonia and primary spermatogonia present growth oocytes present [Fig. 3(a)] tidily arranged, without space between oocytes or ovigerous lamellae [Fig. 2(a)] Developing Gonad developing for the Enlarging ovaries, starting Whitish testes; first time or restarting the to become opaque due to spermatogonia, primary development onset of vitellogenic and secondary process; oogonia, spermatocytes, few primary growth oocyte, spermatids and scarce pre-vitellogenic oocyte spermatozoa are present and few vitellogenic [Fig. 3(b)] oocyte are present [Fig. 2(b)] Spawning capable Fishareabletospawnin Large and yellowish to Large, firm and opaque this cycle orange ovaries; individual testes. oocytes visible Spawning capable 1: all macroscopically. stages of spermatogenesis Spawning capable 1: (spermatogonia, primary growth and few spermatocyte, spermatid, pre-vitellogenic and spermatozoa) are present. mature oocyte are Great amount of present, with vitellogenic secondary spermatocyte, oocyte as the main spermatids and germinal cell in the spermatozoa [Fig. 3(c)]. gonad [Fig. 2(c)]. Spawning capable 2: all Spawning capable 2: stages of spermatogenesis primary growth and few are present; secondary pre-vitellogenic or spermatocytes still vitellogenic oocytes are abundant, but present; mature oocyte spermatozoa are the main are the main germinal cell germinal cells in the in the gonad [Fig. 2(d)] gonad [Fig. 3(d)]. Spawning capable 3: testes full of spermatozoa, with a number of Spermatogonia, spermatocytes and spermatids present, but less abundant [Fig. 3(e)]

Table I. cotinued

Developmental stage Female Male Actively spawning Ovulation and spawning in progress; large number of mature oocytes; atretic follicle and postovulatory follicle complex are present [Fig. 2(e)] Regressing Cessation of spawning Flacid ovaries, blood vessels Small and flaccid testes; prominent and ovigerous little to no active lamellae distended; spermatogenesis; oogonial proliferation; spermatogonial great amount of primary proliferation; great growth oocytes, atretic amount of follicles and spermatogonia, few postovulatory follicle primary or secondary complex are present; spermatocytes; residual some vitellogenic or spermatozoa are present mature residual oocytes [Fig. 3(f)] present [Fig. 2(f)] Regenerating Sexually mature, Small ovaries, blood vessels Small and translucent reproductively inactive reduced but present; only testes; proliferation of oogonia and primary spermatogonia growth oocytes present; throughout testes; small few atretic follicle or old amount of residual postovulatory follicle spermatozoa occasionally complex may be present present in the gonad or in [Fig. 2(g)] sperm 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 of standard length LS1 and LS2 had a red-to-yellow intermediate coloration, were considered as transitioning from nondimorphic to dimorphic states and were excluded from the 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 related to have a non-dimorphic fin, whereas larger classes (LS5, LS6 and LS7) were related a dimorphic fin [Fig. 9(a) and Table S1, Supporting information]. The IMMAT andDEV maturation phases were highly related to having a non-dimorphic anal fin, whereas the SC2 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 standardP length( < 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 sizesL ( S6, LS7

(a) (b)

pg pv m pv

pg pg vt vt

pv (c)m (d)

pg pv m m

pv vt vt pg vt m vt

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, previtellogenic oocyte; 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 to red-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 all fins of males were strongly associated (P < 0·001). Only males had hooks on pelvic and anal fins. The number of rays with hooks was 2 < significantly correlated with LS (r = 0·85; P 0·01) and associated with maturation < phases (F21,6 = 78·83, P 0·01). Males in phases SC2 and SC3 had a significantly higher 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 increase < in standard length (F2,7 = 242·45, P 0·01). The development of hooks was also associated with maturation phases (𝜒2 = 232.160a, P < 0·01; Table S5, Supporting

(a)pg (b) (c) pg m

* pg pg * at pg pg pg m m

Fig. 4. Histological section of Hyphessobrycon igneus ovary illustrating maturation phases: (a) actively spawning, (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 with DEV phases, the developed hooks (D) with SC2 and with SC3 phases and the greatly geveloped hooks (GD) only with SC2 phase. The relationships between the number and development of hooks and LS, and maturation phases are shown in Fig. 10. Hooks started their development at LS3 size class and DEV maturation phase, increasing in both number and size as individuals became larger and entered more advanced maturation phases. Males in SC2 and SC3 phases had the highest values of IG and the greatest capability of releasing sperm and had the most numerous and developed fin-ray hooks. The REGRES phase was not well defined in relation to thenumber and developmental degree of hooks due to low sample size; nevertheless, a tendency toward hook regression was noted. < 𝜒2 The number (F21,2 = 7·924; P 0·01) and developmental degree ( = 125·584; P < 0·01) of hooks significantly differed among months (Fig. 11). Males witha large number of hooks were significantly more frequent in September, October and November than in April, May, June and March. These last months were those when males showed the fewest hooks (Fig. 12). There was an association of April and March with the absence of hooks, of April and May with the presence of WD hooks, of July and 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 about 80% of the derived Characidae species. Among small characins, just over 15% of species restrict their reproduction to 3 months at most, 50% take 4 to 6 months to reproduce, >20% extend their reproductive period beyond 6 months, whereas about 13% 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, together with an all-year-round spermatozoid production, probably explains the low number of

(a) (b) (c) sc1 * * * * sc1 st sc2 * * sz sz * sc1 * st

(d) (e)

sz sz

* sz sz sc2 * st sc1 sc1 * * * (f) (g) sc1 sz * * *

* * * * sz

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 male gonads enable the fertilization of oocytes spawned during the two reproductive peaks of the species. Hyphessobrycon igneus is sexually dimorphic in size, with males longer than females. This difference is probably due to a tendency for larger males to mate more successfully and defend their territory from rivals (Forsgren et al., 2002; Braga et al., 2006). In Hyphessobrycon Durbin 1908, there are territorial species, such as Hyphessobrycon eques (Steindachner 1882) (Matheus, 2006) and Hyphessobrycon panamensis Durbin 1908 (Kramer, 1978), wherein males are larger than females. In contrast, the genus also includes species in which females are larger than males [as

14 (a)

0 G I 1·8 (b) 1·6 1·4 1·2 1 0·8 0·6 0·4 0·2 0 APRMAYJUNE JULY AUG SEPT OCT NOV DEC JAN FEB MAR Month

Fig. 6. Monthly mean ± S.D. gonado-somatic index (IG) for (a) female and (b) and male Hyphessobrycon igneus. 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 on population structure and the reproductive biology of Hyphessobrycon spp. are needed to assess whether males and females have different growth rates and if these rates are related 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 inmany fishes (Forsgren et al., 2002), with fin variation being commonly observed in various fish groups (Py Daniel & Cox-Fernandes, 2005). The differences in male andfemale anal-fin shape observed herein is often used as a character in the study of Characidae systematics (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 intrageneric variation in this pattern; for example, anal-fin shape is similar in males and females of H. togoi (Oyakawa et al., 2006) and Hyphessobrycon nicolasi Miquelarena & López, 2010 (Miquelarena & López, 2010).

(a) 100 90 80 70 60 50 40 30 20

(%) 10 y

enc 0 u (b)

e freq 100 v 90

Relati 80 70 60 50 40 30 20 10 0 APR MAY JUNE JULY AUG SEPT OCT NOV DEC JAN FEB MAR Month

Fig. 7. Monthly variation in the relative frequency of gonadal maturation phase of (a) female and (b) male Hyphessobrycon igneus. , Developing; , spawning capable 1; , spawning capable 2; , spawning capable 3 (male); actively spawning (female); , regressing; , regenerating.

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 inthe species H. bifasciatus, wherein both caudal and anal-fins are red in young, but are lemon-yellow in adults (Oyakawa et al., 2006); in Hyphessobrycon brumado Zanata & Camelier, 2010 and Hyphessobrycon negodagua Lima & Gerhard 2001, males and females differ in the colour of the caudal region (Zanata & Camelier, 2010). Sexual

(a) (b)

Fig. 8. Hyphessobrycon igneus male (a) and female (b) specimens.

45 (a) 90 (b) 40 80 35 70 30 60 25 50 20 40 15 30

(%) 10 20 y 5 10 enc

u 0 0 45 (c) 80 (d) e freq

v 40 70 35 60 30 Relati 50 25 40 20 30 15 10 20 5 10 0 0 8 T S1 S2 S3 S4 S5 S6 S7 S V L L L L L L L L SC1 SC2 SC3 DE REGR MMA REGEN.

Fig. 9. Relative frequency distribution by (a) standard length (LS) classes and (b) gonad maturation stage for male Hyphessobrycon igneus with dimorphic ( ) or nondimorphic ( ) anal-fin morphology. Similarly, relative frequency distribution by (c) LS classes and (d) gonad maturation stage for male H. igneus but with red ( )or yellow 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 fins are more intensely coloured in sexually mature males than females in Glandulocauda melapleurus (Ellis 1911) and in Glandulocauda caerulea Menezes & Weitzman, 2009, whereas in Mimagoniates lateralis (Nichols 1913), Mimagoniates microlepis (Steindachner 1877), Mimagoniates rheocharis Menezes & Weitzman 1990 and Mimagoniates sylvicola Menezes & Weitzman 1990, sexually mature males have the whole body more intensely coloured than females (Menezes & Weitzman, 2009). In males of Hemigrammus tocantinsi Carvalho et al., 2010 the body and fins are red or orange, whereas females exhibit yellow coloration (Carvalho et al., 2010). Astyanax vermilion Zanata & Camelier, 2009 also present sexually dimorphic colour patterns with 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 and coloration of all fins as females. The differentiation of males from females begins at c.26mmLS, when they are able to release spermatozoids; i.e. they are sexually mature. Thus, the characters of fin shape and colour are related to sexual maturation in males and, once established, remain throughout adulthood rather than being lost 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

© 2017 The Fisheries Society of the British Isles, Journal of Fish Biology 2018, 92, 131–149 DIMORPHISM, ONTOGENY AND REPRODUCTION IN HYPHESSOBRYCON IGNEUS 143 15 mm; , greatly · 95–30 · ,26 S4 , developed; L by (a) different standard 94 mm; · 74–26 e · s ,23 , weakly developed; S3 L Hyphessobrycon igneus ration pha u SC1 SC2 SC3 REGR Mat , absent; 73 mm; · 20) in male 53–23 · > V , ,20 DE S2 L T , 15–19; 52 mm; · IMMA (b) 32–20 · , 10–14; 99 mm. Maturity stages: IMMAT, immature; DEV, developing; SC1, spawning capable · ,17 8 S S1 L L ,5–9; 79–42 · ,39 S7 S8 L L ,0–4; S6 78 mm; L · 38–39 · S5 ss L ,36 S7 ht cla L g S4 Len L 37 mm; · S3 L 37–36 · ,33 S2 S6 L L classes and (d) gonadal maturation phase. Length classes: S L S1 36 mm; · L (a) (c) (d) ) classes and (b) gonadal maturation phases. Similarly, relative frequency of the degree of hook development ( S 0 0 0 0

16–33 8 60 40 20 8 60 40 20 L

· 100 100

(%) y enc u freq e v Relati ,30 S5 L 1; SC2, spawning capable 2; SC3, spawning capable 3; REGRES, regressing; REGEN, regenerating. developed) by (c) length ( Fig. 10. Relative frequency of the number of anal-fin rays having hooks (

(a) 100

20 (%) y 0 enc u (b) 100 e freq v 80 Relati

0 APR MAY JUNE JULY AUG SEPT OCT NOV DEC JAN FEB MAR Month

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 most unusual in the family (Miquelarena & López, 2006; Bertaco et al., 2007). Fin-ray hooks are often associated with reproductive behaviour, although their specific function is yet to be demonstrated. In H. igneus, the number and development of hooks were associated with both LS and stage of maturity. Hook development started when males were about 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 developed in the largest specimens (c.40mmLS) that were able to release sperm and were collected during the species reproductive period. The months when male IG values were highest (indicating greater gonadal development) were also those when the proportion of specimens with well-developed hooks was highest, decreasing thereon. Thus, the results 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, but their maintenance or loss after the reproductive period is still debated. In characins, some studies have concluded that hooks develop until the fish reaches sexual maturity and 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 males with weakly developed, or lacking, hooks present after the reproductive period can be

18 1·2 16 1·0 14

12 0·8 10 G N 0·6 I 8

6 0·4 4 0·2 2

0 0 APR MAY JUNE JULY AUG SEPT OCT NOV DEC JAN FEB MAR Month

Fig. 12. Monthly variation in number (N)ofHyphessobrycon 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 of hooks in mature males (Azevedo, 2004). In contrast, there are reports for many other species 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 and pelvic fins of six characid species; however, seasonal variation in hook occurrence was not examined. Most H. igneus specimens collected after the breeding period showed weakly developed hooks, although large adult males lacking hooks were also recorded. This suggests that in most males the fin hooks regress after reproduction but do not entirely disappear, remaining present though less developed. The long reproductive period with a short interval before the subsequent period may explain the maintenance of weakly developed hooks throughout the year. These structures begin to regress after reproduction, but with the beginning of a new reproductive period they resume their development before disappearing completely, increasing in number and size once again. These results suggest that long reproductive periods might make it less likely to observe the development and regression of bony hooks in anal and pelvic-fin rays of 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 (process 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 length 𝛼 classes (LS) in males of Hyphessobrycon igneus by Fisher’s exact test ( = 0·05).

Table S2. Association between the morphology of the anal fin and the maturation stage 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 lengthL ( S) 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 in males of Hyphessobrycon igneus, by Fisher’s exact test (𝛼 = 0·05). Table S5. Association between maturation stage and the development of the hooks of males of Hyphessobrycon igneus, by Fisher’s exact test (𝛼 = 0·05). Table S6. Association between month and degree of development of hooks in males of Hyphessobrycon igneus, by Fisher’s exact test (𝛼 = 0·05).

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