, Volume 808, Issue 1, pp 5–22 | Cite as

Dimorphic male squid show differential gonadal and ejaculate expenditure

  • Lígia H. Apostólico
  • José E. A. R. Marian


Under intense sexual selection, males less successful in fighting and mate guarding often adopt alternative reproductive tactics. In many species, males employing alternative tactics show not only behavioral differences, but also divergences in morphology and physiology, a phenomenon called intrasexual male dimorphism. Herein, we investigated intrasexual male dimorphism in the loliginid squid Doryteuthis plei, associated with alternative mating tactics. We show that small males (sneakers) have spermatophores with discontinuously smaller sperm mass and longer spermatozoa than large males (consorts). Moreover, sneakers produce club-like spermatangia, whereas consorts produce hook-like spermatangia, each type of spermatangia associated with a different female storage site and adoption of a distinct mating position. We also show that dimorphic males have different gonadal investment, sneakers showing higher increment rates in testis mass and higher investment in spermatophoric complex than consorts. Under the complex squid mating system, with two distinct fertilization environments, we hypothesize that sneakers may maximize their reproductive success by both investing more in gonad growth and partitioning ejaculates into extra mating opportunities, whereas consorts may benefit from investment in somatic growth. Squids may be the first example of animals that, even with distinct sperm storage sites, fit the general predictions of sneaks and guards theoretical models.


Allocation trade-off Allometry Alternative reproductive tactics Conditional strategy Intrasexual male dimorphism Male–male competition Sperm competition Status dependence 


Sexual selection exerts a major influence on the reproductive success of each individual (Andersson, 1994). Under intense male–male competition for female access, a high variance in relative fitness between males is expected. Thus, males that are unsuccessful in mate guarding and fighting contests with dominant or territorial males may avoid direct rivalry and adopt sneaking behaviors as a way to ensure some mating success, leading to the evolution of alternative reproductive tactics within the same sex (reviewed in Gross, 1996; Shuster & Wade, 2003; Taborsky et al., 2008). Such alternative mating tactics are thought to selectively favor discretely divergent phenotypes within males, characterized by the expression of distinct life histories and discontinuous morphological and physiological traits between conspecifics, a phenomenon known as male intrasexual dimorphism (Gadgil, 1972; Gross, 1996; Tomkins et al., 2005).

Divergent male morphs generally exhibit alternative reproductive tactics (e.g., sneaking vs. dominant tactics), and in this case they often face asymmetrical risks related to sperm competition (i.e., competition of ejaculates from two or more males for fertilization of a given set of eggs; Parker, 1970). According to theoretical models of sneaks and guards (Parker, 1990b), sneaker males always experience high risk of sperm competition, as they mate with females that had already mated or will most likely mate with a dominant male. Territorial males, on the other hand, are usually successful in preventing females from copulating with other males, so they generally face lower risks of sperm competition, which depends on the frequency of sneakers in the population (Parker, 1990a, b; Parker et al., 1997). As a consequence of this asymmetry, males that adopt disfavored roles are expected to present greater gonadal expenditure, such as increased relative testis mass and sperm production, as a way of compensating their behavioral disadvantage (e.g., Taborsky, 1994, 1998; Gage et al., 1995; Simmons et al., 1999, 2007).

Loliginid squids form dense spawning aggregations of sexually mature individuals during their reproductive season. They present remarkably complex mating behaviors, including courtship, agonistic contests, mate guarding, and communal egg-laying (Hanlon & Messenger, 1996). Both males and females copulate with several partners, so sperm competition seems to be a common feature in their mating systems (Hanlon, 1998). Alternative reproductive tactics include primarily two sets of behavioral display, typically associated with differences in male body size. Large (consort) males, which engage in physical combats with rivals and guard females before and after mating and during egg-laying, mate with females preferably in the male-parallel position, depositing spermatophores inside the female’s mantle cavity, near the oviduct opening. Small (sneaker) males, in turn, avoid fighting contests and instead adopt surreptitious behaviors to access paired females, adopting the head-to-head mating position and placing spermatophores near or in the seminal receptacle, below the mouth (e.g., Loligo pealeii, Hanlon, 1996; L. vulgaris reynaudii, Hanlon et al., 2002; Heterololigo bleekeri, Iwata & Sakurai, 2007; Doryteuthis opalescens, Zeidberg, 2009).

Intrasexual dimorphism among males in loliginid squids has only been thoroughly investigated in Heterololigo bleekeri (Iwata & Sakurai, 2007; Iwata et al., 2011, 2015; Hirohashi & Iwata, 2013; Hirohashi et al., 2013, 2016). In this species, size-dependent alternative mating tactics were associated with a morphological switch point in spermatophore length, with large consort males producing discontinuously longer spermatophores than small sneakers (Iwata & Sakurai, 2007). Interestingly, some findings on H. bleekeri were contrary to the expectations of sneaks and guards theoretical models (Parker, 1990b). Sneakers, which face higher risks of sperm competition than consorts, should have higher ejaculate expenditure. However, compared to consorts, they produce shorter spermatophores that contain a smaller amount of longer sperm cells (Iwata & Sakurai, 2007; Iwata et al., 2011). It is still not known, however, if squid male morphs also differ in gonadal investment, i.e., in testis mass and sperm and spermatophore production.

The loliginid squid Doryteuthis plei (Blainville, 1823) is one of the most abundant cephalopods on the continental shelf off southern and southeastern Brazil, and constitutes an important fishery resource (Perez et al., 2002). Previous studies regarding population dynamics and reproductive biology of D. plei have reported that, within this species, there is a polimodal distribution of male body size and the coexistence of two maturation groups, comprising a class of small-sized and another of large-sized mature individuals (Perez et al., 2002; Postuma & Gasalla, 2014). Behavioral data obtained both in the field and in captivity reveal that, in this species, males may adopt both head-to-head and parallel mating positions, typical of other loliginids (Hanlon & Messenger, 1996). Morphological assessments have also shown that spermatangia placed near the oviduct opening inside the female presented a distinct morphology from those placed near the seminal receptacle (Marian, 2012).

Despite the promising lines of evidence of alternative mating tactics and the presence of different-sized mature males in D. plei, no studies have been conducted to associate alternative reproductive behaviors with morphologically dimorphic males in this species. Here, we investigated the existence of intrasexual male dimorphism in D. plei associated with alternative mating tactics by examining the presence of a body size morphological switch point between male morphs. Moreover, we compare gonadal expenditures between alternative morphs to test if they fit the predictions of sneaks and guards theoretical models.

Materials and methods

Data collection

We collected a total of 287 males of D. plei, ranging from 88 to 327 mm of mantle length (ML), during the summer months of 2014–2016 off the northern coast of São Sebastião Island, between 23o44′00″S and 45o17′50″W (Ilhabela municipality, São Paulo state, Brazil). We captured all individuals by hand-jigging and transported them to the Marine Biology Center of the University of São Paulo (CEBIMar-USP), where we kept them in tanks with a continuous flow of fresh seawater. Before dissection, we over-anesthetized all males in an isotonic solution of MgCl2, following guidelines for the welfare of cephalopods (Moltschaniwskyj et al., 2007). We classified all captured individuals as fully mature according to the maturity scale for D. plei proposed by Perez et al. (2002).

For all collected males, we measured dorsal mantle length and hectocotylus (i.e., left ventral arm, specialized for spermatophore transfer during mating) length to the nearest millimeter, and total body weight to the nearest 0.01 g. After dissection, we also recorded testis and spermatophoric complex weight (also to the nearest 0.01 g). Spermatophoric complex comprises two distinct structures, the spermatophoric organ and the Needham’s sac (or spermatophoric sac), which are responsible for spermatophore production and storage, respectively. We fixed spermatophores from Needham’s sac in a modified Karnovsky’s fixative (see Marian, 2012) for 4 h at 4°C. After fixation, we randomly selected five intact spermatophores from each male for assessments of total spermatophore length (SpL), sperm mass length (SML), and sperm mass diameter (SMD) (Fig. 2a, b), and then calculated the sperm mass length index, i.e., SMLI = (SML/SpL)(100), and estimated the volume of the sperm mass (SMV = π(SMD/2)2SML). Then, we calculated mean and standard deviation of all measurements. We obtained all measurements using NIH ImageJ Software, based on pictures taken under a Discovery V20 Zeiss stereomicroscope equipped with an AxioCam MRc 5 Zeiss digital camera.

Statistical analysis

We used the method developed by Eberhard & Gutiérrez (1991) to test for the existence of male intrasexual dimorphism. This approach relies on a series of statistical models for detecting the position of a size-dependent morphological switch point between two male morphs. Basically, this method comprises first a test for nonlinearity between mantle length and each tested characteristic (a potential indicative of male dimorphism), and then tests for the existence of a switch point (i.e., a mantle length value at which individuals would switch between two morphs), which was determined as the value where (1) the linear slope of the relationship between mantle length and each characteristic was altered or (2) the change in the characteristic was discontinuous, rather than continuous. A detailed description of the regression model (and equations used) is provided as a supplemental material (see Online Resource 1).

If the Eberhard & Gutiérrez (1991) analysis detected that the relationship between mantle length and any characteristic tested (i.e., hectocotylus length, spermatophore length, sperm mass length, sperm mass length index, and sperm mass volume) was nonlinear, allowing us to demonstrate the existence of male dimorphism, our next step would be to compare gonadal investment between males of different morphs. Based on the body size switch point obtained in the analysis (and confirmed by the morphology of spermatangia; see below), we separated males into two categories: individuals with mantle length above the switch point were categorized as consorts, whereas those below the switch point were classified as sneakers. In order to compare gonadal investment between male morphs, we examined the static allometric relationships of reproductive organs weight (i.e., testis weight and spermatophoric complex weight) on soma weight (total body weight minus reproductive organs weight). Static allometry shows the relative growth of a certain characteristic in relation to body size across individuals of the same species, in the same life stage (e.g., Stern & Emlen, 1999; Bonduriansky, 2007). We calculated the reduced standardized major axis regression slopes and intercepts from log-transformed variables using the R package smatr Version 3.2.3 (Warton et al., 2006, 2012), which was developed specifically for allometry analyses. The principles of this approach are identical to ANCOVA, a procedure that has been recommended for comparisons of slopes and elevations (or intercepts) of testis allometry between males performing alternative mating tactics (Tomkins & Simmons, 2002), but assuming error on both axes of the allometry. First, we tested whether the reduced major axis slopes of reproductive organs allometries (i.e., testis and spermatophoric complex) were significantly different between male morphs. If no significant differences between sneaker and consort males slopes were detected, we fitted a common slope to the data and compared the allometric intercepts between groups using a Wald test (Warton et al., 2006). This approach allows us to test for differences in reproductive organs weight between males standardized to a common body size.

To test for differences in sperm size between sneakers and consorts (see section Spermatozoa morphometry), we built linear mixed models (LMMs) using the R package lme4 (Bates et al., 2005). For the LMMs, we used head and flagellum length as dependable variable, male phenotype (sneaker vs. consort) as fixed effect, and male identity as random effect. We used a likelihood ratio test (χ 2) to test the significance of fixed effect (male phenotype) on dependent variables (head and flagellum length). We conducted all statistical analyses in R 3.1.2 (R Development Core Team, 2008).

Spermatophoric reaction and spermatangia morphology

The spermatophoric reaction typically occurs during mating, when males transfer spermatophores to females. It encompasses a series of complex evagination processes of spermatophore membranes and tunics, resulting in the formation of the so-called spermatangium (i.e., everted spermatophore containing the sperm mass), which ends attached to the female body (Drew, 1919; Marian, 2012). During dissection of the spermatophore sac to extract intact spermatophores for morphometry (see Data Collection), several spermatophores were triggered and underwent the spermatophoric reaction, producing spermatangia. For 215 males (ca. 75% of our sample), we have preserved these spermatangia in Karnovsky’s fixative. From this sample, we randomly selected 32 spermatangia for morphometry.

To illustrate club-like and hook-like spermatangia formation during the spermatophoric reaction, we have also selected fresh spermatophores for in vitro experiments. We randomly removed spermatophores from the spermatophoric sac of anesthetized males and placed them on a Petri dish with filtered seawater. We induced the spermatophoric reaction by rapidly pulling the cap thread of the spermatophore, aided by dissecting forceps. We performed these analyses under a Zeiss SV-11 stereomicroscope and recorded them using a Sony NEX FS700 digital camera coupled to the stereomicroscope system.

Histology of the sperm mass

We selected 18 spermatophores preserved in Karnovsky’s fixative (consorts, N = 3; sneakers, N = 3; 3 spermatophores per male) for histological analyses of the sperm mass. The sperm masses were dissected from the spermatophores, dehydrated, and embedded in glycol methacrylate resin (Leica Historesin Embedding Kit, Leica Microsystems Nussloch GmbH, Germany). Serial transverse and sagittal Sects. (3 µm) were stained with Hematoxylin–Eosin and photographed with a DS-Ri1 camera coupled to a Nikon Eclipse 80i microscope.

Spermatozoa morphometry

We obtained samples of spermatozoa from spermatangia of a total of 60 anesthetized males (consorts, N = 30; sneakers, N = 30). After the formation of the spermatangium (see above), the contained sperm is released from its tip. We fixed a suspension of sperm released from a male’s spermatangia in 1.5 ml tubes containing 4% paraformaldehyde in phosphate-buffered saline (PBS 0.1 M, pH 7.2, at 4°C). After fixation, we subjected a volume of 60 μl of the sample to a cytocentrifuge technique (10,000 rpm, 5 min). This procedure allowed the concentration of sperm cells and their adhesion to a microscopic slide. After the slides were dried, we submerged them in Toluidine Blue stain for 2 min to enhance visualization of the flagella. We measured head and flagella length (from 20 spermatozoa per individual) using Nikon NIS-Elements Basic Research 3.2 software, based on pictures taken under a Nikon Eclipse 80i microscope (DIC microscopy technique, 630× magnification) equipped with a DS-Ri1 digital camera.


Male intrasexual dimorphism

The results of Eberhard & Gutiérrez (1991) analysis for detection of intrasexual dimorphism are summarized in Online Resource 2. Hectocotylus length showed a linear relationship with mantle length, indicating that there was no dimorphism for this characteristic (Fig. 1a). On the contrary, all internal characteristics (i.e., spermatophore length, sperm mass length, sperm mass length index, and sperm mass volume) presented a nonlinear relationship with mantle length, each one with a different body size switch point (Fig. 1b–e). For spermatophore length (SpL) and sperm mass length (SML), there was a change on slope between male morphs at the switch point, but the relationship was not discontinuous. The best-fit switch point for SpL was at ML 220.2 mm (sneakers: N = 143, SpL = 8.11 ± 1.32 mm; consorts: N = 144, SpL = 10.64 ± 0.58 mm; Fig. 1b). For SML, the best-fit switch point was at ML 215.1 mm (sneakers: N = 135, SML = 4.79 ± 1.29 mm; consorts: N = 152, SML = 7.12 ± 0.47 mm; Fig. 1c). However, for sperm mass length index (SMLI) and sperm mass volume (SMV), the relationship was discontinuous. The best-fit switch point for SMLI was at ML 169.3 mm (sneakers: N = 77, SMLI = 54.06 ± 7.26; consorts: N = 210, SMLI = 66.47 ± 2.92; Fig. 1d), and for SMV it was at 205.0 mm (sneakers: n = 126, SMV = 0.18 ± 0.12 mm3; consorts: n = 161, SMV = 0.51 ± 0.12 mm3; Fig. 1e).
Fig. 1

Test for the existence of male intrasexual dimorphism in males of Doryteuthis plei based on Eberhard & Gutiérrez (1991). a Linear relationship between mantle length and hectocotylus length. b Relationship between mantle length and spermatophore length (sneakers: Y = 2.056 + 0.037X; consorts: Y = 7.118 + 0.014X), showing no discontinuity but a change in linear slope at the switch point. c Relationship between mantle length and sperm mass length (sneakers: Y = −1.127 + 0.036X; consorts: Y = 4.600 + 0.010X), showing no discontinuity but a change in linear slope at the switch point. d Relationship between mantle length and sperm mass length index (SMLI) (sneakers: Y = 27.459 + 0.191X; consorts: Y = 60.58 + 0.025X), showing discontinuity at the switch point. e Relationship between mantle length and sperm mass volume (sneakers: Y = −0.317 + 0.003X; consorts: Y = −0.179 + 0.003X), showing discontinuity at the switch point. Dashed vertical lines show body size switch point b at 220.2 mm, c at 215.1 mm, d at 169.3 mm, and e at 205.0 mm of mantle length

The unforeseen detection of different body size switch points led us to investigate the morphology of spermatangia produced by different-sized males in order to clarify our findings. Previous studies on D. plei have shown that spermatangia placed near the oviduct and near the seminal receptacle of females have dimorphic morphologies (Fig. 3c, d; Marian, 2012), which, for other loliginid species, correspond to different mating tactics (i.e., male-parallel and head-to-head, respectively; Fig. 3a, b). Based on the spermatangia obtained from fresh spermatophores (Table 1; see videos in Online Resources 3 and 4), we then aimed at seeing if dimorphic spermatangia were possibly associated to (1) a switch point around 205.0–220.2 mm of mantle length (i.e., switch points obtained for spermatophore length, sperm mass length, and sperm mass volume; Fig. 1b, c, e) or to (2) a switch point around 169.3 mm of mantle length (i.e., to different sperm mass proportions within each spermatophore—SMLI; Fig. 1d). Spermatangia were clearly dimorphic: all males larger than 169.3 mm produced hook-like spermatangia (Fig. 2b, left; Table 1), whereas 90% of specimens smaller than 169.3 mm (52 out of 58) produced club-like spermatangia (Fig. 2b, right; Table 1), the only exception being six males with mantle lengths around the switch point (ML = 153, 161, 163, 165, 166, 168 mm) that had hook-like spermatangia.
Table 1

Morphology of spermatangia per size class

Size class (mm)

Total sample

Males with sampled spermatangia


n (%)


n (%)

ML < 169.3


58 (75%)


52 (90%)


6 (10%)

169.3 < ML < 220.2


56 (85%)


56 (100%)

ML > 220.2


101 (70%)


101 (100%)



215 (75%)


Spermatangia were obtained from fresh spermatophores (see text for details)

Fig. 2

Typical morphology of spermatophores and spermatangia from different male morphs of Doryteuthis plei. a Spermatophore of consort males (ML > 169.3 mm; above) and sneaker males (ML < 169.3 mm; below), observed under the stereomicroscope (×8.0). Arrowheads point to the cap thread. b Long hook-like spermatangium from consorts (left), and short club-like spermatangium from sneakers (right), observed under the stereomicroscope (×10.0). Arrowheads point to the opening, from where spermatozoa are released. CB cement body, EA ejaculatory apparatus, SM sperm mass, SML sperm mass length, SpL spermatophore length

Observations of different attachment sites inside females were consistent with these findings: spermatangia placed near the seminal receptacle had a club-like format (Fig. 3d), whereas those found near the oviduct membranes were hook-like shaped (Fig. 3c; see also Marian, 2012). Coupling our morphological observations of spermatangia sampled from males and females with our statistical data, we decided to establish the value of 169.3 mm of mantle length, rather than around 205.0–220.3 mm, as the switch point responsible for intrasexual dimorphism in this species (see also Discussion). Given that the ML 169.3 mm switch point was effective for ca. 97% of the sample for which we analyzed spermatangia morphology (209 out of 215 males), we classified all individuals smaller than this switch point as sneakers (N = 77; ML 88.0–169.0 mm) and the remaining ones as consorts (N = 210; ML 170.0–327 mm) for the analyses of gonadal investment, sperm mass volume, and spermatozoa morphometry.
Fig. 3

Alternative mating tactics and spermatophore deposition sites in Doryteuthis plei. a Male-parallel mating employed by consort males, which transfer spermatophores to the female mantle cavity near the oviduct opening. b Head-to-head mating employed by sneaker males, which transfer spermatophores to the female buccal membrane, near the seminal receptacle. c Female oviduct opening bordered by membranous lips, where spermatangia (arrowheads) are attached during male-parallel mating. d Ventral region of the female buccal membrane, where spermatangia (arrowheads) are attached during head-to-head mating. Spermatangia are placed near the opening of the female storage organ (arrow). bm buccal membrane, h hectocotylus, la left ventral arm, ll left lip of the oviduct opening, ra right ventral arm, rl right lip of oviduct opening, su sucker. Originally published in Marian (2012) and reproduced with permission

Gonadal investment

Standardized major axis regression slopes and their 95% confidence interval for both testis and spermatophoric complex allometries are presented in Table 2. Based on a likelihood ratio test, testis allometric slope showed a significant difference between morphs (Fig. 4a; Table 2): sneakers, which showed a steeper slope than consorts, presented hiperallometry (slope >1), whereas consorts presented hipoallometry (slope <1). Contrarily, allometric slopes for spermatophoric complex showed no significant difference between male morphs (Table 2). This result allowed us to compare the intercepts of these allometries in dimorphic males, fitting a common slope to the data (Table 3). We found a significant difference in the intercept (elevation) for spermatophoric complex of dimorphic males, with sneakers presenting higher elevation than consorts (Fig. 4b).
Table 2

Allometric relationships between soma and reproductive organs weight for sneaker and consort males of Doryteuthis plei


Sneakers (<ML 169.3 mm)

Consorts (>ML 169.3 mm)

Slope homogeneityb









χ 2













Spermatophoric complex











ML mantle length, ns not significant

aThe allometric slope is the standardized major axis slope (±95% confidence interval, CI). Slopes greater than 1 indicate hiperallometry, and slopes less than 1 indicate hipoallometry

bThe likelihood ratio test (χ 2) compares homogeneity of slopes (i.e., whether sneaker and consort males slopes are significantly different from each other)

Fig. 4

Allometric relationships between soma weight and a testis weight and b spermatophoric complex weight in consort (black circles) and sneaker males (gray circles) of the squid Doryteuthis plei. Values were log-transformed prior to the analysis. Reduced major axis slopes and elevations were calculated using SMATR package (see text)

Table 3

Allometric relationships between soma and spermatophoric complex weight for sneaker and consort males of Doryteuthis plei when a common slope is fitted to the data





Sneakers (<ML 169.3 mm)

Consorts (>ML 169.3 mm)

Δ elevationb











Spermatophoric complex















ML mantle length

aDifferences in elevations between sneaker and consort males were tested using Wald test

b“Δ elevation” is the change in the elevations of morphs (consorts–sneakers); hence negative values indicate greater investment by sneaker males

Sperm mass structure and volume

The sperm mass of sneaker and consort spermatophores was similar in structure. Within a homogenous eosinophilic matrix, spermatozoa are densely packed in layers, displaying a helical organization, i.e., with sperm being present in the central axis, around which the layers of sperm wind spirally (Fig. 5). The estimated total volume of the sperm mass, however, differed between the morphs, with consorts showing significantly larger volume (0.45 ± 0.15 mm3; N = 210) than sneakers (0.12 ± 0.09 mm3; N = 77) (unpaired t test, 22.35, P < 0.001).
Fig. 5

Sagittal (a, b) and cross-sections (c, d) of the sperm masses of consort (a, c) and sneaker (b, d) males of Doryteuthis plei. Sperm masses of both sneaker and consort males are composed of a homogenous eosinophilic matrix, within which spermatozoa are densely packed in layers, displaying a helical organization, i.e., with sperm being present in the central axis (arrowheads), around which the layers of sperm wind spirally

Spermatozoa morphometry

We found that consort males produced shorter spermatozoa (head length = 8.49 ± 0.19 µm; flagellum length = 71.44 ± 2.28 µm; N = 600; Figs. 6a, 7) than sneaker males (head length = 9.27 ± 0.20 µm; flagellum length = 81.78 ± 1.98 µm; N = 600; Figs. 6b, 7). These differences in spermatozoa size between male morphs were statistically significant (head length: χ 2 = 97.58; P < 0.001; flagellum length: χ 2 = 117.56; P < 0.001).
Fig. 6

Spermatozoa morphology from dimorphic males of the squid Doryteuthis plei. Samples collected from a consort and b sneaker males. Images taken using DIC microscopy technique (×630 magnification)

Fig. 7

Frequency distribution of spermatozoa length sampled from spermatangia of dimorphic males in the squid Doryteuthis plei. Size distribution of a head length and b flagellum length. Consort males are represented in dark gray, and sneaker males in light gray


Within conditional strategies (Hazel et al., 1990, 2004; Gross, 1996; Tomkins & Hazel, 2007), an individual status, which is often influenced by its physical or social condition (e.g., body size, age, nutritional condition, and competitive ability) or by external environmental variables (e.g., density of females, frequency of rival males, resource availability and quality), is the key determinant of male fitness and phenotype expression (Gross, 1996). The status-dependent model (Repka & Gross, 1995; Gross, 1996; Gross & Repka, 1998) suggests that, within a population, adoption of a certain tactic over its alternative typically involves a decision based on the individual’s relative condition, aiming at maximizing its fitness. For instance, males above a certain critical switch point have a higher fitness pay-off by adopting the dominant tactic (e.g., fighting), whereas those below this value benefit from expressing an alternative tactic (e.g., sneaking) (Emlen, 1997; Simmons et al., 1999; Hunt & Simmons, 2001). Within loliginid squids, alternative tactics may be an example of a conditional strategy: larger consorts benefit from territorial behavior, defending females from rival males, whereas small sneakers, which do not engage in contests or mate guarding, may achieve higher success from seeking sneaking copulation with paired females (Hanlon, 1996, 1998; Hanlon et al., 1997, 2002; Iwata et al., 2005; Shashar & Hanlon, 2013).

Our results with Doryteuthis plei show that spermatophore characteristics (i.e., spermatophore length, sperm mass length, sperm mass length index, and sperm mass volume) present a nonlinear relationship with mantle length, evidencing the existence of morphological switch points possibly associated with intrasexual dimorphism and alternative reproductive tactics within males. Previous studies on loliginids have shown that small sneakers are more likely to mate in the head-to-head position and place their spermatophores near the seminal receptacle of females (Fig. 3b, d), whereas consorts mate preferably in male-parallel position, placing their spermatophores near the oviduct (Fig. 3a, c) (Hanlon, 1996, 1998; Hanlon et al., 1997, 2002; Iwata et al., 2005; Shashar & Hanlon, 2013). Our data show that long hook-like spermatangia (Fig. 2b, left) found near the oviduct of females were similar to those obtained in vitro from males larger than ML 169.3 mm (i.e., switch point value obtained for SMLI), males which also presented discontinuously larger proportion of sperm mass comprised within each spermatophore (Fig. 2a, above). In turn, short club-like spermatangia (Fig. 2b, right) attached near the seminal receptacle of females were morphologically identical to those produced in vitro by most males smaller than ML 169.3 mm, males which also presented discontinuously lower values of SMLI (Fig. 2a, below). Our findings are consistent with the previous observations on dimorphic morphology of spermatangia attached to different sites of females (Heterololigo bleekeri, Iwata & Sakurai, 2007; Doryteuthis plei, Marian, 2012), showing that these different phenotypes are associated with alternative reproductive tactics. These results suggest that, in D. plei, males smaller than ML 169.3 mm act preferably as sneakers (i.e., adopt head-to-head copulations and place spermatophores near the seminal receptacle; Fig. 3b, d), whereas larger males act as consorts (i.e., adopt male-parallel mating and place spermatophores inside the female’s mantle cavity; Fig. 3a, c).

We found no morphological discontinuity for hectocotylus length, similar to the previous studies on the species H. bleekeri, which presented a linear relationship between all tested external characteristics and mantle length (Iwata & Sakurai, 2007). Despite that, in H. bleekeri, a morphological dimorphism in spermatophore length was detected: consorts produced discontinuously larger spermatophores and rope-like spermatangia, while sneakers produced smaller spermatophores and drop-like spermatangia (Iwata & Sakurai, 2007; Iwata et al., 2015). Although we have also detected a morphological dimorphism in spermatophore length (and also in sperm mass length and volume) for D. plei, combined evidence of spermatangia and spermatophores morphology did not allow us to associate a switch point at around ML 205.0–220.3 mm with alternative mating tactics, as it seems that all males within this body size range act as consorts.

One possible explanation for two distinct switch points (Fig. 1b–e) may be the coexistence of three male morphs in this species. In the lizard Uta stansburiana, for example, orange- and blue-throated males are territorial (orange males being more aggressive and maintaining larger territories), while yellow-throated males act opportunistically to acquire mates (Rhen & Crews, 2002). Both males and females exhibit alternative phenotypes derived from polymorphic genotypes, one locus with three alternative alleles being responsible for both throat color and behavior (Sinervo & Zamudio, 2001). Polymorphic genotypes, i.e., expression of different alleles at one or several loci, have also been associated to three alternative reproductive phenotypes within males in other animal groups, with or without dimorphic phenotypic expression within females (e.g., Shuster & Sassaman, 1997; Farrell et al., 2013; Lank et al., 2013). Although alternative reproductive tactics are widely known in loliginid squids (e.g., Hanlon & Messenger, 1996), the existence of three male phenotypes has never been suggested. We highlight that further investigation on this hypothesis and on whether there would be additional morphological and/or behavioral differences between male morphs is required. Moreover, genetic or environmental factors that may determine male phenotypes are still completely unexplored in loliginid squids, and further research on whether alternative male morphs are conditionally expressed or determined by polymorphic genotypes is necessary.

Theoretical games between sneaks and guards (Parker, 1990b) predict that, within a species, there is an asymmetry in sperm competition risk between males that employ different tactics. According to this model, sneakers should have a greater gonadal expenditure, as a way of compensating their disadvantageous behavioral role (Parker, 1990b). In the squid D. plei, the allometric slope of testis weight on soma weight differed between male morphs (Fig. 4a; Table 2): sneakers showed hiperallometry (slope >1), whereas consorts showed hipoallometry (slope <1). This finding might indicate a resource allocation trade-off, suggesting that, as males increase in body size, they invest differently in sexually selected structures (e.g., Simmons & Emlen, 2006). Sneakers show higher increments in testis mass (hence, sperm production), rather than overall body size. Moreover, they show relatively larger spermatophoric complexes than consorts, demonstrated by allometric differences on elevation coefficients between morphs (Fig. 4b; Table 3). These data suggest that sneakers present higher levels of spermatophore production (i.e., higher relative tissue growth in the spermatophoric organ) and/or that they may store more spermatophores (i.e., due to a relatively larger spermatophoric sac) than consorts per body mass. Our data on testis and spermatophoric complex weight seem to agree with the general concepts of sperm competition games between sneaks and guards, a framework which has gathered extensive support from a wide range of taxa (e.g., Dominey, 1980; Stockley & Purvis, 1993; Taborsky, 1994, 1998; Gage et al., 1995; Scaggiante et al., 1999; Simmons et al., 1999, 2007; Alonzo & Warner, 2000; Mazzoldi et al., 2000; Tomkins & Simmons, 2000; Rudolfsen et al., 2006).

Although sneakers have higher gonadal expenditure, we found that these males invest less in sperm mass per spermatophore than consorts (i.e., discontinuously lower SMLI values), a finding that seems like a contradiction to sperm competition models. Under high sperm competition risk, males are expected not only to have higher expenditures on spermatogenesis and testis size, but also transfer ejaculates containing more sperm during copulation (e.g., Gage et al., 1995; Stockley et al., 1997; Simmons et al., 1999; Tomkins & Simmons, 2000). If sneakers were shown to produce a much higher number of tiny sperm when compared to consorts, it could compensate a smaller size of sperm mass within each spermatophore. However, herein we have shown that sneakers produce spermatozoa ca. 15% longer than consorts, congruently to previous studies on the loliginid squid H. bleekeri, in which sneaker males were reported to present ca. 50% longer sperm than consort males (Iwata et al., 2011).

Traditionally, sperm competition theory assumes that, under a limited budget for gamete production, males should face a trade-off between sperm number and sperm size (Parker, 1993). Thus, when sperm competition follows a “fair raffle” (i.e., the probability of fertilization for each male is proportional to the relative number of his sperm in the female tract), males facing high risks of sperm competition are predicted to maximize sperm numbers, hence minimizing sperm size, as a way of increasing their chances of fertilization success (Parker, 1982, 1993, 1998). Although such trade-off does not seem to be an obligatory condition (e.g., Gomendio & Roldan, 1991, 1993; Gage & Morrow, 2003; Parker & Pizzari, 2010; but see Pitnick, 1996; Immler et al., 2011), in loliginid squids, spermatozoa are stored within a limited space inside each spermatophore. Thus, there must exist a trade-off between sperm length and total sperm number compacted within a single spermatophore. In H. bleekeri, sneakers pack ca. five times less (but longer) spermatozoa per spermatophore than consorts (Iwata et al., 2011). Herein, we have shown that sneakers produce smaller sperm mass per spermatophore (Fig. 1c, 2), longer spermatozoa (Figs. 6, 7), sperm organization, and density being apparently similar within the sperm mass (Fig. 5). These findings suggest lower contents of spermatozoa packaged per spermatophore when compared to consorts. Therefore, such trade-off between sperm number and size very likely occurs in D. plei as well.

Previous studies have shown that sperm length may increase (e.g., Gomendio & Roldan, 1991; Pitnick, 1996; Simmons et al., 1999; Morrow & Gage, 2000; Byrne et al., 2003), decrease (e.g., Stockley et al., 1997; Gage & Morrow, 2003; Immler & Birkhead, 2007), or be unrelated to sperm competition risk (e.g., Briskie et al., 1997; Hosken, 1997; Gage & Freckleton, 2003). According to Parker (1993), sperm length should evolve independently of sperm competition—except under specific conditions such as when sperm size boosts sperm survivorship and sperm competition risk increases with the delay between mating and fertilization. Additionally, sperm size may also be optimized independently from sperm competition pressures and be related, for example, to female tract extension (e.g., insects, Dybas & Dybas, 1981, Morrow & Gage, 2000; birds, Briskie & Montgomerie, 1992; Briskie et al., 1997). In loliginid squids, the existence of two different spermatangia deposition sites inside the female’s body may pose additional environmental pressures on both sperm and spermatophore morphology (e.g., Iwata et al., 2011).

Different sperm mass contents between sneakers and consorts could be related, for example, to anatomical constrains imposed by both size and location of the spermatangia deposition sites. The seminal receptacle membrane, located more externally on the female’s body, may grant some advantage for attachment of sneakers short club-like spermatangia, whereas longer and hook-like spermatangia would be more easily displaced or flushed. Additionally, as a consequence of their smaller size, sneaker spermatangia have their distal tips (from where sperm is released) close to the opening of the female sperm storage organ (Fig. 3d), which would clearly not be the case for consort spermatangia. Although the dynamics of sperm attraction and storage in the seminal receptacle are still obscure (e.g., Hirohashi & Iwata, 2013), the distance between the site of sperm release (spermatangium’s distal tip) and the opening of the sperm storage organ could constrain spermatangium size (and hence, sperm mass size) of sneakers. This would be in agreement with the sperm-swarming phenomenon observed in H. bleekeri sneakers, a strategy hypothesized to retain sperm near the buccal membrane and the seminal receptacle (Hirohashi et al., 2013).

Longer sperm, on the other hand, may be advantageous in preventing displacement or dilution near the buccal membrane of females when compared to consorts, which attach their spermatophores internally at the oviduct membranes. Also, longer sperm may enhance their competitiveness by conferring higher motility, superior viability or by exhibiting greater advantage in filling the limited space within or near their storage site or in displacing competing sperm (e.g., Gomendio & Roldan, 1991; Briskie & Montgomerie, 1992; Parker, 1993, 1998; Gage, 1994; Ball & Parker, 1996; Radwan, 1996; LaMunyon & Ward, 1998, 1999). In the squid H. bleekeri, sperm velocity was similar between different-sized spermatozoa from dimorphic males (Iwata et al., 2011), although longer sperm from sneakers showed higher longevity when compared to sperm from consorts (Hirohashi et al., 2016). Further research in D. plei is required to evaluate if sperm from dimorphic males differ in velocity and/or viability, and also how sperm competition and local environmental characteristics from different deposition sites may influence the evolution of different-sized spermatozoa in this squid species.

Another issue related to distinct fertilization environments and dimorphic sperm size, as far as we know not yet considered elsewhere, is the fact that sneaker sperm (both from spermatangia and from the seminal receptacle) should be able to cross the gelatinous envelopes surrounding the egg capsule to achieve fertilization. Egg capsule formation in squids is still poorly known, but oviducal and nidamental glands contribute with several layers of jellies to form inner and outer protective envelopes, respectively (Boletzky, 1986). Egg capsule formation is probably initiated within the mantle cavity as the eggs leave the oviduct opening (Boletzky, 1986), the egg capsule being then passed to the space between the arms to be finally deposited on the substrate (e.g., Fields, 1965). Therefore, it is reasonable to suppose that sneaker sperm may encounter additional resistance to reach the eggs than consort sperm, given the location and timing of egg capsule formation. Although Iwata et al. (2011) found no difference in velocity between different-sized sperm in H. bleekeri, future studies should investigate if other traits could be related to this difference in sperm size, such as traction or swimming efficiency in viscous fluids.

Finally, we hypothesize that lower sperm mass content in sneakers may also be related to a tactic of partitioning sperm among numerous mating opportunities. According to sperm competition games theory, there must be a trade-off between expenditure on ejaculate components (e.g., sperm number, sperm mass load, and spermatophore size) and expenditure on acquiring matings (e.g., mate searching). Thus, higher investments on each ejaculate generally increase fertilization success per mating, but decrease the number of matings achieved by a male (Parker, 1990a, 1998; Parker & Pizzari 2010). In loliginid squids, sneakers are assumed to present lower fertilization success than consorts (Buresch et al., 2001; Hanlon et al., 2002; Iwata et al., 2005; Shashar & Hanlon, 2013), possibly as a consequence of different deposition sites and fertilization timing. During egg release, sperm from hook-like spermatangia attached inside the female’s mantle cavity likely have privileged access to the egg capsules as they are expelled from the oviduct. Consequently, there will likely be fewer eggs available to be fertilized by sperm located near the seminal receptacle, as they will only contact the eggs when these pass through the female’s mouth region (Hanlon et al., 1997; Buresch et al., 2009). Hence, investing numerically on transferring larger numbers of (tiny) sperm to a single female may not enhance fertilization success for sneakers. However, if sneakers invest less ejaculate resources within a single mating, they may reduce their chances of becoming sperm depleted, thus optimizing the number of sired offspring by acquiring a higher number of mating opportunities (e.g., Warner et al., 1995; Wedell et al., 2002; Cornwallis & Birkhead, 2006).

Observations of D. plei reproductive behaviors in captivity (personal observation) seem to support the hypothesis raised above. Sneakers copulate much more frequently than consorts, which usually invest their time and energy into mate guarding of a single female, protecting her before, during, and after mating. Such behavioral divergence between male morphs is congruent with the previous reports for other loliginids (Hanlon, 1998; Hanlon et al., 1997, 2002). Combining these results, we suggest that, under higher risks of sperm competition, it may be advantageous for sneakers to present higher increment rates in testis mass and higher investment in spermatophore production. However, given the unusual complex aspects of squids mating systems (e.g., two sites for spermatangia deposition, different fertilization timing), other factors may influence sperm allocation patterns in this species. Sperm (and spermatophores) are costly (Dewsbury, 1982; Olsson et al., 1997), so males must allocate their ejaculates strategically across successive matings and within a single female. Consequently, partitioning sperm investments into several spermatophores and several copulation opportunities could be the best tactic for sneakers to increase fertilization success. Consorts, which face lower risks of sperm competition, may benefit from relatively higher investments in somatic growth, instead of gonads. Larger bodies may be more effective on precopulatory male–male competition, influencing the outcomes of physical contests between rival males and mate guarding, therefore guaranteeing higher reproductive success (Petrie, 1988; Warner et al., 1995; DiMarco & Hanlon, 1997; Taborsky, 1998; Alonzo & Warner, 2000; Mazzoldi et al., 2000; Hanlon et al., 2002; Parker et al., 2013).


This study provided the first evidence of intrasexual dimorphism in the squid Doryteuthis plei associated with alternative reproductive tactics. Small sneaker males produce spermatophores with discontinuously smaller sperm mass content and place short club-like shape spermatangia near the seminal receptacle of females through head-to-head mating. Large consort males, in turn, produce spermatophores almost completely filled by sperm mass and place long hook-like spermatangia near the oviduct opening by male-parallel copulation. This study was also the first to compare gonadal investment between males that adopt alternative tactics in cephalopods, revealing that sneakers show greater gonadal expenditure, which may be associated to a relatively higher rate of spermatophore production and/or storage, consistently with sneaks and guards models of sperm competition (Parker, 1990b). Contrastingly, however, sneakers seem to invest less sperm per spermatophore and produce longer spermatozoa than consorts. Under the complex scenario of squids mating system, we hypothesize that sneakers may increase their reproductive success by partitioning their ejaculates into extra mating opportunities, whereas consorts may benefit from higher investment rates on somatic growth, thus maximizing their chances of defeating rival males and mate guarding. Additionally, smaller sperm mass and longer spermatozoa in sneakers may not only be related to sperm competition pressures, but also to anatomical constrains related to different sites of spermatangia deposition and fertilization within the female’s body. In this context, squids may indeed fit the general predictions of sperm competition theoretical models, even with the existence of two very distinct sperm storage sites. Finally, we highlight that future research should be performed to investigate if dimorphic males adjust their investments on other ejaculate features that may increase their reproductive success, such as aspects of functional morphology of spermatophore and spermatangia, to evaluate how both squid mating systems and sperm competition influence the evolution of intrasexual dimorphism in squids.



This study was conducted as part of the first author’s Master dissertation in the Graduate Program in Zoology of the Department of Zoology, at the University of São Paulo (USP). The authors appreciate the financial support and grants provided by CAPES (Coordination for the Improvement of Higher Education Personnel), CAPES/PROEX and CNPq (National Council for Scientific and Technological Development—Proc. 477233/2013-9). The authors are also grateful for the support from the following laboratories and institutions: “Centro de Biologia Marinha” (logistic support for animal collection and maintenance, light microscopy facilities), “Laboratório de Cultivo e Estudos de Cnidaria” (light microscopy facilities), “Laboratório de Entomologia e Aracnologia” (light microscopy facilities), and “Laboratório de Biologia Celular de Invertebrados Marinhos” (citocentrifuge facilities for spermatozoa preparation). The authors specially thank Dr. Alvaro E. Migotto, from “Centro de Biologia Marinha,” for the invaluable assistance during in vitro experimentation and digital filming of the spermatophoric reaction, Dr. Glauco Machado (USP) for his helpful critical reading of the research proposal, Dr. Bruno Buzatto (University of Southwestern Australia) for the inestimable assistance and critical comments concerning statistical analyses, and the colleagues at CIAC 2015 Symposium (Hakodate, Japan) for all comments and suggestions that helped to improve this study. Glauco Machado, Bruno Buzatto, Vlad Laptikhovsky, and an anonymous reviewer helped to improve the quality of the manuscript and are greatly appreciated. This is a contribution of NP-BioMar (Research Center for Marine Biodiversity, USP).

Supplementary material

10750_2017_3145_MOESM1_ESM.pdf (103 kb)
Online Resource 1 Detailed description of the regression model (and equations used), adapted from Eberhard & Gutierrez (1991) original article. This analysis was applied herein for the detection of a body-size switch point (i.e., detection of male intrasexual dimorphism) in the squid Doryteuthis plei. (PDF 104 kb)
10750_2017_3145_MOESM2_ESM.pdf (142 kb)
Online Resource 2 Results of the analysis for detection of male intrasexual dimorphism in Doryteuthis plei, based on the models described in Eberhard & Gutierrez (1991). Regression coefficients and significance tests for each model are provided. For spermatophore length, the best-fit switch point was at 220.2 mm of mantle length (Model 3, adjusted R 2 = 0.891); for sperm mass length, the best-fit switch point was at 215.1 mm of mantle length (Model 3, adjusted R 2 = 0.874); for sperm mass length index (SMLI), the best-fit switch point was at 169.3 mm of mantle length (Model 2, adjusted R 2 = 0.690); and for sperm mass volume, the best-fit switch point was at 205.0 mm of mantle length (Model 2, adjusted R 2 = 0.789). (PDF 142 kb)

Online Resource 3 Digital filming of in vitro experiment of spermatophoric reaction and hook-like spermatangium formation in a consort male (mantle length = 252.0 mm) of the squid Doryteuthis plei, conducted under a Zeiss SV-11 stereomicroscope coupled to a Sony NEX FS700 digital camera. The spermatophore was dissected from the spermatophoric sac of the anesthetized male and placed in a Petri dish filled with filtered seawater. The spermatophoric reaction was induced by rapidly pulling the cap thread, aided by dissecting forceps. The reaction encompasses a series of complex evagination processes of membranes and tunics of the spermatophore, resulting on the formation of a hook-like spermatangium (i.e., everted spermatophore containing the sperm mass). (WMV 26364 kb)

Online Resource 4 Digital filming of in vitro experiment of spermatophoric reaction and club-like spermatangium formation in a sneaker male (mantle length = 106.0 mm) of the squid Doryteuthis plei, conducted under a Zeiss SV-11 stereomicroscope coupled to a Sony NEX FS700 digital camera. The spermatophore was dissected from the spermatophoric sac of the anesthetized male and placed in a Petri dish filled with filtered seawater. The spermatophoric reaction was induced by rapidly pulling the cap thread, aided by dissecting forceps. The reaction encompasses a series of complex evagination processes of membranes and tunics of the spermatophore, resulting on the formation of a club-like spermatangium (i.e., everted spermatophore containing the sperm mass). (WMV 9283 kb)


  1. Alonzo, S. H. & R. R. Warner, 2000. Allocation to mate guarding or increased sperm production in a Mediterranean wrasse. The American Naturalist 156: 266–275.CrossRefGoogle Scholar
  2. Andersson, M., 1994. Sexual Selection. Princeton University Press, Princeton.Google Scholar
  3. Ball, M. A. & G. A. Parker, 1996. Sperm competition games: external fertilization and “adaptive” infertility. Journal of Theoretical Biology 180: 141–150.CrossRefPubMedGoogle Scholar
  4. Bates, D., M. Machler, B. M. Bolker & S. C. Walker, 2005. Fitting linear mixed-effects models using lme4. Journal of Statistical Software 67: 1–48.Google Scholar
  5. Boletzky, S. V., 1986. Encapsulation of cephalopod embryos: a search for functional correlations. American Malacological Bulletin 4: 217–227.Google Scholar
  6. Bonduriansky, R., 2007. Sexual selection and allometry: a critical reappraisal of the evidence and ideas. Evolution 61: 838–849.CrossRefPubMedGoogle Scholar
  7. Briskie, J. V. & R. Montgomerie, 1992. Sperm size and sperm competition in birds. Proceedings of the Royal Society of London B: Biological Sciences 247: 89–95.CrossRefGoogle Scholar
  8. Briskie, J. V., R. Montgomerie & T. R. Birkhead, 1997. The evolution of sperm size in birds. Evolution 51: 937–945.CrossRefPubMedGoogle Scholar
  9. Buresch, K. M., R. T. Hanlon, M. R. Maxwell & S. Ring, 2001. Microsatellite DNA markers indicate a high frequency of multiple paternity within individual field-collected egg capsules of the squid Loligo pealeii. Marine Ecology Progress Series 210: 161–165.CrossRefGoogle Scholar
  10. Buresch, K. C., M. R. Maxwell, M. R. Cox & R. T. Hanlon, 2009. Temporal dynamics of mating and paternity in the squid Loligo pealeii. Marine Ecology Progresss Series 387: 197–203.CrossRefGoogle Scholar
  11. Byrne, P. G., L. W. Simmons & J. D. Roberts, 2003. Sperm competition and the evolution of gamete morphology in frogs. Proceedings of the Royal Society of London B: Biological Sciences 270: 2079–2086.CrossRefGoogle Scholar
  12. Cornwallis, C. K. & T. R. Birkhead, 2006. Social status and availability of females determine patterns of sperm allocation in the fowl. Evolution 60: 1486–1493.CrossRefPubMedGoogle Scholar
  13. Dewsbury, D. A., 1982. Ejaculate cost and mate choice. The American Naturalist 119: 601–610.CrossRefGoogle Scholar
  14. DiMarco, F. P. & R. T. Hanlon, 1997. Agonistic behavior in the squid Loligo plei (Loliginidae, Teuthoidea): Fighting tactics and the effects of size and resource value. Ethology 103: 89–108.CrossRefGoogle Scholar
  15. Dominey, W. J., 1980. Female mimicry in male bluegill sunfish - a genetic polymorphism? Nature 284: 546–548.CrossRefGoogle Scholar
  16. Drew, G. A., 1919. Sexual activities of the squid, Loligo pealii. II. The spermatophore; its structure, ejaculation and formation. Journal of Morphology 32: 379–435.CrossRefGoogle Scholar
  17. Dybas, L. K. & H. S. Dybas, 1981. Coadaptation and taxonomic differentiation of sperm and spermathecae in featherwing beetles. Evolution 35: 168–174.CrossRefPubMedGoogle Scholar
  18. Eberhard, W. G. & E. E. Gutiérrez, 1991. Male dimorphisms in beetles and earwigs and the question of developmental constraints. Evolution 45: 18–28.CrossRefPubMedGoogle Scholar
  19. Emlen, D. J., 1997. Alternative reproductive tactics and male-dimorphism in the horned beetle Onthophagus acuminatus (Coleoptera: Scarabaeidae). Behavioral Ecology and Sociobiology 41: 335–341.CrossRefGoogle Scholar
  20. Farrell, L. L., T. Burke, J. Slate, S. B. McRae & D. B. Lank, 2013. Genetic mapping of the female mimic morph locus in the ruff. BMC Genetics 14: 1–4.CrossRefGoogle Scholar
  21. Fields, W. G., 1965. The structure, development, food relations, reproduction, and life history of the squid Loligo opalescens Berry. Fisheries Bulletin 131: 1–108.Google Scholar
  22. Gadgil, M., 1972. Male dimorphism as a consequence of sexual selection. The American Naturalist 106: 574–580.CrossRefGoogle Scholar
  23. Gage, M. J. G., 1994. Associations between body size, mating pattern, testis size and sperm lengths across butterflies. Proceedings of the Royal Society of London B: Biological Sciences 258: 247–254.CrossRefGoogle Scholar
  24. Gage, M. J. G. & R. P. Freckleton, 2003. Relative testis size and sperm morphometry across mammals: no evidence for an association between sperm competition and sperm length. Proceedings of the Royal Society of London B: Biological Sciences 270: 625–632.CrossRefGoogle Scholar
  25. Gage, M. J. G. & E. H. Morrow, 2003. Experimental evidence for the evolution of numerous, tiny sperm via sperm competition. Current Biology 13: 754–757.CrossRefPubMedGoogle Scholar
  26. Gage, M. J. G., P. Stockley & G. A. Parker, 1995. Effects of alternative male mating strategies on characteristics of sperm production in the Atlantic salmon (Salmo salar): theoretical and empirical investigations. Philosophical Transactions of the Royal Society B: Biological Sciences 350: 391–399.CrossRefGoogle Scholar
  27. Gomendio, M. & E. R. S. Roldan, 1991. Sperm competition influences sperm size in mammals. Proceedings of the Royal Society of London B: Biological Sciences 243: 181–185.CrossRefGoogle Scholar
  28. Gomendio, M. & E. R. S. Roldan, 1993. Coevolution between male ejaculates and female reproductive biology in eutherian mammals. Proceedings of the Royal Society of London B: Biological Sciences 252: 7–12.CrossRefGoogle Scholar
  29. Gross, M. R., 1996. Alternative reproductive strategies and tactics: diversity within sexes. Trends in Ecology & Evolution 11: 92–98.CrossRefGoogle Scholar
  30. Gross, M. R. & J. Repka, 1998. Stability with inheritance in the conditional strategy. Journal of Theoretical Biology 192: 445–453.CrossRefPubMedGoogle Scholar
  31. Hanlon, R. T., 1996. Evolutionary games that squids play: fighting, courting, sneaking, and mating behaviors used for sexual selection in Loligo pealei. Biological Bulletin 191: 309–310.CrossRefPubMedGoogle Scholar
  32. Hanlon, R. T., 1998. Mating systems and sexual selection in the squid Loligo: How might commercial fishing on spawning squids affect them? California Cooperative Oceanic Fisheries Investigations Reports 39: 92–100.Google Scholar
  33. Hanlon, R. T. & J. B. Messenger, 1996. Cephalopod Behaviour. Cambridge University Press, Cambridge.Google Scholar
  34. Hanlon, R. T., M. R. Maxwell & N. Shashar, 1997. Behavioral dynamics that would lead to multiple paternity within egg capsules of the squid Loligo pealei. Biological Bulletin 193: 212–214.CrossRefPubMedGoogle Scholar
  35. Hanlon, R. T., M. J. Smale & W. H. H. Sauer, 2002. The mating system of the squid Loligo vulgaris reynaudii (Cephalopoda, Mollusca) off South Africa: fighting, guarding, sneaking, mating and egg laying behavior. Bulletin of Marine Science 71: 331–345.Google Scholar
  36. Hazel, W. N., R. Smock & M. D. Johnson, 1990. A polygenic model for the evolution and maintenance of conditional strategies. Proceedings of the Royal Society of London B: Biological Sciences 242: 181–187.CrossRefGoogle Scholar
  37. Hazel, W. N., R. Smock & C. M. Lively, 2004. The ecological genetics of conditional strategies. The American Naturalist 163: 888–900.CrossRefPubMedGoogle Scholar
  38. Hirohashi, N. & Y. Iwata, 2013. The different types of sperm morphology and behavior within a single species: Why do sperm of squid sneaker males form a cluster? Comunicative & Integrative Biology 6: e26729.CrossRefGoogle Scholar
  39. Hirohashi, N., L. Alvarez, K. Shiba, E. Fujiwata, Y. Iwata, T. Mohri, K. Inaba, K. Chiba, H. Ochi, C. T. Supuran, N. Kotzur, Y. Kakiuchi, U. B. Kaupp & S. A. Baba, 2013. Sperm from sneaker male squids exhibit chemotactic swarming to CO2. Current Biology 23: 1–7.CrossRefGoogle Scholar
  40. Hirohashi, N., M. Tamura-Nakano, F. Nakaya, T. Iida & Y. Iwata, 2016. Sneaker male squid produce long-lived spermatozoa by modulating their energy metabolism. The Journal of Biological Chemistry: jbc-M116.Google Scholar
  41. Hosken, D. J., 1997. Sperm competition in bats. Proceedings of the Royal Society of London B: Biological Sciences 264: 385–392.CrossRefGoogle Scholar
  42. Hunt, J. & L. W. Simmons, 2001. Status-dependent selection in the dimorphic beetle Onthophagus taurus. Proceedings of the Royal Society of London B: Biological Sciences 268: 2409–2414.CrossRefGoogle Scholar
  43. Immler, S. & T. R. Birkhead, 2007. Sperm competition and sperm midpiece size: no consistent pattern in passerine birds. Proceedings of the Royal Society of London B: Biological Sciences 274: 561–568.CrossRefGoogle Scholar
  44. Immler, S., S. Pitnick, G. A. Parker, K. L. Durrant, S. Lupold, S. Calhim & T. R. Birkhead, 2011. Resolving variation in the reproductive trade-off between sperm size and number. Proceedings of the National Academy of Sciences 108: 5325–5330.CrossRefGoogle Scholar
  45. Iwata, Y. & Y. Sakurai, 2007. Threshold dimorphism in ejaculate characteristics in the squid Loligo bleekeri. Marine Ecology Progress Series 345: 141–146.CrossRefGoogle Scholar
  46. Iwata, Y., H. Munehara & Y. Sakurai, 2005. Dependence of paternity rates on alternative reproductive behaviors in the squid Loligo bleekeri. Marine Ecology Progress Series 298: 219–228.CrossRefGoogle Scholar
  47. Iwata, Y., P. Shaw, E. Fujiwara, K. Shiba, Y. Kakiuchi & N. Hirohashi, 2011. Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours. BMC Evolutionary Biology 11: 1–9.CrossRefGoogle Scholar
  48. Iwata, Y., Y. Sakurai & P. Shaw, 2015. Dimorphic sperm-transfer strategies and alternative mating tactics in loliginid squid. Journal of Molluscan Studies 81: 147–151.CrossRefGoogle Scholar
  49. LaMunyon, C. W. & S. Ward, 1998. Larger sperm outcompete smaller sperm in the nematode Caenorhabditis elegans. Proceedings of the Royal Society of London B: Biological Sciences 265: 1997–2002.CrossRefGoogle Scholar
  50. LaMunyon, C. W. & S. Ward, 1999. Evolution of sperm size in nematodes: sperm competition favours larger sperm. Proceedings of the Royal Society of London B: Biological Sciences 266: 263–267.CrossRefGoogle Scholar
  51. Lank, D. B., L. L. Farrell, T. Burke, T. Piersma & S. B. McBae, 2013. A dominant allele controls development into female mimic male and diminutive female ruffs. Biology Letters 9: 1–4.CrossRefGoogle Scholar
  52. Marian, J. E. A. R., 2012. Spermatophoric reaction reappraised: novel insights into the functioning of the loliginid spermatophore based on Doryteuthis plei (Mollusca: Cephalopoda). Journal of Morphology 273: 248–278.CrossRefPubMedGoogle Scholar
  53. Mazzoldi, C., M. Scaggiante, E. Ambrosin & M. B. Rasotto, 2000. Mating system and alternative male mating tactics in the grass goby Zosterisessor ophiocephalus (Teleostei: Gobiidae). Marine Biology 137: 1041–1048.CrossRefGoogle Scholar
  54. Moltschaniwskyj, N. A., K. Hall, M. R. Lipinski, J. E. A. R. Marian, M. Nishiguchi, M. Sakai, D. J. Shulman, B. Sinclair, D. L. Sinn, M. Staudinger, R. Van Gelderen, R. Villanueva & K. Warnke, 2007. Ethical and welfare considerations when using cephalopods as experimental animals. Reviews in Fish Biology and Fisheries 17: 455–476.CrossRefGoogle Scholar
  55. Morrow, E. H. & M. J. G. Gage, 2000. The evolution of sperm length in moths. Proceedings of the Royal Society of London B: Biological Sciences 267: 307–313.CrossRefGoogle Scholar
  56. Olsson, M., T. Madsen & R. Shine, 1997. Is sperm really so cheap? Costs of reproduction in male adders, Vipera berus. Proceedings of the Royal Society of London B: Biological Sciences 264: 455–459.CrossRefGoogle Scholar
  57. Parker, G. A., 1970. Sperm competition and its evolutionary consequences in the insects. Biological Reviews 45: 525–567.CrossRefGoogle Scholar
  58. Parker, G. A., 1982. Why are there so many tiny sperm? Sperm competition and the maintenance of two sexes. Journal of Theoretical Biology 96: 281–294.CrossRefPubMedGoogle Scholar
  59. Parker, G. A., 1990a. Sperm competition games: raffles and roles. Proceedings of the Royal Society of London B: Biological Sciences 242: 120–126.CrossRefGoogle Scholar
  60. Parker, G. A., 1990b. Sperm competition games: sneaks and extra-pair copulations. Proceedings of the Royal Society of London B: Biological Sciences 242: 127–133.CrossRefGoogle Scholar
  61. Parker, G. A., 1993. Sperm competition games: sperm size and sperm number under adult control. Proceedings of the Royal Society of London B: Biological Sciences 253: 245–254.CrossRefGoogle Scholar
  62. Parker, G. A., 1998. Sperm competition and the evolution of ejaculates: towards a theory base. In Birkhead, T. R. & A. P. Møller (eds), Sperm Competition and Sexual Selection. Academic Press, San Diego: 3–54.CrossRefGoogle Scholar
  63. Parker, G. A. & T. Pizzari, 2010. Sperm competition and ejaculate economics. Biological Reviews 85: 897–934.PubMedGoogle Scholar
  64. Parker, G. A., M. A. Ball, P. Stockley & J. G. Gage, 1997. Sperm competition games: a prospective analysis of risk assessment. Proceedings of the Royal Society of London B: Biological Sciences 264: 1793–1802.CrossRefGoogle Scholar
  65. Parker, G. A., C. M. Lessells & L. W. Simmons, 2013. Sperm competition games: a general model for precopulatory male-male competition. Evolution 67: 95–109.CrossRefPubMedGoogle Scholar
  66. Perez, J. A. A., D. C. Aguiar & U. C. Oliveira, 2002. Biology and population dynamics of the long-finned squid Loligo plei (Cephalopoda: Loliginidae) in southern Brazilian waters. Fisheries Research 58: 267–279.CrossRefGoogle Scholar
  67. Petrie, M., 1988. Intraspecific variation in structures that display competitive ability: large animals invest relatively more. Animal Behavior 36: 1174–1179.CrossRefGoogle Scholar
  68. Pitnick, S., 1996. Investments in testes and the cost of making long sperm in Drosophila. The American Naturalist 148: 57–80.CrossRefGoogle Scholar
  69. Postuma, F. A. & M. A. Gasalla, 2014. Reproductive activity of the tropical arrow squid Doryteuthis plei around São Sebastião Island (SE Brazil) based on a 10-year fisheries monitoring. Fisheries Research 152: 45–54.CrossRefGoogle Scholar
  70. R Development Core Team, 2008. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL
  71. Radwan, J., 1996. Intraspecific variation in sperm competition success in the bulb mite: a role for sperm size. Proceedings of the Royal Society of London B: Biological Sciences 263: 855–859.CrossRefGoogle Scholar
  72. Rhen, T. & D. Crews, 2002. Variation in reproductive behavior within a sex: neural systems and endocrine activation. Journal of Neuroendocrinology 14: 517–531.CrossRefPubMedGoogle Scholar
  73. Repka, J. & M. R. Gross, 1995. The evolutionarily stable strategy under individual condition and tactic frequency. Journal of Theoretical Biology 176: 27–31.CrossRefPubMedGoogle Scholar
  74. Rudolfsen, G., L. Figenschou, I. Folstad, H. Tveiten & M. Figenschou, 2006. Rapid adjustments of sperm characteristics in relation to social status. Proceedings of the Royal Society of London B: Biological Sciences 273: 325–332.CrossRefGoogle Scholar
  75. Scaggiante, M., C. Mazzoldi, C. W. Petersen & M. B. Rasotto, 1999. Sperm competition and mode of fertilization in the grass goby Zosterisessor ophiocephalus (Teleostei: Gobiidae). Journal of Experimental Zoology 283: 81–90.CrossRefGoogle Scholar
  76. Shashar, N. & R. T. Hanlon, 2013. Spawning behavior dynamics at communal egg beds in the squid Doryteuthis (Loligo) pealeii. Journal of Experimental Marine Biology and Ecology 447: 65–74.CrossRefGoogle Scholar
  77. Shuster, S. M. & C. Sassaman, 1997. Genetic interaction between male mating strategy and sex ratio in a marine isopod. Nature 388: 373–377.CrossRefGoogle Scholar
  78. Shuster, S. M. & M. J. Wade, 2003. Mating systems and strategies. Princeton University Press, Princeton.Google Scholar
  79. Simmons, L. W. & D. J. Emlen, 2006. Evolutionary trade-off between weapons and testes. Proceedings of the National Academy of Sciences 103: 16346–16351.CrossRefGoogle Scholar
  80. Simmons, L. W., D. J. Emlen & J. L. Tomkins, 2007. Sperm competition games between sneakers and guards: a comparative analysis using dimorphic male beetles. Evolution 61: 2684–2692.CrossRefPubMedGoogle Scholar
  81. Simmons, L. W., J. L. Tomkins & J. Hunt, 1999. Sperm competition games played by dimorphic male beetles. Proceedings of the Royal Society of London B: Biological Sciences 266(145): 150.Google Scholar
  82. Sinervo, B. & K. R. Zamudio, 2001. The evolution of alternative reproductive strategies: fitness differential, heritability, and genetic correlation between the sexes. The American Genetic Association 92: 198–205.Google Scholar
  83. Stern, D. L. & D. J. Emlen, 1999. The developmental basis for allometry in insects. Development 126: 1091–1101.PubMedGoogle Scholar
  84. Stockley, P. & A. Purvis, 1993. Sperm competition in mammals: a comparative study of male roles and relative investment in sperm production. Functional Ecology 7: 560–570.CrossRefGoogle Scholar
  85. Stockley, P., M. J. G. Gage, G. A. Parker & A. P. Møller, 1997. Sperm competition in fishes: the evolution of testis size and ejaculate characteristics. The American Naturalist 149: 933–954.CrossRefPubMedGoogle Scholar
  86. Taborsky, M., 1994. Sneakers, satellites, and helpers: parasitic and cooperative behavior in fish reproduction. Advances in the study of behavior 23: 1–100.CrossRefGoogle Scholar
  87. Taborsky, M., 1998. Sperm competition in fish: ‘bourgeois’ males and parasitic spawning. Trends in Ecology & Evolution 13: 222–227.CrossRefGoogle Scholar
  88. Taborsky, M., R. F. Oliveira & J. Brockmann, 2008. The evolution of alternative reproductive tactics: concepts and questions. In Oliveira, R. F., M. Taborsky & H. J. Brockmann (eds), Alternative Reproductive Tactics: An Integrative Approach. Cambridge University Press, Cambridge: 1–21.Google Scholar
  89. Tomkins, J. L. & L. W. Simmons, 2000. Sperm competition games played by dimorphic male beetles: fertilization gains with equal mating access. Proceedings of the Royal Society of London B: Biological Sciences 267: 1547–1553.CrossRefGoogle Scholar
  90. Tomkins, J. L. & L. W. Simmons, 2002. Measuring relative investment: a case study of testes investment in species with alternative male reproductive tactics. Animal Behaviour 63: 1009–1016.CrossRefGoogle Scholar
  91. Tomkins, J. L. & W. Hazel, 2007. The status of the conditional evolutionarily stable strategy. TRENDS in Ecology and Evolution 22: 522–528.CrossRefPubMedGoogle Scholar
  92. Tomkins, J. L., J. S. Kotiaho & N. R. LeBas, 2005. Matters of scale: positive allometry and the evolution of male dimorphisms. The American Naturalist 165: 389–402.CrossRefPubMedGoogle Scholar
  93. Warner, R. R., D. Y. Shapiro, A. Marcanato & C. W. Petersen, 1995. Sexual conflict: males with highest mating success convey the lowest fertilization benefits to females. Proceedings of the Royal Society of London B: Biological Sciences 262: 135–139.CrossRefGoogle Scholar
  94. Warton, D. I., I. J. Wright, D. S. Falster & M. Westoby, 2006. Bivariate line-fitting methods for allometry. Biological Reviews 81: 259–291.CrossRefPubMedGoogle Scholar
  95. Warton, D. I., R. A. Duursma, D. S. Falster & S. Taskinen, 2012. SMATR 3 - an R package for estimation and inference about allometric lines. Methods in Ecology and Evolution 3: 257–259.CrossRefGoogle Scholar
  96. Wedell, N., M. J. G. Gage & G. A. Parker, 2002. Sperm competition, male prudence and sperm-limited females. Trends in Ecology & Evolution 17: 313–320.CrossRefGoogle Scholar
  97. Zeidberg, L. D., 2009. First observations of “sneaker mating” in the California market squid, Doryteuthis opalescens, (Cephalopoda: Myopsida). Marine Biodiversity Records 2: 1–4.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2017

Authors and Affiliations

  • Lígia H. Apostólico
    • 1
  • José E. A. R. Marian
    • 1
  1. 1.Departamento de Zoologia, Instituto de BiociênciasUniversidade de São PauloSão PauloBrazil

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