Mar Biol DOI 10.1007/s00227-008-1059-zORIGINAL PAPER Is Hippolyte williamsi gonochoric or hermaphroditic? A multi-approach study and a review of sexual systems in Hippolyte shrimps Nuxia L. Espinoza-Fuenzalida ? Martin Thiel ? Enrique Dupre ? J. Antonio Baeza Received: 12 March 2008 / Accepted: 29 August 2008 ? Springer-Verlag 2008 Abstract Sexual systems vary considerably among caridean shrimps and while most species are gonochoric, others are described as sequential protandric hermaphro- dites or simultaneous hermaphrodites with an early male phase. At present, there is confusion about the sexual system exhibited by several species mostly because those studies attempting to reveal their sexual system draw inferences solely from the distribution of the sexes across size classes. Here we investigated the sexual sys- tem of the shrimp Hippolyte williamsi from Chile to determine if the species is protandric or gonochoric with sexual dimorphism (males smaller than females). Mor- phological identiWcation and size frequency distribu- tions indicated that the population comprised small males, small immature females, and large mature females, which was conWrmed by dissections. No transi- tional individuals were found. Males maintained in the laboratory molted 1?8 times, and many grew up to reach sizes observed in only a small fraction of males in the Weld. No indication of sex change was recorded. Our results indicate that H. williamsi is a sexually dimorphic gonochoric species and emphasizes the importance of using several kinds of evidence (size measurements, growth experiments, morphological dissections, and his- tological studies) to reveal the sexual system of Hippo- lyte species. Whether the observed size dimorphism between males and females in many species of Hippolyte is expression of contrasting sexual and natural selection, and whether divergent sexual Wtness functions can con- tribute to the evolution of hermaphroditism remains to be revealed in future studies. Introduction Sexual systems vary considerably in the Caridea. Most shrimps are gonochoric, with individuals reproducing as male or female during their entire life (Bauer 2001, 2004). Examples include Rhynchocinetes typus (Thiel and Hinojosa 2003), Macrobrachium rosenbergii (Karplus 2005), Hippolyte obliquimanus (Terossi et al. 2008), and most species from the diverse genus Alpheus (Correa and Thiel 2003; Anker et al. 2006). Other species are protandric her- maphrodites, in which individuals change from male to female (e.g., Pandalus?Butler 1964, 1980; HoVman 1972, see also review by Bergstr?m 2000). Several variants of protandry have been reported, such as mixed protandry with primary females in Processa edulis (No?l 1976) and Crangon crangon (Boddeke et al. 1991; Schatte and Sabo- rowski 2006), or with primary males in Thor manningi Communicated by B. Notes. N. L. Espinoza-Fuenzalida ? M. Thiel ? E. Dupre ? J. A. Baeza Facultad Ciencias del Mar, Universidad Cat?lica del Norte, Larrondo 1281, Coquimbo, Chile e-mail: nef001@ucn.cl M. Thiel Center for Advanced Studies in Arid Systems, CEAZA, Coquimbo, Chile e-mail: thiel@ucn.cl J. A. Baeza Smithsonian Marine Station at Fort Pierce, 701 Seaway Drive, Fort Pierce, FL 34949, USA J. A. Baeza (&) Smithsonian Tropical Research Institute, Apartado Postal 0843-03092, Balboa, Anc?n, Republic of Panama e-mail: baezaa@si.edu123 Mar Biol(Bauer and VanHoy 1996). No study so far has reported protogyny (female Wrst) among shrimps (e.g., as in the iso- pod Gnorimosphaeroma oregonense?Brook et al. 1994). Finally, simultaneous hermaphroditism with an early pro- tandric phase, i.e. adolescent protandry sensu Ghiselin (1974) or protandric simultaneous hermaphroditism sensu Bauer and Holt (1998), where individuals initially mature as males and later become simultaneous hermaphrodites, has been described for various species of Lysmata and might be more common than previously assumed (Baeza 2008a). Interestingly, within the Caridea there is also consider- able variation of sexual systems at the generic level. For instance, while Thor manningi has been recognized as a protandric species (Bauer 1986), studies of its congeners, T. dobkini and T. Xoridanus, indicated that they are gonochoric (Bauer and VanHoy 1996; Bauer 2004). This variability in gender expression among closely related species suggests that caridean shrimps might be used as models to test the role of the environment in explaining evolutionary innovations in gender in marine invertebrates. However, at present, comparative studies are precluded due to the scar- city of information about the sexual system of many species in diverse families. While the sexual system of most shrimp species is well known, for some species the distribution of the sexes among individuals is not clear. For several species there is controversy with respect to their sexual system. For instance, initial studies on the population structure of Cran- gon franciscorum indicated that this species was gonoch- oric (Siegfried 1980, 1989). The body size of the males was almost invariably smaller than that of the females, a condi- tion explained at that time by sex-speciWc mortality or migratory behavior (op. cit.). The sexual dimorphism reported for this species might also indicate strong selective pressures among males (but not females) favoring a small male condition (see review in Baeza and Thiel 2007). On the other hand, recent studies combining population dynamics, gonad dissections, and rearing of shrimps in cap- tivity have demonstrated that C. franciscorum is actually protandric (Gavio et al. 2006). A similar sexual system (i.e., facultative protandry) has been reported for the conge- ner Crangon crangon, where a small proportion of males in the population change sex later in life (Boddeke et al. 1991; Schatte and Saborowski 2006). To the best of our knowledge, the caridean family with the highest diversity of sexual systems is the Hippolytidae. It contains species that are gonochoric, protandric hermaph- rodites and simultaneous hermaphrodites (Bauer 2004; Bauer and VanHoy 1996; Baeza 2006; Terossi et al. 2008). Furthermore, there are species whose sexual system remains controversial such as Hippolyte inermis, which is reported either as protandric (Zupo 2000, 2001; Zupo and Buttino 2001; Zupo and Messina 2007) or gonochoric (Cobos et al. 2005). Because the two studies examining the sexual system of this species were conducted in localities hundreds of kilometers apart (i.e., Italy and Spain), this controversy might well be explained if the two sampled populations pertain to two diVerent cryptic species, each one with a diVerent sexual system. Data on gender size fre- quency, gonad anatomy, secondary sexual characters and development in laboratory culture are necessary to deter- mine the sexual system of marine shrimps (e.g. Terossi et al. 2008). The aim of the present study was to elucidate the sexual system of Hippolyte williamsi Schmitt 1924, a small spe- cies that inhabits seaweed meadows and seagrass beds of Heterozostera chilensis (0?8 m depth) in northern-central Chile (Gonz?lez 1992; Stotz and Gonz?lez 1997). Studies on the general ecology of this shrimp are lacking. However, preliminary observations indicated that males were smaller than females. Although this size diVerence between the sexes is suggestive of protandry, as reported for its conge- ner Hippolyte inermis (Zupo and Messina 2007; but see Cobos et al. 2005), gonochorism cannot be ruled out as an alternative pattern of gender expression (see Terossi et al. 2008). To properly examine the sexual system of H. wil- liamsi, we sampled in diVerent seasons to document the population structure and sexual characters. Additionally, we reared males in the laboratory in three diVerent social environments (in isolation, together with females, or in presence of other males), recording possible sex change and death. Materials and methods Collection and maintenance of shrimps Shrimps were collected with kick nets at low tides from a shallow subtidal sea grass bed of Heterozostera chilensis at Puerto Aldea (S 30? 17, W 71? 36), Tongoy, Coquimbo, Chile, during December 2005, October 2006 and January 2007. The species was identiWed using d?Udekem d?Acoz (1996, 1997, 2007) and the key of Wicksten (1990), and our original identiWcation was kindly conWrmed by Sammy De Grave (Oxford University Museum of Natural History, accession number of the specimen deposited at the museum: OUMNH 2007-12-0002). Immediately after col- lection, shrimp were either preserved in 5% formalin or kept alive and placed in large plastic buckets and/or ice chests (only for samples from 2006 and 2007) to be trans- ported to the seawater laboratories of Universidad Cat?lica del Norte, Coquimbo, Chile. Shrimps were maintained in 55 L plastic containers with circulating Wltered seawater at water temperatures ranging123 Mar Biolfrom 12 to 20?C (14?18?C during October?December 2006; 17?20?C in January?February 2007; 14?18?C in March-April 2007; 12?16?C May?June 2007) and 34.3? 34.7 ppt salinity and were fed daily with planktonic micro- algae (Chaetoceros spp.), benthic diatoms (Grammatophora marina, Melosira nummuloides, Navicula sp. Nitzschia longissima) scraped from the surface of large tanks main- tained in the laboratory, Wsh food (TretraFin?), and/or chopped fresh Emerita analoga, before being selected for experiments. Previous studies had highlighted the impor- tance of food in development of hippolytid shrimp (see e.g. Zupo 2001; Zupo and Messina 2007). Thus, in order to avoid potential biases caused by a highly speciWc diet (e.g. only diatoms, or only particular strains of microalgae), we pro- vided a diet of microalgae and animal food representative of the diet of H. williamsi in its natural environment. Measurements and morphological examination of shrimps Preserved shrimps were used for the examination of repro- ductive anatomy and measurements of body length. Under the dissecting microscope, the carapace length (CL, mea- sured from the orbital margin to the posterior border of the carapace) of each preserved shrimp was measured. Before measuring the shrimps, all preserved specimens were iden- tiWed as males, ovigerous and non-ovigerous females, using the presence or absence of embryos beneath the abdomen and of male gonopores on the coxae of the Wfth pereiopods. The examination of these characters permitted rapid identi- Wcation of males and females. A total of 50 males (CL = 2.1?4.2 mm) and 50 females (CL = 3.1?4.4 mm) were selected for a more detailed examination of their sex- ual anatomy to conWrm gender identity based on external characters. Mature specimens were deWned as males or females by the presence (females) or absence (males) of brooded embryos and a large rostrum (rostrum long, reach- ing beyond the Wrst third of the scaphocerite in females, rostrum shorter than the eyes in males?Wicksten 1990), and by the presence (males) or absence (females) of a gono- pore on the coxae of the Wfth pair of legs, vas deferentia connected to the gonads, and an appendix masculina on the endopod of the second pleopod. The sex of 306 shrimps could not be reliably identiWed as either male or female by using only external morpholog- ical characters. These 306 individuals were dissected (together with the other 50 males and 50 females above) to reveal the presence or absence of male and female internal characters. Gonads were extracted and examined for the presence or absence of testes, ovaries, vas deferens, and oviducts. Then, the Wrst and second pleopods of each one of these shrimps were dissected and the presence or absence of appendices masculinae was recorded. During dissec- tions, we were particularly interested in recognizing ?inter- mediate? individuals, i.e. males with maturing ovaries and/ or shrimps with both male and female characteristics. For instance, individuals carrying embryos beneath the abdo- men (female character) but with appendices masculinae on the second pleopods (male characters) would have been considered intermediate shrimps. The existence of interme- diates has been reported before for protandric and protan- dric-simultaneous hermaphroditic shrimps (Bauer and Holt 1998; Bauer 2004; Gavio et al. 2006). An ideal character denoting ?transitional? individuals in a protandric species is the presence of maturing ovaries that can be observed through the carapace in a certain propor- tion of the males. Unfortunately, it was not possible to look for such a character before dissections in our preserved specimens because their carapace became white and opaque. This color change caused by preservation impedes any observation of internal organs (including the gonads). In order to characterize the external and internal charac- teristics of males and females, a sample was collected in October 2007 and immature and mature shrimps were dis- sected and prepared for scanning electron microscopy. The samples were Wxed in 3% glutaraldehyde in Wltered sea water (1.0 m, Millepore?) at room temperature for 10 hours, rinsed 3 times, dehydrated using an ethanol series of 50, 80 and 100%, critical-point dried with CO2 in a Samdri- 780-A (Tousimis, USA), sputter-coated with gold and ana- lyzed on a JEOL T-300 scanning electron microscope (SEM). Experimental test of sex change To determine whether males change to females later in life, two diVerent experiments were conducted with shrimps collected in October 2006 and January 2007. In the Wrst experiment, 20 ?focal? males (1.9 < CL < 3.5 mm) were maintained in isolation in plastic containers (3.5 L) until death or for a maximum time period of 6 months. They were fed with benthic diatoms and Wsh food (Tretra- Fin?). In the second experiment, pairs of focal males were maintained in plastic containers (3.5 L) with either two other males (MO for Male-Only treatment) or with two females (MF for Male?Female treatment) (n = 10 repli- cates per treatment with 40 diVerent focal shrimps). The experiment lasted until death or for a maximum time period of 4 months. They were fed fresh chopped Emerita analoga. The rationale for the second experiment was to subject focal male shrimp to diVerent social environments that might promote sex change (as reported before for Lysmata wurdemanni?Baeza and Bauer 2004; Baeza 2007a). All experiments and treatments were examined daily for the presence of exuvia from molting shrimps. The pos- sibility of sex change by focal males was determined every123 Mar Biolseventh day when gently placing and manipulating the focal males under the dissecting microscope to examine for the presence of newly developed female characters (e.g., enlargement of the rostrum, second abdominal pleura). Growth rates of shrimps in experiment 1 were measured to compare the maximum size of males retrieved from the Weld to that attained by males in the laboratory. This comparison allowed us to conWrm whether laboratory conditions were appropriate for maintenance and growth of male shrimps. The body size of the experimental male shrimps was measured from digital images taken (Canon? PowerShot G3) at the start of the experiment and imme- diately after every molt. The total length of each shrimp (TL, mm) was measured from the tip of the scaphocerite to the tip of the uropods using the software Image-Pro Plus 4.5 for Windows. Next, TL was transformed to CL using the equation: TL = 6.905 (CL) ? 1.859, or corre- spondingly CL = (TL + 1.859)/6.905 (r2 = 0.844), obtained by measuring the TL and CL of 20 male shrimps. This transformation permitted us to compare the size of males attained in the Weld and laboratory using the same proxy (CL). Results Population structure and dissections A total of 3,540 shrimps were collected during the study; 1,762 shrimps were classiWed as males under the dissecting microscope while the remaining 1,778 shrimps were classi- Wed as females. Of these 1,778 females, 978 individuals (55%) carried embryos beneath the abdomen. Sex ratio (male to females) did not deviate signiWcantly from a ran- dom distribution of 1:1 for each of the sampling dates (December 2005; 0.46:0.54, 12 = 0.32, P = 0.5712, October 2006; 0.41:0.59, 12 = 1.633, P = 0.2012, January 2007; 0.56:0.44, 12 = 0.723, P = 0.3952). No intermediate shrimps with both male and female external or internal char- acteristics were observed at either sampling date. Dissections and SEM images demonstrated that all shrimps classiWed as males based on external characters had male gonopores on the coxae of the Wfth pair of pereio- pods and the absence of female gonopores on the coxae of the third pair of legs (Fig. 1a?c). Similarly, although the female gonopore was not visible under the dissecting microscope, all shrimps classiWed as females based on their external features had female but no male gonopores (Fig. 2a?c). In males, the gonopore was clearly deWned and the posterior border protruded greatly, forming a rounded lip (Fig. 1c). In females, the gonopores were not clearly delimited and consisted of a slit-like opening closed by a thickened valve-like Xap or cover (Fig. 2a?d). In large brooding (mature) females, the section of the gonopore closer to the coxae was more clearly marked than the sec- tion distal to the coxae (Fig. 2a, b). The gonopore of small non-brooding (immature) females was smaller and with thinner borders than that of brooding females (Fig. 2c, d). Various setae surrounding the gonopores were visible in both mature and immature females (Fig. 2a?d). In contrast, the gonopores of males were not Xanked by these setae (Fig. 1a?c; Fig. 2a, b). In one of the studied females, the gonopore appeared open because the valve-like Xap was folded inward in contrast to that observed in the other stud- ied females. The inward position of this cover suggests that this single female was close to molting and spawning (Fig. 2e, f). In males, the exopods from the Wrst pair of pleopods was approximately twice the length of the endopods (Fig. 1d; Fig. 3a). The endopods from the Wrst pair of pleo- pods had no appendix interna and no cincinulli (Fig. 3a). In contrast, the endopods from the second pair of pleopods had an appendix interna with numerous cincinulli and an appendix masculina with straight setae at the distal end that were about twice as long as the appendix interna itself (Fig. 1d?g; Fig. 3c). In small, most probably immature males, the appendix masculina was reduced in size, not even reaching half the length of the appendix interna, and had a reduced number of distal setae (Fig. 1g). In females, the endopods from the Wrst pair of pleopods had no appen- dix interna and cincinulli (Fig. 3b). The endopods of the second pair of pleopods had an appendix interna with numerous cincinulli but lack an appendix masculina (Fig. 3d). Dissection of the gonads of both ovigerous and non- ovigerous shrimps classiWed as females conWrmed the pres- ence of paired ovaries lying above the hepatopancreas, below the heart and extending into the Wrst abdominal somite (Fig. 2b, c; Fig. 4a?c). The gonads dissected from male shrimps were paired testes located above the hepato- pancreas and below the heart (Fig. 4d). The carapace length of male and female shrimps varied between 1.6 and 4.8 mm (mean ? SD; 2.8 ? 0.57) and between 1.7 and 5.0 mm (3.7 ? 0.59), respectively (Fig. 5). SigniWcant diVerences in CL between the sexes were detected for each sampling date (Mann?Whitney Rank Sum Test; December 2005, T = 13,276, n(males) = 150, n(females) = 173, P < 0.001, October 2006; T = 195094.5, n(males) = 547, n(females) = 705, P < 0.001, January 2007: T = 1189779.5, n(males) = 877, n(females) = 1,096, P < 0.001), indicating sexual dimorphism with respect to body size (Fig. 5). Also, the carapace length of (mature) females car- rying embryos beneath the abdomen was signiWcantly larger than that of (immature) females with no eggs (4.1 ? 0.29 and 3.3 ? 0.52 mm, for ovigerous and123 Mar Biolnon-ovigerous females, respectively; Mann?Whitney Rank Sum Test; T = 364505, n(non-ovigerous) = 800, n(ovigerous) = 877, P < 0.001). Experimental test of sex change None of the males maintained in isolation in experiment 1 changed to females nor did they acquire any ?intermediate? morphology; no widening of the second abdominal pleura or enlargement of the rostrum was noticed. Males molted between 1 and 8 times during the Wrst experiment (3.5 ? 1.9 times during the experiment; 2.5 ? 1.2 times during 3 months, considering all shrimps alive until the third month; 5.0 ? 1.9 times during 6 months considering only those shrimps alive until the sixth month) (Fig. 6). The Wnal body size attained by these focal males (3.7 ? 0.38 mm CL) was greater than that observed for males collected from the Weld (2.8 ? 0.57 mm CL) (Mann?Whitney Rank Sum Test; T = 31855.5, n(experiment males) = 20, n(Weld males) = 1,762; P ? 0.001) (Fig. 6). These data suggest that males in the laboratory were in good condition and that the absence of sex change was not due to any experimental artifact such as detrimental food conditions. In the second experiment, males molted several times (number of molts not recorded during this experiment) before either dying or until the experiment was terminated. Fig. 1 Scanning electron microscopy (SEM) view of male Hippolyte williamsi. a Ventral view of the pereiopods; gonopores (arrows). b Detail of the gonopores. c Detail of the posterior lip (arrow) of the gonopore. d?g First and second pleopods. d Exopod (ex) and endopod (en) of the Wrst pleopod. e Endopod of the second pleopod (en2), with appendix masculinae (am) in the inner border. f Distal end of the appendix interna (ai) and setae (s) of the appendix masculina. g Short appendix masculina (am) of immature male. Scale bar a 0.5 mm, b?e 100 m, f 20 m, g 10 m123 Mar BiolIn this last experiment, insemination of females by males was occasionally observed in the MF treatment, conWrming the good condition of male shrimp. The time to death regis- tered for focal males in the second experiment was similar to that of males in the Wrst experiment: eight out of 20 (40%) focal males were alive at the end of the fourth month in the Wrst experiment while 12 out of 60 (20%) of the focal males were alive at the end of the second experi- ment (this experiment lasted 4 months) (Chi-square of independence; 12 = 3.841, P > 0.05). The somewhat elevated mortality observed in the second experiment was due to cannibalism, mostly observed in the MO treatment. On more than one occasion, some individuals were observed feasting on the Xesh of another shrimp that had recently molted. Discussion In Hippolyte williamsi, the population is composed of both males and females. On average, females were larger than the males, and ovigerous females were larger than non- ovigerous females. Although this size frequency distribu- tion of the sexes agrees with expectations for protandric hermaphrodites (Bauer 2004), no transitional individuals were collected in the Weld. Also, laboratory experiments demonstrated that males might live and grow steadily for months before dying without experiencing any morphologi- cal change that might denote sex change. All this informa- tion represents strong evidence that the studied species is not a hermaphrodite but a gonochorist in contrast to that reported for other congeners (Zupo and Messina 2007; but see Cobos et al. 2005). Sex change may depend on extrinsic factors such as food (Zupo and Messina 2007) or the social environment (Baeza and Bauer 2004; Baeza 2007b). It could be argued that males in the Wrst experiment might not have changed to females due to the absence of environmental stimuli, including social cues, which trigger sex change. The need for such cues triggering changes between ontogenetic phase is recognized for many marine invertebrates, including shrimps (e.g., during settlement?do Santos et al. 2004). On the other hand, experiments conducted in the sequen- tial-simultaneous hermaphroditic shrimp Lysmata wurde- manni have demonstrated that males invariably change to the terminal sex phase (hermaphrodites) before dying, even when not exposed to any social cues (Baeza 2007b). The results from our second experiment where males were exposed to diVerent social environments conWrmed that none of the males changed to females. Also, many of the males in our Wrst rearing experiment attained sizes larger than that reported for males from the Weld. That males lived for a long time and grew steadily in the laboratory further supports the idea that Hippolyte williamsi is gonochoric. On the other hand, sex change in H. williamsi might not be obligatory but facultative, as suggested for C. crangon where less than 2% of the males in the population turn to females (laboratory observations extending over 8 months? Schatte and Saborowski 2006). Also, in the putatively obligatory sex changing C. franciscorum, laboratory exper- iments have demonstrated morphological evidence of sex change only in 2 out of 40 experimental males (Gavio et al. Fig. 2 SEM view of female Hippolyte williamsi. a Mature female gonopore (g), fourth (cx4) and Wfth (cx5) coxae showing no sign of male gonopore. b Detail of the gonopore of mature females (arrow points to the gonopore). c Gonopore of immature female. d Detail of c. e Open gonopore of purportedly spawning female. f Detail of e. Scale bar a 200 m, b, d and f 20 m, c?e 100 m123 Mar Biol2006). If sex change in the studied species is facultative rather than obligatory and the probability of males chang- ing sex in H. williamsi is as low as in C. crangon or C. fran- ciscorum, then our low number of replicates and males (a total of 20 and 60 males in the Wrst and second experiment, respectively) could easily lead to sex change in this species going undetected. Furthermore, although mortality did not diVer between our Wrst and second experiment, the chances of detecting facultative sex change in the second experiment might have also been reduced because several recently molted males were consumed by conspeciWcs. Despite the frequency of sex change in Crangon species was extremely low, careful morphological and anatomical examination in C. crangon and C. franciscorum had conWrmed the existence of intermediate individuals with both male and female characteristics (either males with developing oocytes or females with atrophied vas deferens?Boddeke et al. 1991; Gavio et al. 2006; Schatte and Saborowski 2006). Fig. 3 Pleopods of Hippolyte williamsi. a First pleopod of male showing exopod (ex) and endopod (en). b First pleopod of female. c Second pleopod of male with appendix masculina (am) and appendix interna (ai) bearing cincinulli (cc). d Second pleopod of ovigerous female (eggs removed) with appendix interna (ai)123 Mar BiolIn H. williamsi, no intermediate shrimps were recorded from relatively large samples (more than 1,000 shrimps in two out of three samples) taken at three diVerent occasions in two diVerent years. Furthermore, careful dissections of selected individuals oVered no indication of intermediate stages based on internal organs. Although future studies need to examine the possibility of facultative sex change in the studied species, all presently available information sug- gests that Hippolyte williamsi is gonochoric. Hippolyte williamsi features marked sexual dimorphism: on average, females from the natural environment were larger than males. This size diVerence between the sexes contrasts with that reported for gonochoric shrimps that live in socially monogamous heterosexual pairs (e.g., in Ponto- nia margarita males are only slightly smaller than females ? Baeza 2008b) or featuring mating systems in which female monopolization by males during mating interactions is common (e.g., in Rhynchocinetes typus males are larger than females ? Correa and Thiel 2003). In socially mono- gamous shrimps, where sexual selection is weak, body size diVerences between the sexes are absent or are compara- tively small (Baeza 2008b). In species with strong hierar- chical dominances, males defend females, and thus, sexual dimorphism in body size and structures that may be potentially used as weapons should be favored by intra- sexual competition (Baeza and Thiel 2007). In Hippolyte williamsi, shrimp were observed as loose dense aggregations in seagrass beds, a very heterogeneous habitat that together with high population density might impede successful monopolization of receptive females by a single dominant male. Under these circumstances, it might be advantageous for males to roam and constantly search for receptive females rather than attempting to monopolize them. This behavior might favor small body size because that leads to an increase in agility and encounter rates with receptive females (Shuster and Wade 2003; Baeza and Thiel 2007). The fact that many males in the laboratory reached sizes found in only a small proportion of the males in the Weld suggests that males have the potential to grow to larger sizes under suitable conditions. Possibly, natural selection (due to Wsh predation) eliminates many males in the Weld before reaching their maximum size. Larger males might be better in defending mates against other males, but might simultaneously be more exposed to visual predators, which are common in seagrass beds (Woods 2002). The actual reasons for the small male size of H. williamsi in the Weld is presently unknown, but this system seems to oVer a unique opportunity to examine the interaction between natural and sexual selection, and to test whether small males have higher mating possibilities than large males in environ- ments with high predation pressure (see also Wellborn and Cothran 2007). Fig. 4 SEM view of the male and female gonads of Hippolyte wil- liamsi. The dorsal carapace was removed to show gonad morphology in each micrograph. a Ovary of mature female; anterior lobes (al), eye (e). b Ovary covered with epithelium (ep). c Lateral view of mature ovary; anterior lobe (al), ventral lobe (vl). d Testes (t) of mature male. All scale bars = 200 m123 Mar BiolOverall, our study represents an example in which a combination of size measurements, growth experiments, examination of external morphology and internal anatomy assisted in characterization of the sexual system of a marine shrimp. This approach will assist in establishing shrimps, in particular the highly diverse family Hippolytidae with the diverse genera Thor, Lysmata and Hippolyte, as model sys- tems to investigate the evolution of sexual systems. A com- parative framework might prove most useful to accomplish this task; after correcting for shared ancestry, it should be possible to reveal the environmental (including social) con- ditions favoring or constraining particular sexual systems (as well as other traits) (see Harvey and Pagel 1991). In particular, the genus Hippolyte might be a worthwhile study system because most species live in seagrass and algae beds, i.e. they face similar habitat factors, including food and structural protection from predators (Table 1). An initial comparison indicates substantial size diVerences between males and females of the species from this genus (Table 1). Future studies might show to which degree sex- ual selection and natural selection inXuence the reproduc- tive success of the sexes and their optimum body size. It remains to be shown whether divergent sexual Wtness functions favor the evolution of protandric hermaphroditism in the species from this genus. At present only three species from the genus Hippolyte have been carefully examined for their sexual system, using more than one kind of evidence (i.e., H. inermis, H. obliq- uimanus and H. williamsi?Table 1). Protandric hermaphro- ditism has been suggested for a number of other Hippolyte species, primarily based on sexual size dimorphism. How- ever, two recent studies demonstrated that size dimorphism is not necessarily a reliable indicator for the sexual system of the shrimp from this genus (Terossi et al. 2008; this study). Collaborative eVorts involving evolutionary biolo- gists, physiologists and molecular ecologists from diVerent continents and countries will not only help to (re)conWrm the sexual system of the species from the genus Hippolyte Fig. 5 Population structure of Hippolyte williamsi at Puerto Aldea, Coquimbo, Chile, during December 2005 (n = 323), October 2006 (n = 1,244), and March 2007 (n = 1,973) Fig. 6 Experimental test for sex change in Hippolyte williamsi. a Car- apace length at the start and at the end of the Wrst laboratory experiment in a total of 20 experimental male shrimps. b The number of molts experienced by shrimps during the experimental period. c The number of days that shrimps remained alive during the experiment. The hori- zontal grey line indicates the average size of males (2.5 mm CL) collected in their natural environment (n = 1,762 males collected in December 2005, October 2006, and January 2007)123 Mar BiolTa bl e 1 So ci o ec o lo gy o f s hr im ps fro m th e ge n u s H ip po ly te Sp ec ie s D ist rib u tio n W D H a R Pb TB L M B L M CL FB L FC L SR SS c EV Id R ef er en ce s H . a cu ta S Ja pa n d? U de ke m d? A co z (19 96 ) H . a u st ra lie n si s SE A u st ra lia 0? 15 A l 25 d? U de ke m d? A co z (19 96 , 2 00 1) H . b iW di ro str is N ew Z ea la n d d? U de ke m d? A co z (19 96 ) H . c al ifo rn ie n si s N E Pa ci W c 0? 10 Sg d? U de ke m d? A co z (19 96 ) an d W ic ks te n (19 90 ) H . c a ra di n a A us tr al ia , N ew C al ed o n ia d? U de ke m d? A co z (19 96 ) H . c a ta gr ap ha So ut h A fri ca 6? 8 Sy 22 d? U de ke m d? A co z (20 07 ) H . c la rk i N E Pa ci W c 0? 10 A l d? U de ke m d? A co z (19 96 ) an d W ic ks te n (19 90 ) H . c o m m e n sa lis In do - Pa ci W c d? U de ke m d? A co z (19 96 ) H . c o e ru le sc en s Ce n tr al A tla n tic 0? 1 A l 16 .5 13 12 . 8 W ill ia m s (19 84 ), d ?U de ke m d? A co z (19 96 ) an d H ac ke r an d M ad in (1 99 1) H . e dm o n ds on i H aw ai i d? U de ke m d? A co z (19 96 ) H . g a rc ia ra so i N E A tla n tic , M ed ite rr an ea n 0? 15 Sg , A l Su , F a 0. 9 15 1. 6 1. 8 d? U de ke m d? A co z (19 96 ) a n d K o u ko ur as a n d A n as ta sia do u, (20 02 ) H . ho lth u is i M ed ite rr an ea n 7? 50 A l Fa 19 1. 4 3. 5 2. 5 d? U de ke m d? A co z (19 95 ) a n d K o u ko ur as a n d A n as ta sia do u, (20 02 ) H . hu n tii E A tla n tic , M ed ite rr an ea n Sy 17 Ce id ig h an d M cG ra th (1 97 8) H . i ne rm is N E A tla n tic , M ed ite rr an ea n 0? 30 Sg Sp -F a 29 4. 1 45 7 1. 6 PH S, G , M , H d? U de ke m d? A co z (19 96 ) a n d K o u ko ur as a n d A n as ta sia do u, (20 02 ) H . ja rvi n e n si s Ce n tr al P ac iW c, In do - Pa ci W c d? U de ke m d? A co z (19 96 ) H . kr a u ss ia n a S A fri ca , M ad ag as ca r Sg 0. 9 1. 8 2. 0 (P H ) S d? U de ke m d? A co z (19 96 ) a n d To rr es e ta l. (20 07 ) H . la ga rd e re i E A tla n tic 0? 1 A l 22 14 16 . 5 1. 2 d? U de ke m d? A co z (19 95 , 1 99 6) H . le pt o c e ru s N E A tla n tic , M ed ite rr an ea n , B la ck S ea 0? 30 Sg , A l Sp -F a 17 2. 5 22 6. 1 1. 3 d? U de ke m d? A co z (19 96 ) a n d K o u ko ur as a n d A n as ta sia do u, (20 02 ) H . le pt o m et ra e N E A tla n tic , M ed ite rr an ea n 95 ?1 30 Sy 18 d? U de ke m d? A co z (19 96 , 2 00 7) H . lo n gi al le x Tr op ic al W es t A fri ca 35 ?4 0 Sy 8 d? U de ke m d? A co z (20 07 ) H . lo n gi ro st ri s N E A tla n tic , M ed ite rr an ea n Sg Su -F a 17 22 1. 3 (P H) S C? id ig h et al . (19 82 ) a n d Sc ha V m ei ste r et al . (20 06 ) H . m u lti c o lo ra ta N ew Z ea la n d d? U de ke m d? A co z (19 96 ) H . n ic ho lso n i Ca rib be an , W es t I nd ie s 0? 28 A l V ar 1. 26 1. 84 1. 5 S Sp ot te et al . (19 95 ), Sp ot te an d Bu bu ci s (19 96 ) an d d? U de ke m d? A co z (19 96 , 20 07 ) H . n ie za bi to w sk ii M ed ite rr an ea n 0. 5? 5 Sg , A l Fa 10 2. 9 20 4 2. 0 (P H ) S d? U de ke m d? A co z (19 96 ), K o u ko u ra s an d A n as ta sia do u (20 02 ) a n d G ar c? a R as o et al . (1 99 8) H . o bl iq ui m a n u s W A tla n tic , Ca rib be an , B ra zi l 0? 2 Sg , A l 15 2. 5 3. 2 1. 3 G C S, G , M d? U de ke m d? A co z (19 96 ) a n d Te ro ss i e ta l. (20 08 ) H . pa lli ol a E A tla n tic 0? 25 A l 10 d? U de ke m d? A co z (19 96 ) H . pl e u ra c a n th us N W A tla n tic Sg , A l Sp - Fa < $ 18 Sh ie ld (19 78 ), W ill ia m s (19 84 ) a n d d? U de ke m d? A co z (19 96 )123 Mar Biolbut also contribute to a better understanding of the evolution of hermaphroditism in caridean shrimps. Acknowledgments JAB thanks for the support from a Smithsonian Tropical Research Institute (STRI) Postdoctoral Fellowship and a Smithsonian Marine Station at Fort Pierce (SMSFP) Postdoctoral Fel- lowship. JAB specially acknowledges support from Valerie Paul and the SMSFP for funding a Weld trip to Chile during which most of this manuscript was written. The constructive comments from three anon- ymous reviewers substantially improved the manuscript. This is con- tribution number 740 from the Smithsonian Marine Station at Fort Pierce. References Anker A, Ahyong ST, Noel PY, Palmer AR (2006) Morphological phylogeny of alpheid shrimps: parallel preadaptation and the ori- gin of a key morphological innovation?the snapping claw. 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