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Mating Systems and Sexual Selection in Male-Pregnant Pipefishes And

Mating Systems and Sexual Selection in Male-Pregnant Pipefishes And

Systems and in Male-Pregnant Pipefishes and : Insights from Microsatellite-Based Studies of Maternity

A. G. Jones and J. C. Avise

In pipefishes and seahorses (family ), the males provide all postzy- gotic care of by brooding on their ventral surfaces. In some , this phenomenon of male ‘‘’’ results in a reversal of the usual direction of sexual selection, such that compete more than males for ac- cess to mates, and secondary evolve in females. Thus the syngnathids can provide critical tests of theories related to the of differences and sexual selection. Microsatellite-based studies of the genetic mating systems of several species of pipefishes and seahorses have provided insights into important aspects of the natural history and evolution of these fishes. First, males of species with completely enclosed pouches have complete confidence of paternity, as might be predicted from theory for species in which males invest so heavily in offspring. Second, a wide range of genetic mating systems have been documented in nature, including genetic in a sea- , in two species of pipefish, and in a third pipefish species. The genetic mating systems appear to be causally related to the intensity of sexual selection, with secondary sex characters evolving most often in females of the more polyandrous species. Third, genetic studies of captive-breeding pipe- fish suggest that the sexual selection gradient (or Bateman gradient) may be a substantially better method for characterizing the than previously available techniques. Finally, these genetic studies of syngnathid mating systems have led to some general insights into the occurrence of clustered at microsatellite loci, the utility of linked loci in studies of parentage, and the use of parentage data for direct estimation of adult size.

In various classes of a few exceptional with typical sex roles (Williams 1966, cases occur, in which the instead of the 1975). male has acquired well pronounced secondary sexual characters, such as brighter colors, greater Male is fairly common in size, strength, or pugnacity. the world, but reversals of the di- , 1871 rection of sexual selection are not. For ex- ample, males of many species partic- One major theme in evolutionary ipate in the rearing of offspring (Lack has been the attempt to identify ultimate 1968), and, in fishes with parental care, the factors responsible for the differences be- male is more often the caregiver than the From the Department, 3029 Cordley Hall, tween males and females (Andersson female (Breder and Rosen 1966). In most Oregon State University, Corvallis, OR 97331 (Jones), 1994; Darwin 1871). Many of the obvious and Genetics Department, University of Georgia, Ath- species with male parental care, males ens, Georgia (Avise). We thank A. Berglund, C. Kvar- external differences between the re- must nevertheless compete for access to nemo, G. Moore, G. Rosenqvist, and L. Simmons for ex- sult from the operation of sexual selection cellent collaborative interactions. A. Berglund, A. mates, and males (more so than females) DeWoody, A. Fiumera, C. Kvarnemo, B. McCoy, D. or from related to parental re- Pearse, B. Porter, and D. Walker provided valuable sponsibilities. Fortunately, evolution has tend to be modified by sexual selection comments on the manuscript. AGJ’s work in the Avise provided certain exceptional groups of (Andersson 1994). These species are of in- lab was supported by the National Science Foundation, terest with respect to parental investment the Pew Foundation, funds from the University of Geor- taxa that, by virtue of unusual biological gia, and by an NIH training grant. Address correspon- features, can facilitate comprehension of theory, which predicts that males should dence to Adam G. Jones at the address above or e-mail: apportion their investment in progeny as [email protected]. This paper was delivered at a more orthodox conditions. With respect symposium entitled ‘‘DNA-Based Profiling of Mating to the evolution of the sexes, species ex- a function of their certainty of paternity Systems and Reproductive Behaviors in Poikilothermic hibiting ‘‘sex-role reversal’’ are especially (Clutton-Brock 1991). However, species of ’’ sponsored by the American Genetic As- sociation at Yale University, New Haven, CT, USA, June important because they allow critical tests special interest to sexual selection theory 17–20, 2000. of theories that were derived primarily to are those in which females compete more 2001 The American Genetic Association 92:150–158 explain patterns of variation in species intensely for access to mates and, conse-

150 Table 1. Definitions of genetic mating systems (following Searcy and Yasukawa 1995) systems in natural of four syngnathid species. Do pipefish and sea- Number of successful Number of successful Gender experiencing more Term mates per mating male mates per mating female intense sexual selection horse males have complete confidence of paternity, as might be expected given their Many One Males Polyandry One Many Females involved parental care? Do genetic mating Monogamy One One Neither systems appear to be linked to the evolu- Polygynandry Many Many Either or neither tion of ? Can improved quantitative methods be used to compare genetic mating systems? After answering quently, the females are most modified by some species, are glued to the male’s these and related questions, we also sum- sexual selection. ventral surface without any outer cover- marize some unexpected genetic results Species that are sex role reversed (with ing, whereas in others, such as the sea- from syngnathids that are relevant to mo- respect to sexual selection intensity) have horse, the male possesses an elaborate lecular studies of parentage in general. been described from only a handful of evo- pouch with a small opening through which lutionary lineages, including some eggs are deposited. Presumably the extent Sexual Selection and the Genetic (Gwynne 1991; Smith 1980), fishes (Ber- of energetic investment by a male in his Mating System glund et al. 1986a,b; Vincent 1992), and offspring also varies among species (Ber- (Jehl and Murray 1986; Oring and glund et al. 1986b), an important consid- The same processes that cause a reversal Lank 1986). Additional role-reversed taxa eration for sexual selection theory (Triv- in the direction of sexual selection across no doubt will be discovered, but for now, ers 1972). taxa probably also function on a smaller three groups stand out as most promising Syngnathid lineages therefore provide scale to affect the intensity of sexual se- for continued study of sexual selection. In excellent opportunities for comparative lection within and among species with katydids (family Tettigonidae), the male analysis. Numerous field and laboratory typical sex roles. Thus an enhanced un- invests in progeny by transferring an edi- studies have shown that two pipefish spe- derstanding of the causes of sexual selec- ble spermatophylax to the female during cies, typhle and ophi- tion in sex-role-reversed taxa should shed (Brown and Gwynne 1997). dion, are sex role reversed, with females light on the principles that govern this im- Populations and species vary with respect competing more intensely for mates than portant evolutionary mechanism in all to the amount of male investment, and in males (Berglund et al. 1986a, 1989). Sexual species. some situations females compete more in- selection appears to be especially intense A topic of special interest is the rela- tensely than males for access to mates in N. ophidion, and females possess sec- tionship between the mating system (def- (Gwynne and Simmons 1990). Shorebirds ondary sexual characters that are impor- initions in Table 1) and the strength of sex- (Charadriiformes) are a second important tant in male choice (Rosenqvist 1990). Fe- ual selection. Here we are concerned with group that includes sex-role-reversed taxa, males with intense blue coloration on the the genetic mating system rather than the and these too have had a large impact on head and flank and with appearance-en- social mating system (Andersson 1994; the development of sexual selection theo- larging skin folds are preferred by males, Emlen and Oring 1977; Searcy and Yasu- ry. The best known of sex-role-reversed demonstrating that these traits probably kawa 1995). These two views of the mating shorebirds are the spotted sandpiper (Ac- arose through sexual selection (Rosenqv- system must be connected to some de- titus macularia), phalaropes ( Phal- ist 1990). Similar sexually dimorphic traits gree, of course, but whereas the social aropus), and jacanas (family Jacanidae). can be seen in females of many other mating system is concerned with pair The shorebirds are of particular interest, (thus far unstudied) pipefish species bonds and other observable interactions because, as in the katydids, they include (Dawson 1985), suggesting that numerous among individuals, the genetic mating sys- species with conventional as well as re- sex-role-reversed pipefish species exist. In tem is concerned with the allocation of bi- versed sex roles (Jehl and Murray 1986). addition, other species in Syngnathidae ological parentage. In principle, a com- A third promising group, the fish family appear not to be sex role reversed. In sea- plete description of the genetic mating Syngnathidae with more than 200 species , for example, males appear to be system for a particular breeding assem- of pipefishes (Dawson 1985) and about 32 the predominant competitors for mates blage would provide data on the number species of seahorses (Lourie et al. 1999), (Vincent et al. 1992). Thus comparisons of mates and the number of offspring pro- is one of the most unusual collections of among syngnathid lineages, particularly in duced by each reproductively active adult animals with respect to paternal invest- light of a molecular phylogeny, should during a well-defined breeding interval. ment. During copulation the female trans- provide insight into factors affecting sex- The genetic mating system is undoubt- fers unfertilized eggs to the male’s ventral ual selection and sex role reversal. edly related to the operation of sexual se- surface, where fertilization takes place. In this article we review microsatellite lection and, indeed, should be seen as an Unlike other attentive fish which data pertaining to the comparative analy- integral component of the process of sex- may build nests, fan eggs, or carry devel- sis of sexual selection in pipefishes and ual selection. Darwin (1871) originally de- oping young in their (Breder and seahorses. A primary goal of these studies fined sexual selection as differential repro- Rosen 1966), pipefish and males has been to resolve possible relationships ductive success of individuals caused by maintain a -like connection that between the genetic mating system and competition over mates (Andersson 1994). permits the transfer of nutrients from a sexual selection in sex role reversed taxa, Hence the variance in fitness due to sexual male to his offspring (Berglund et al. so we begin with a short introduction to selection comes mainly from differential 1986b; Haresign and Shumway 1981). these topics. A brief description of genetic mating success (although other factors Brood pouches vary considerably among parentage analysis follows, and empirical such as mate quality also may contribute; species (Dawson 1985; Herald 1959). In examples are provided of genetic mating Andersson 1994). A description of the ge-

Jones and Avise • Syngnathid Mating Systems 151 Table 2. Comparison of microsatellite in syngnathid species

Number of Range of Four-locus alleles per observed exclusion Species Loci used locus heterozygosities probabilitya

S. scovelli micro11.1, micro22.3, micro25.10, 19–29 0.88–0.95 .999 micro25.22 S. floridae micro11.1, micro22.3, micro25.10, 18–44 0.78–0.95 .999 micro25.22 S. typhle typh04, typh16, typh18, typh12 15–39 0.55–0.95 .999 H. angustus Han03, Han05, Han06, Han15 9–22 0.73–0.95 .997

Pipefish and seahorse primer sequences are available in Jones et al. (1998, 1999b). a The expected proportion of individuals in a population that would be rejected as possible parents of a typical Figure 1. Results of computer simulations investigat- offspring, given that one of the offspring’s parents is known. ing the power of two seahorse microsatellite loci (Han03 and Han05) to detect multiple maternity within a male’s pouch (after Jones et al. 1998). Each simulated brood contained 458 embryos that were the progeny netic mating system includes data regard- sexual selection under this mating system of two mothers. Each mother was assigned a genotype based on Hardy–Weinberg expectations, and the first ing both mating and , may affect either sex or may be nonexis- mother contributed from 50 to 99% of the male’s eggs. and therefore describes the majority of tent, depending on the relative distribu- Samples of 15, 30, or 60 embryos were drawn at ran- the variance in fitness resulting from sex- tions of mating success for each gender. dom from the brood and if three or more maternal al- leles appeared in the progeny at either locus, then mul- ual selection (Arnold 1983). Given the im- Sexual selection in polygynandrous mat- tiple maternity was deemed to have been detected. portance of the mating system to sexual ing systems may occur, but in general is Almost all failures to detect multiple mating by males were because embryos from one of the females went selection, a more precise understanding of expected to be weaker than in strictly po- unsampled rather than because of insufficient variation mating systems should be informative lygynous or polyandrous populations (be- at the marker loci. with regard to sexual selection (Arnold cause polygynandry will tend to reduce and Duvall 1994). The above argument ap- the ability of successful individuals to mo- plies only to the term genetic mating sys- nopolize access to mates). served heterozygosities ranging from tem as it is defined here. Broader uses of These simple hypotheses have motivat- 0.55–0.95 (Table 2). The power of these the term mating system typically do not ed our genetic studies of mating systems marker systems for parentage assessment possess so clear a relationship to sexual in pipefishes and seahorses. Various spe- can be gauged by the four-locus exclusion selection (Andersson 1994; Emlen and Or- cies can be ranked a priori with respect to probabilities (Chakraborty et al. 1988), ing 1977). level of sexual dimorphism. Then the hy- which always were greater than 0.997 (Ta- For species with typical gender roles, pothesis to be tested is whether differenc- ble 2). Darwin (1871) observed that highly polyg- es among species in sexual dimorphism Available evidence suggests that most or ynous species tend to be more sexually di- are related to the underlying genetic mat- all individuals even in large syngnathid morphic than less polygynous ones, with ing systems, perhaps with the most di- populations have unique multilocus geno- secondary sexual characters evolving in morphic species also being the most poly- types. For example, calculated expected males (Table 1). Numerous attempts to androus (Table 1). frequencies of observed four-locus geno- quantify this relationship have yielded types across all species ranged from 3.3 ϫ mixed results (e.g., Bjo¨rklund 1990; Clut- 10Ϫ7 to 1.8 ϫ 10Ϫ11. We never observed two Microsatellites and Mating ton-Brock et al. 1977, 1980; Ho¨glund 1989; individuals that shared even a two-locus Measures Oakes 1992; Payne 1984), perhaps in part genotype in population samples of adult S. because, until recently, molecular markers Microsatellite markers (Goldstein and scovelli (n ϭ 79), S. floridae (n ϭ 65), or H. were not available to assess genetic mat- Schlo¨tterer 1999; Hughes 1998; Tautz 1989) angustus (n ϭ 41). Three pairs of individu- ing systems reliably. However, even now are particularly suited to studies of genet- als with identical two-locus genotypes were that scores of studies have quantified ic parentage because they are highly poly- observed in a sample of 293 adult S. typhle rates of extrapair fertilization (notably in morphic, codominant, single locus, and (42,778 pairwise comparisons), but no two birds), the data have yet to be integrated PCR based. Thus individuals in a popula- individuals were observed to share a three- in ways that definitively address relation- tion often exhibit unique DNA profiles, pa- locus genotype. These considerations in- ships between the genetic mating system rental genotypes can be reconstructed dicate that the microsatellite markers were and the strength of sexual selection. from progeny array data, and the markers more than adequate for the parentage as- While these issues are being sorted out can be assayed from small sources sessments. In fact, simulations by Jones et in species with typical sex roles, we can such as individual embryos. al. (1998) show that two seahorse micro- also examine the predictions for species One drawback of microsatellites is that satellite loci are sufficient to detect essen- with reversed sex roles. In such - the primers often are species or genus tially all instances of multiple mating. With isms, stronger sexual selection is expected specific, and we have had to generate this level of , multiple ma- in species that are more polyandrous, with primers independently on several occa- ternity is difficult to detect only when re- secondary sexual characters evolving in sions for the three species of pipefish (ge- productive skew between two mates is females (Jehl and Murray 1986). Thus the nus Syngnathus) and one seahorse (Hip- high and the number of offspring sampled expectations regarding the strength of sex- pocampus subelongatus) assayed to date from a brood is low (Figure 1), but even in ual selection for polygyny, polyandry, and (Table 2). High levels of microsatellite this case the failing is not in the markers monogamy are fairly simple (Table 1). Po- polymorphism were observed for all spe- but in the sampling strategy (Jones et al. lygynandry is more problematic because cies, with 9–44 alleles per locus and ob- 1998).

152 The Journal of Heredity 2001:92(2) Figure 2. An example from S. floridae showing the logic behind reconstruction of maternal genotypes us- ing two microsatellite loci. By examining a pregnant male and the embryos in his brood pouch, maternal alleles in each are deduced by subtraction. Then a dilocus table is constructed, each cell of which reports the number of progeny with a particular com- bination of alleles at the two loci. The maternal dilocus genotype is evident from these nonrandom allelic as- sociations. In this case, one mother had the genotype 150/160 at micro25.10 and 96/100 at micro25.22, while the other mother’s genotype was 146/182, 102/108. The diagram at right shows a spatial map of these maternal alleles in the male’s brood pouch. In this case, the pouch was divided into 14 sections, with three embry- os sampled from each. Embryos of the same pattern are full sibs and those of different patterns are half sibs (had different mothers).

Beyond the mere detection of multiple mating, the particular genotypes of par- ents contributing to a brood also can be reconstructed. Pipefish and seahorses are convenient in this regard because a preg- nant male carries an entire brood that may include hundreds of offspring. Once the male is established as the true genetic sire of all progeny within his pouch (see be- low), the maternal contribution to each embryo can be deduced by subtraction. Figure 3. Diagrammatic representation of the patterns of parentage observed during microsatellite-based par- Then, with the maternal alleles known for entage studies of four syngnathid species. Males are shown above the brood pouch diagrams, females below. each embryo, a table can be constructed Notice the brood pouches on the ventral surfaces of depicted males. Also shown are spatial maps of full- and half- sib embryos (as in Figure 2) within males’ pouches. These diagrams are intended to present a comparative over- that reveals the unique dilocus genotype view of the patterns of mating in these species. Therefore they are not drawn to scale and only portray a small of each mother. An example from S. flori- subset of the assayed progeny per pouch. Arrows indicate patterns of depositions () by females. In S. scovelli, S. floridae, and S. typhle, females mate with multiple males, but not so in H. subelongatus. Males of S. dae is shown in Figure 2. In this case, the floridae and S. typhle often receive eggs from multiple females, whereas S. scovelli and H. subelongatus males male evidently mated with two females. typically do not. As described in the text, available data indicate a positive relationship between polyandry and Positions of the embryos were mapped the evolution of sexual dimorphism in these taxa. during dissection of the brood pouch, and the reconstituted maternal genotypes the high levels of polymorphism at the mi- closed, fully covers all embryos (see Fig- were projected onto this map. This exam- crosatellite loci (see above), that particu- ure 3). During in S. typhle, recep- ple reveals another convenient feature of lar female most likely mothered the brood. tive males present an opening at the an- Syngnathus brooding: In each of 43 multi- Similarly if the same multilocus maternal terior end of the pouch through which mated pipefish males genetically assayed, genotype appeared in the progeny of more females deposit unfertilized eggs. Males embryos invariably proved to be clumped than one male, then it is highly likely that release directly into the pouch and by maternity within the brood pouch, a the identified female had mated with mul- then shake the posteriorly to finding that lends additional support to tiple males. In this way, multiple mating by make room for additional eggs. The ven- the validity of the reconstructed maternal females can be deduced solely from the tral seam opens only when the male gives genotypes. contents of male brood pouches—the several weeks later. Seahorse males Deduced maternal genotypes also can adult females need not be available for as- possess a pouch that is fully enclosed ex- be used to document instances of multiple say. cept for a small opening through which fe- mating by females, as well as to match males deposit eggs, and the sequence of particular collected specimens of adult fe- Parental Investment and Pipefish egg transfer resembles that of pipefish. males to their mates. If the genotype of an Paternity Thus in these species it is difficult to imag- assayed female is the same as the inferred Pipefish in the genus Syngnathus possess ine how rival males might steal any fertil- genotype from a male’s brood, then given a pouch with a ventral seam that, when izations.

Jones and Avise • Syngnathid Mating Systems 153 Indeed, every pregnant syngnathid male Table 3. Relevant reproductive information on three pipefish species (genus Syngnathus) and one whose brood has been genetically assayed seahorse species (genus ) that have been the subject of molecular parentage analyses to date has proved to be the true genetic S. scovelli S. floridae S. typhle H. subelongatus of all embryos within his pouch. Thus males with enclosed pouches appear Sexual dimorphism Sexual size dimor- Female Ͼ male Female Ͼ male Female Ͼ male Female ϭ male to have complete confidence of paternity, phism a result consistent with expectations Sexual color dimor- Permanent female Some yellow on fe- Transient female or- Body color frequen- based on pouch configuration and paren- phism stripes male nament cies differ Sexual shape dimor- Female keeled abdo- Slightly larger female Slightly larger female Female :trunk ra- tal investment theory, which predicts that phism men trunk trunk tio slightly less a male’s devotion to offspring should be Results of parentage studies positively related to paternity confidence Percent males multiply Ͻ573900 (Clutton-Brock 1991). However, not all spe- mated Average number of 1.0 1.9 3.1 1.0 cies of pipefish have enclosed pouches. In mates per male Nerophis ophidion, unfertilized eggs are Multiple mating by fe- Common Common Common None loosely attached to the male’s outer ven- males tral surface where he fertilizes them by Reproductive floating through a cloud of sperm that he Typical male brood 30–120 250–700 25–130 200–700 size range releases into the water (Rosenqvist 1993). Egg maturation Continuous Continuous Continuous Batch Of interest, males of this species invest Length of breeding Year-round Year-round ϳ3 months ϳ4 months less energy per than females, season Site fidelity No data No data None Yes whereas in S. typhle, a species with com- plete paternity confidence, males and fe- males invest equally in progeny (Berglund et al. 1986b). These observations raise sayed (Table 3, Figure 3). On the other os in pregnant dusky pipefish (Jones and two interesting questions for future re- hand, the genetic data documented mul- Avise 1997b), males proved to have mated search: Do syngnathid species without en- tiple mating by females (Table 3, Figure 3). with one to three females each (Table 3, closed pouches have complete confidence On two separate occasions, a single de- Figure 3). In addition, there was clear evi- of paternity? And, did the brood pouch duced female was the mother of embryos dence for multiple mating by females. evolve as a structure to nurture and pro- in more than one male’s pouch. Thus the Quantitative estimates of the breeding tect embryos, or as a barrier to prevent genetic data showed that females are able population size and multiple maternity cuckoldry? to split a batch of eggs among multiple demonstrated that the rate of multiple males, and that multiple mating by fe- mating by females of dusky pipefish is males was common in this population. probably quite high (see Jones and Avise Genetic Mating Systems in Natural The logic behind the latter statement 1997b). Because both males and females Populations of Syngnathids stems from the fact that only a small frac- often have multiple successful partners For each syngnathid species genetically tion of the breeding population was sam- during a single , this spe- surveyed, field collections of pregnant pled from an extensive sea grass meadow. cies is genetically polygynandrous. males, their brooded embryos, and a sam- Thus females probably deposited eggs in ple of adult females were analyzed for mi- the pouches of males not sampled, as well The Broad-Nosed Pipefish (S. typhle) crosatellite genotypes. Here we summa- as in the males documented in our sam- This western European species is sexually rize the results species by species. ple. Given the low power to detect multi- dimorphic, but less so than Gulf pipefish. ple mating by a particular female, the fact Females are larger than males on average The Gulf Pipefish (S. scovelli) that we detected the phenomenon at all and have slightly deeper abdomens. They This North American coastal species is suggests that multiple mating by females also possess a transient ornament that be- the most sexually dimorphic of the syng- was common. Overall the Gulf pipefish comes visible during courtship and has nathids genetically examined thus far. Fe- mating system is best described as poly- been shown to be a target of sexual selec- males are larger than males and possess a androus, with a male typically receiving tion (Berglund et al. 1997). Nevertheless, deeply keeled abdomen, silvery stripes on eggs from only one female, and a female the sexual dimorphism displayed by S. ty- the trunk, and an enlarged dorsal fin (Fig- often mating with multiple males during phle is not nearly as dramatic as that in S. ure 3) that probably are the result of sex- the time of male pregnancy. scovelli. ual selection (Brown 1972). These char- A population from Sweden’s west coast acteristics are absent in other Syngnathus The Dusky Pipefish (S. floridae) was the focus of the genetic parentage as- species, and hence S. scovelli appears to This western Atlantic species also is sex- sessment. Ninety percent of the males car- be the member of the genus most modi- ually dimorphic, but to a much lesser ex- ried broods with two to six mothers each, fied by sexual selection (Brown 1972; Daw- tent than the Gulf pipefish (Brown 1972). indicating a much higher rate of multiple son 1985). Mature females are larger than males and mating than in previously studied syng- Jones and Avise (1997a) collected spec- possess a slightly deeper and more yellow nathid species (Jones et al. 1999b). As in imens from one locale on Florida’s north- abdomen, probably due to the presence of the Gulf and dusky pipefishes, multiple ern Gulf coast. Despite high statistical ripe ova (Brown 1972). This species does mating by females also was documented. power, the molecular appraisal found evi- not possess obvious secondary sexual These results are consistent with labora- dence for multiple maternity in only one characters. tory and field observations suggesting brood among the 40 pregnant males as- In a genetic analysis of brooded embry- that both males and females frequently

154 The Journal of Heredity 2001:92(2) have multiple mates (Berglund et al. ger interval (marginally significant) be- ly similar to those in S. scovelli. These ob- 1986a; Vincent et al. 1995). Thus S. typhle tween the production of successive servations should be interpreted with cau- is polygynandrous. broods. Overall, results indicate that sea- tion until they can be verified in nature horses frequently maintain long-term pair using molecular markers, but current data The Western Australian Seahorse bonds throughout the breeding season, appear to bolster the conclusion that (Hippocampus subelongatus) and investigation into the costs associated more polyandrous species have experi- Motivation for the study of seahorse par- with mate switching should be a fruitful enced stronger sexual selection on fe- entage was twofold. First, seahorses tend area for future investigation. males. to be the least sexually dimorphic of all If the genetic mating system is indeed syngnathids, so the mating system might Overview of Syngnathid Mating causally related to the evolution of sexual reflect an apparently weak intensity of sex- Systems dimorphism, an important goal is to de- ual selection (Vincent et al. 1992). Second, The parentage analyses described above termine the ecological factors and evolu- several seahorse species studied intently can be considered in light of hypotheses tionary constraints that may shape the from a behavioral standpoint are socially relating the genetic mating system to the mating system. Although too little is monogamous, with long-term pair bonding evolution of sexual dimorphism. The Gulf known about syngnathid ecology to rig- (Masonjones and Lewis 1996; Vincent pipefish is polyandrous and also is the orously address causal hypotheses about 1994b, 1995; Vincent et al. 1992; Vincent most sexually dimorphic species studied mating system evolution in this group, and Sadler 1995). to date. The western Australian seahorse some potentially important differences The species H. subelongatus, previously is monogamous with long-term pair among species are listed in Table 3. For known as H. angustus, occurs along Aus- bonds, a situation that should reflect weak example, S. scovelli and S. floridae occur tralia’s west coast (Lourie et al. 1999), and sexual selection, and this species displays in relatively warm waters where breeding males and females do not differ in obvious very little sexual dimorphism. The two can occur year-round, whereas S. typhle secondary sexual characters (Table 3; un- other Syngnathus species are polygynan- occurs in northern latitudes where breed- published data). Females and males do dif- drous and they exhibit intermediate levels ing is sharply seasonal, with individual fer slightly in body proportions and in the of sexual dimorphism. Thus available data pipefish experiencing many fewer repro- frequency of body color morphs (Table 3; indicate a positive correlation between ductive episodes per lifetime. These con- Kvarnemo C and Moore GI, unpublished the strength of sexual selection and the ditions might promote a bet-hedging strat- data), but so far no evidence suggests that extent of polyandry in sex-role-reversed egy in S. typhle, whereby males and these differences are related to sexual se- pipefishes. This comparative result is females are inclined to seek multiple ma- lection. Jones et al. (1998) used microsat- based on observations of only four spe- tes. ellite markers to provide the first evidence cies, however, so it must be considered In addition, female pipefish in the genus that seahorses are, indeed, genetically mo- provisional. Nevertheless, these genetic Syngnathus mature eggs continuously and nogamous. A population near Perth, Aus- studies provide important baseline infor- appear able to hold them until a mate be- tralia, is the subject of ongoing study. In mation for future comparative analyses of comes available. In contrast, a seahorse all cases examined (n ϭ 43), a pregnant syngnathid mating systems. female prepares a batch of eggs all at once male carried eggs from only one female Additional support for a connection be- and, if not to be in vain, must deposit (Jones et al. 1998; Kvarnemo et al. 2000, tween sexual selection and the mating sys- them in a male’s pouch within a short time unpublished data). Furthermore, in con- tem comes from observational studies on (Vincent 1994a). This feature of seahorse trast to the previous studies of pipefish other syngnathid species. For example, is certainly related to the mating systems, no evidence was found the seahorses H. zosterae, H. whitei, and evolution of monogamy, but whether it is for multiple mating by females. Thus mo- H. fuscus all appear to be monogamous a cause or consequence remains un- lecular evidence supports the notion that and exhibit almost no sexual dimorphism known. Additional factors possibly related H. subelongatus is strictly monogamous (Masonjones and Lewis 1996; Vincent to the evolution of mating systems in during a single reproductive interval (Fig- 1994b, 1995; Vincent et al. 1992; Vincent syngnathids include site fidelity (Table 3), ure 3, Table 3). and Sadler 1995). Two pipefish species, male brood size (Table 3), variation in An additional question motivated by the penicillus and mate quality (Jones et al. 2000b), popula- unusual social system of seahorses was intestinalis, appear to be monogamous as tion density (Clutton-Brock et al. 1997), whether males mate with the same female well (Gronell 1984; Watanabe et al. 1997) (Emlen and Oring 1977), poten- repeatedly within a breeding season. Field and exhibit little or no sexual dimorphism tial reproductive rates of the sexes (Clut- and laboratory observations suggest that (Dawson 1985; Vincent et al. 1992). Within ton-Brock and Vincent 1991), predation other species of seahorses do so (Vincent Syngnathus, two other pipefish species (S. risk (Fuller and Berglund 1996), resource et al. 1992; Vincent 1995). Kvarnemo et al. acus and S. rostellatus) appear from field abundance (Orians 1969), and phylogenet- (2000) addressed this by marking males in observations to be polygynandrous (Vin- ic constraints. the field and sampling their broods re- cent et al. 1995). Neither species displays peatedly throughout the breeding season. obvious secondary sexual traits, but S. ros- The Bateman Gradient and Sexual Of 14 males successfully sampled more tellatus is sexually size dimorphic. Finally, Selection than once, the genetic data document that the pipefish N. ophidion seems to be poly- eight remained faithful to their original androus and is extremely sexually dimor- That a relationship between the genetic mates and six changed mates between phic (Rosenqvist 1990), with females pos- mating system and the evolution of sexual broods. Males who switched mates moved sessing secondary sexual characters (i.e., dimorphism is clear for the syngnathid much greater distances during the breed- appearance-enlarging traits and anterior species studied thus far may be due to the ing season and displayed a somewhat lon- coloration in females) that are qualitative- fact that the assayed species covered a

Jones and Avise • Syngnathid Mating Systems 155 wide spectrum of mating systems, from polyandry to monogamy. Qualitative defi- nitions of the mating system (Table 1) are useful verbal summaries, but they are in- adequate for detailed quantitative com- parisons. A major shortcoming is that po- lygynandry, probably the most common mating system in animal populations, is an overly broad category. Expectations for the strength of sexual selection under this system are not always clear, and the bor- ders between polygynandry and other mating systems can be indistinct. Thus a major goal of research on syngnathid mat- ing systems has been to test other meth- Figure 5. Path diagram showing the vital role of the ods of mating system quantification with sexual selection gradient in the sexual selection pro- respect to sexual selection. cess. Traditional measures or correlates of sexual se- Arnold and Duvall (1994) present a new lection remain important, but the sexual selection gra- dient is the final path to fitness for all sexually selected theoretical framework that considers mat- traits (Arnold and Duvall 1994). ing systems in the context of formal selec- tion theory. This integration points to the relationship between mating success (the the sexes to be quantified. Results of the number of times an individual mates) and Figure 4. Sexual selection (Bateman) gradients for experiment were consistent with expecta- reproductive success (the number of off- (top) and S. typhle (bottom) tions based on the Bateman gradient mod- spring produced) as a central feature of (after Jones et al. 2000a). The sexual selection gradient el: The gradient of females was significant- is given by a weighted least squares regression line the genetic mating system with respect to relating reproductive success to mating success. Each ly steeper than that of males (Figure 4). In sexual selection. In his classic article, A. J. symbol in the graph represents a mean (with one stan- addition, the slopes of the lines responded Bateman (1948) perceived the difference dard error for S. typhle). Males are shown as open as predicted to changes in the operational squares, females as closed circles. In Drosophila, males in this relationship in males compared to and females exhibit sexual selection gradients that are sex ratio. These results lend additional females as the cause of sexual selection. steep and shallow, respectively, a pattern consistent support to the Bateman gradient concept, with strong sexual selection on males. In pipefish, fe- In species with typical sex roles, such as males have a significantly steeper sexual selection gra- suggesting that sexual selection gradients Drosophila melanogaster, males exhibit a dient than males (P ϭ .004), a pattern consistent with do provide a superior method for the strong positive relationship between mat- a reversal in the intensity of sexual selection. quantification of mating systems with re- ing success and reproductive success spect to the strength of sexual selection. (Bateman 1948), whereas in females this Thus a goal in future studies of sexual se- relationship is weaker (Figure 4). Thus a ual selection gradient (Arnold and Duvall lection in Syngnathidae, as well as in other male benefits greatly in terms of offspring 1994). Thus if the gradient is zero, vari- taxa, should be to quantify Bateman gra- number from having multiple mates, ance in mating success has no effect on dients of natural populations in a compar- whereas a female does not, a situation that the strength of sexual selection and there ative context. should promote stronger sexual selection can be no sexual selection due to differ- in males than in females. The relationship ential mating success. However, if the gra- Additional Insights from Studies of between mating success and reproductive dient is steep, very strong sexual selection Syngnathid Mating Systems success can be quantified (and compared is possible. Under this view the sexual se- statistically) using a least-squares regres- lection gradient concept does not dimin- Microsatellite-based studies of parentage sion approach (Arnold and Duvall 1994). ish the importance of other methods re- in pipefishes and seahorses have also pro- The resulting line is referred to as the sex- lated to the measurement of sexual vided novel perspectives on the evolution ual selection gradient (Arnold and Duvall selection, but rather should be seen as a of the markers themselves, and on the 1994), or Bateman gradient (Andersson quantitative framework that can be used broader use of molecular parentage anal- and Iwasa 1996). to sort out the relative importance of the yses in population studies. The following One simple conceptualization of the sex- multifarious factors that contribute to sex- sections will highlight three such applica- ual selection gradient’s position in the sex- ual selection. tions. ual selection process is presented in Fig- Pipefish provide a unique opportunity ure 5. The operational sex ratio, potential to test the validity of the sexual selection Clustered Microsatellite Mutations in S. reproductive rates of the sexes, and rela- gradient concept. In sex-role-reversed spe- typhle tive parental investment, among other fac- cies, a steep sexual selection gradient is In a large study of parentage involving 110 tors, no doubt interact in complex ways expected in females and a shallow gradi- S. typhle families and 3195 progeny, Jones to affect the strength of sexual selection ent in males. Jones et al. (2000a) tested et al. (1999a) obtained direct estimates of (Andersson 1994), and they typically do this hypothesis in popu- rates at two highly polymorphic so by influencing patterns of mating suc- lations of S. typhle. Microsatellite markers microsatellites. Both loci contained tetra- cess. In the context of sexual selection, were used to describe parentage com- nucleotide microsatellite repeats (motif variance in mating success can contribute pletely for small pipefish breeding groups, GGAT or GGTT), and mutation rates were to variance in fitness only through the sex- allowing the sexual selection gradients of between 9.4 ϫ 10Ϫ4 and 4.4 ϫ 10Ϫ3 per

156 The Journal of Heredity 2001:92(2) . The pattern of mutations con- pregnant adult males, their intact broods, struction, and the use of parentage as- formed well to a stepwise mutation model and adult females, and it takes advantage sessment for the estimation of adult pop- (Shriver et al. 1993), with all mutations re- of the fact that microsatellite markers can ulation size. sulting from the insertion or deletion of match particular collected females to par- Much research on syngnathids remains one or a few microsatellite motif units. ticular broods with great precision (see to be done. For example, all of the genetic Another interesting result of the study above). The method by which adult pop- studies conducted to date have dealt with was that mutations often occurred in clus- ulation size is estimated can be described mating patterns in only one population ters. Combining data from both loci, a to- by analogy to traditional mark-recapture per species. Geographic variation in mat- tal of 35 mutant progeny were assayed, procedures routinely employed by field ing systems probably exists, the charac- but they apparently were produced by ecologists ( et al. 1990). terization of which should lead to pro- only 26 independent mutation events Under the Lincoln–Peterson method of gress in understanding the ecological (Jones et al. 1999a). Thus multiple off- a conventional mark-recapture experi- causes and consequences of mating sys- spring in a single brood sometimes re- ment, an initial sample of n1 animals is tem evolution. In addition, all molecular ceived the same mutant allele, a pattern captured, marked, and released. A second studies of syngnathids have focused on indicative of multiple carrying sample of n2 animals is later caught, and species with completely enclosed brood copies of one premeiotic mutation that the number of marked animals (m2) in the pouches, yet patterns of mating and risks arose in a parent’s germline (Woodruff sample gives an indication of the popula- of cuckoldry may well differ in species and Thompson 1992). The phenomenon of tion size. In the pipefish parentage study, where the pouch is absent or less en- clustered mutations may have conse- the initial ‘‘marks’’ were provided by the closed. Comparative studies are also need- quences for the reconstruction of parent- inferred genotypes of females that contrib- ed on species in other genera. Such stud- age using microsatellite-based markers uted to the broods of pregnant males. ies will not only broaden the scope of (Jones et al. 1999a), and also, under some Thus, in essence, a female marked herself analysis, but when interpreted in the con- conditions, for theory by mating with one of our sampled males text of syngnathid phylogeny should also (Woodruff et al. 1996; see also Jones et al. (Jones and Avise 1997b). The genetically help to identify possible phylogenetic con- 1999a). assayed adult females were considered straints on mating system evolution. the second sample, and those females that Future studies of pipefishes and sea- Linked loci in the Study of Parentage matched particular broods were counted horses would also benefit from integrating While documenting genetic monogamy in as ‘‘recaptures’’ of the marked individuals. genetic information with that from other seahorses, Jones et al. (1998) encountered Then an estimate of the population size is areas relating to sexual selection and mat- ϭ ϩ ϩ ϩ two loci that failed to segregate indepen- obtained by nˆ (n1 1)(n2 1)/(m2 1) ing systems, including reproductive ecol- dently within families but displayed no Ϫ 1 (Pollock et al. 1990). Application of ogy, parent-offspring conflict, sexual con- at the population this methodology to the dusky pipefish flict, and brood reduction. Overall these level. This raised the question of whether population led to a surprisingly small pop- small fishes likely will continue to make tightly linked loci can be useful for par- ulation size estimate of only 85–192 adult disproportionately large contributions to entage reconstruction. The authors found females (Jones and Avise 1997b). the scientific understanding of sexual se- that linked loci can be useful if (1) the link- lection and reproductive evolution. age phase of the parents can be ascer- Summary and Future Directions tained with certainty, (2) the loci are sit- uated close enough to one another that The study of pipefish and seahorse mating References parental linkage relationships can be ob- systems is in its early stages but has al- Andersson M, 1994. Sexual selection. Princeton, NJ: tained reliably from progeny arrays, and ready provided some exciting results. A Princeton University Press. (3) the loci are not in severe linkage dis- wide range of genetic mating systems Andersson M and Iwasa Y, 1996. Sexual selection. Trends Ecol Evol 11:53–58. equilibrium at the population level. If have been uncovered in populations of Arnold SJ, 1983. 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In ad- mentation predicts reproductive success in female Use of Parentage Assessment in Direct dition, studies of pipefish have provided pipefish. Behav Ecol Sociobiol 40:145–150. Population Size Estimation the first characterization of Bateman gra- Berglund A, Rosenqvist G, and Svensson I, 1986a. Mate Neutral molecular markers are frequently dients in a sex-role-reversed organism. choice, fecundity and sexual dimorphism in two pipe- fish species (Syngnathidae). Behav Ecol Sociobiol 19: used to calculate indirect estimates of ef- The sexual selection gradient passed this 301–307. fective population size (Hartl and Clark important test, and it appears to be a Berglund A, Rosenqvist G, and Svensson I, 1986b. Re- 1989). In the context of microsatellite- promising new approach for the quantita- versed sex roles and parental energy investment in zy- based studies of parentage, a more direct tive characterization of genetic mating gotes of two pipefish (Syngnathidae) species. Mar Ecol Prog Ser 29:209–215. measure of adult population size can be systems. Studies of seahorses and pipe- Berglund A, Rosenqvist G, and Svensson I, 1989. Repro- obtained using a ‘‘mark-recapture’’ frame- fishes also have offered novel insight into ductive success of females limited by males in two work (Jones and Avise 1997b). The pro- the occurrence of clustered mutations, the pipefish species. Am Nat 133:506–516. cedure requires a collection from nature of utility of linked loci in parentage recon- Bjo¨rklund M, 1990. A phylogenetic interpretation of

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