ORIGINAL ARTICLE doi:10.1111/j.1558-5646.2007.00035.x SEQUENCE EVOLUTION OF THE SPERM LIGAND ZONADHESIN CORRELATES NEGATIVELY WITH BODY WEIGHT DIMORPHISM IN PRIMATES Holger Herlyn1,2 and Hans Zischler1,3 1Institute of Anthropology, University of Mainz, Colonel-Kleinmann-Weg 2 (SB II), D-55099, Germany 2E-mail: [email protected] 3E-mail: [email protected] Received August 24, 2006 Accepted October 9, 2006 Sexual selection has repeatedly been shown to be the probable driving force behind the positive Darwinian evolution of genes affecting male reproductive success. Here we compare the sequence evolution of the sperm ligand zonadhesin with body mass di- morphism in primates. In contrast to previous related studies, the present approach takes into account not only catarrhine primates, but also platyrrhines and lemurs. In detail, we analyze the sequence evolution of concatenated zonadhesin fragments (555 bp) of four Lemuroidea, five Platyrrhini, and seven Catarrhini, using the rate ratio of nonsynonymous to synonymous substitutions (dn/ds = ␻). Unexpectedly, subsequent regression analyzes between ␻ estimates for the terminal branches of a primate phy- logeny and residual male body mass reveal that sequence evolution of zonadhesin decreases with increasing sexual dimorphism in body weight. Mapping published mating system classifications onto these results illustrates that unimale breeding species show a tendency for rather slow sequence evolution of zonadhesin and comparably pronounced sexual dimorphism in body weight. Female choice and sperm competition can be assumed to drive the evolution of zonadhesin. We speculate that the level of sperm competition is lower in more sexually dimorphic primates because males of these species monopolize access to fertile females more successfully. Thus, variation in sperm competition may be driving the observed negative correlation of sequence evolution and sexual dimorphism in body weight. KEY WORDS: Breeding system, female choice, mating system, sexual dimorphism, sperm competition, sperm–egg interaction, zonadhesin. With the increasing amounts of comparative sequence data, evi- tion of genes that influence the reproductive success of males. In- dence is growing that positive selection plays a crucial role in the creased levels of sexual selection should thus accelerate sequence evolution of genes involved in mating behavior, fertilization, sper- evolution of genes affecting male reproductive success. The first matogenesis, and sex determination (reviewed in Swanson and suggestion of such a relationship came from comparative analysis Vacquier 2002). As it affects the evolution of physical traits such of the protamine gene cluster in primates (Wyckoff et al. 2000; as testis size, sperm morphology, and baculum length (Harcourt see also Rooney and Zhang 1999). Subsequently, Dorus et al. et al. 1981; Anderson and Dixson 2002; for review see Dixson (2004) reported a positive correlation of sequence evolution of and Anderson 2004), sexual selection may also drive the evolu- semenogelin 2, a main component of copulatory plugs, and the C 2007 The Author(s) 289 Journal compilation C 2007 The Society for the Study of Evolution H. HERLYN AND H. ZISCHLER level of sperm competition, a form of sexual selection. However, ability (for a compilation of body mass data in primates see, e.g., the estimates of sperm competition used by Dorus et al. (2004), Kappeler 1991, Lindenfors 2002). Initially, a combination of hap- that is, the number of peri-ovulatory partners and residual testis lorhine and strepsirhine data in one statistical approach might size are not available for most primate species. Moreover, ex- appear problematical because body mass dimorphism is much cept for one platyrrhine monkey serving as outgroup (Dorus et al. less common in strepsirhines than in haplorhines (Kappeler 1991, 2004), previous studies were confined to Hominoidea (Wyckoff 1997; Lindenfors and Tullberg 1998; Plavcan 2001; Lindenfors et al. 2000) and Catarrhini (Dorus et al. 2004). Yet seasonality of 2002). However, in the context of the present study the compared breeding (Rowe 1996) suggest high levels of sexual selection in species do not have to be sexually dimorphic. Instead, the sexes Lemuroidea. Finally, sexual selection may be primarily behavioral may be of similar or equal size, and females may even be larger in many species, and successful males may prevent access to re- than males. The only prerequisite is that the sample covers vari- ceptive females. Such behavior could thus reduce the opportunity ance of dimorphism within primates. Another concern could be for interindividual sperm competition. that body mass is under the influence of different selective pres- sures and constraints. Though this is certainly the case, recent ZONADHESIN studies have shown a strong correlation between body mass di- Based on a broad primate sampling, including Platyrrhini and morphism and other indirect measures of sperm competition, such Lemuroidea, we investigate possible correlations between the se- as operational sex ratio, mating systems, and male–male compe- quence evolution of the sperm ligand zonadhesin and sexual se- tition levels (Mitani et al. 1996; Plavcan 2001, 2004; Lindenfors lection of males. Pseudogenization of zonadhesin might occur 2002; Lindenfors et al. 2004). Therefore, we consider sexual di- sporadically (see Hillier et al. 2003). However, we found no evi- morphism in body mass as another surrogate measure of sexual dence for such a process in previous studies (Herlyn and Zischler selection. 2005a,b) and, thus, consider zonadhesin a suitable subject for evolutionary studies. The ∼62 kb of the human zonadhesin locus stretch over 48 exons coding for the 2811 amino acids of precur- Materials and Methods sor zonadhesin (see ENSEMBL Gene: ENSG00000146839). In PCR, SEQUENCING, AND ALIGNING primates, Sus scrofa, and Oryctolagus cuniculus, precursor zon- As it is very difficult to obtain zonadhesin expressing tissue such adhesin encodes for two MAM (meprin/A5 antigen/mu receptor as testis, new sequences were generated on the basis of genomic tyrosine phosphatase) domains, a mucin-like tandem repeat, and DNA rather than cDNA. Given the extensive intronic information five so-called D domains, homologous to von Willebrand factor D covered by the zonadhesin locus (∼53 kb, see ENSEMBL Gene: (Hardy and Garbers 1995; Gao and Garbers 1998; Lea et al. 2001; ENSG00000146839), we confined the present approach to the Herlyn and Zischler 2005a,b). The mucin-like domain of zonad- exonic information upstream and downstream of three exception- hesin consists of tandemly arranged seven amino acid motifs that ally short introns, that is, intron 9–10 (108 bp, taking the human evolve in a concerted fashion. Contrary to this, zonadhesin MAM ortholog as reference), intron 20–21 (110 bp), and intron 32–33 and D domains evolve divergently (Herlyn and Zischler 2006), (93 bp). Exons 9 and 10 encode for fragments of MAM domains apparently driven by positive selection at the level of both single 1 and 2. Exons 20 and 21 and exons 32 and 33 are located within codon sites (Swanson et al. 2003) and posttranslational modifica- domains D1 and D3, respectively. tions (Herlyn and Zischler 2005a,b). The processing of precursor Genomic DNA was extracted from different tissues of the zonadhesin leads to the formation of four subunits that partly bind following primate representatives: Lemuroidea (Eulemur fulvus, to the egg’s zona pellucida (see Hardy and Garbers 1995; Gao and Lemur catta, Microcebus murinus, Varecia variegata), Platyrrhini Garbers 1998; Lea et al. 2001; Bi et al. 2003). (Aotus azarai, Ateles belzebuth, Callicebus cupreus, Saguinus fus- cicollis, Saimiri sciureus), and Cercopithecoidea (Cercopithecus BODY MASS DIMORPHISM mitis, Erythrocebus patas, Macaca mulatta, Mandrillus sphinx, In most primate species, long life spans, low reproductive rates, Papio hamadryas, Pygathrix nemaeus). Primers specific for Cer- and practical difficulties in the field render it impossible to directly copithecoidea, Platyrrhini, and Lemuroidea (Table 1) were de- measure sexual selection on males (Plavcan 2004). Moreover, as signed using published data from GenBank (U40024, AF244982, mentioned above, surrogate measures of sperm competition such AF332975, AY428845-58).Fragments of 310–385 bp length com- as residual testis size and number of peri-ovulatory partners are prising introns 9–10, 20–21, and 32–33, and part of the adjacent not available for most primate species. For both reasons, it is de- exons were amplified by wax-mediated hotstart PCR using the sirable to employ more available estimates when analyzing sexual Taq PCR Core Kit (Qiagen). Each reaction (30 ␮L) contained selection of males on the basis of more comprehensive samplings. 0.5 U Taq polymerase (AmpliTaq, Perkin Elmer), 15 pmol dNTPs, Data on body mass dimorphism fulfil the criterion of easy avail- 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 0.1% Triton 290 EVOLUTION FEBRUARY 2007 SEQUENCE EVOLUTION AND SEXUAL DIMORPHISM Table 1. Primers used for amplification of the three analyzed zonadhesin fragments. 1 ◦ Location Taxon Primer-sequence (5 -3 ) T m ( C) Exons 9, cercopithecoidea-1 for CGTGGT(AG)GGCGTTTTTG 54.0 Intron 9-10, rev TGCATTAGGGAAACCTCC 53.7 Exon 10 platyrrhini-1 for TCGTGGTGGG(CT)(AG)TTTTTG 53.7 rev CTG(CT)(CA)CT(AG)GGGAAATCTC 54.8 strepsirhini-1 for1 CGTGGTGGG(CT)(AG)T(ATG)TTTG 53.6 rev1 CTCAAGGTAGATATAGTG 49.1 for2 GGAC(AG)GATACAGTT(CT)ACCGTG 59.8 rev2 TGGAGGGGTACATTT(CT)TATGTC