Genotyping and AnalysisAll 20 loci were screened in 41 adults from Marrakai Creek to estimate allele frequencies in the population and to test for adherence to Hardy Weinberg equilibrium. These assays revealed unexpectedly low diversity in the population as 18 of the 20 markers were fixed at the cloned allele. Two loci, Kg019 and Kg275, did elicit some diversity (6 and 5 alleles, respectively). These two loci were amplified in a subsample of embryos from each egg mass (8-65 embryos, depending on the total number of embryos present) and in all net-associated males. Simple paternity exclusion followed as embryo genotypes were compared to associated males. All legitimate offspring must share a common allele at each locus with each parent. Divergence from this pattern may indicate the presence of cuckoldry.

J. A. SOMMER1, C. LI1, J. BROZEK1, M. L. BESSERT1,2, G. ORTÍ1,3, T. M. BERRA4

1University of Nebraska; 2University of Wisconsin-Stout; 3George Washington University, 4The Ohio State University

Fig. 1 Male nurseryfish with egg mass; actual size

Microsatellite parentage in the nurseryfish Microsatellite parentage in the nurseryfish ((Kurtus gulliveriKurtus gulliveri) of northern Australia) of northern Australia

Materials and methodsField sitesThe majority of specimens utilized for this study were collected by gill netting in Marrakai Creek (12o40.950’S, 131o20.030’E), a major freshwater tributary of the Adelaide River about 65 km east of Darwin (Fig. 2, 3). Three additional samples from other isolated populations in Northern Australia (Daly River, South Alligator River, and East Alligator River) and four samples from New Guinea were acquired for comparison with the Adelaide River population. Tissue samples of adults and egg masses were preserved in 98% ethanol immediately upon capture. Collections occurred in 2001, 2003, 2004 and 2009 during the dry season between late June and early November, coinciding with the Nurseryfish spawning season.

Marker DevelopmentTotal genomic DNA was extracted from a single adult and microsatellite loci were isolated according to the method described by An et al. (2004). Briefly, DNA was digested with restriction enzymes into fragments averaging 500 base pairs in length. Resulting products were enriched for CA and CAGA repeat motifs, then cloned into bacteria. A total of 475 colonies containing inserts were amplified and sequenced in one direction using BigDye Terminator chemistry (Applied Biosystems) and M13F primer. Forty-six positive clones were sequenced in the opposite direction with M13R. Complementary forward and reverse primers were designed for 24 that possessed adequate flanking regions and a repeat section >12 base pairs. PCR conditions were optimized using a gradient of annealing temperatures from 48-62 °C. Of these 24, only 3 failed to amplify a product of the predicted size and one was a duplicate microsatellite. Sequences of the 20 loci were deposited in GenBank.

Maternity was examined via multiple techniques. First, attempts were made to extract and genotype DNA from the fibrous network (‘raceme’) that holds egg clusters together. If more than one dam contributed, this would be revealed in genotypic data from these conspicuous fibers.

In addition, parental genotype reconstruction was performed with the program Gerud 2.0, which uses an algorithm to describe a minimum number of parental genotypes contributing to an array of typed offspring. This approach is based on Mendelian rules of inheritance but assumes that no parental genotypes are known.

DiscussionOur study is the first to address questions about the genetic mating system of K. gulliveri, a species that is difficult to study because of its preferred habitat of turbid estuarine waters and co-occurence with large saltwater crocodiles. One of the safest ways to study these fishes is to draw inferences from previously collected specimens via hypervariable genetic markers. Microsatellites are typically an ideal marker for such endeavors but in this case yielded very low analytical power. The paucity of variation in the loci examined is puzzling, but has been corroborated with mitochondrial sequence data that also showed extremely low variation. At best, our data is not consistent with the idea of an open water spawning act that provides an opportunity for multiple paternity. Clearly, there are many unresolved questions about the mating system. We hope to gain resolution via the development and use of additional genetic markers. In addition, we hope to develop additional collaborations for a concerted captive rearing effort. In this way, we may be able to safely witness the act that leads to this anomalous form of male parental care.

IntroductionThe Nurseryfish, Kurtus gulliveri, is a freshwater fish distributed in Northern Australia and Southern New Guinea that exhibits forehead brooding, a unique form of male parental care. Mature males develop a fleshy, highly vascularized, dorsal hook upon which they carry egg masses (see Fig. 1, 3). How the egg masses are acquired is unknown. Indeed, the spawning act has never been observed because these fishes occur in turbid streams inhabited by large estuarine crocodiles. At best, field work is difficult and at worst, dangerous. In addition, all captive rearing attempts have failed.

Here, we developed microsatellite genetic markers to shed some light on these heretofore clandestine behaviors. Four particular questions of interest were addressed:1)Are males captured in close proximity to egg masses the sire of those embryos? 2)Do egg-carrying males sire all the embryos they carry or is there any evidence multiple paternity in a mass? 3)Does spawning occur externally or internally? If external, then some males may carry egg masses that are unfertilized.4)Does each egg mass originate from a single female?

A total of 20 microsatellite were developed and screened for parentage analysis. Of these, only two exhibited length variation. These two were scored in all egg masses captured in gill nets along with putative egg-carrying males that were captured in close proximity. The microsatellite data are consistent with the hypotheses that such ‘egg-carrying’ males sire the egg masses they carry and that no form of cuckoldry occurs. These results should be taken with caution, however, given the limited genetic variation at these loci. Although analytical power is limited (i.e., low exclusion probablility), these results are an important first step toward understanding this truly unique brooding behavior.

Fig. 2 Collection sites in Australia and New Guinea

Fig. 3 a. Net-captured male with associated egg mass; b. closeup of fibrous raceme connecting eggs

aa

b

ResultsOf the 20 loci isolated for this work, 18 were fixed at a single allele and only two, Kg 019 and Kg275, yielded any length variation (see Table 1 above). These two loci combined for a total exclusion probability of only 0.44, admittedly low for this type of study. When applied to a sample of males collected adjacent to egg masses (n=7 males), they revealed no illegitimate embryos (n=440

Table 1. Variable loci (red) and two of eighteen fixed loci. T=annealing temperature; k = # alleles; H = heterozygosity; HWE = p(deviation from HW equilibrium)

embryos). All were consistent with the hypothesis that the associate male was the true sire. Genotypes of embryos from three additional egg masses (n=119 embryos) were used to estimate a minimum number of parents contributing to the mass. In all three cases, the resulting minimum number was two. Finally, attempts to extract DNA from the fibrous raceme did not yield any measurable amount of DNA; therefore, we were unable to assay the material for multiple maternity.

AcknowledgementsThis work was supported by small grants from The National Geographic Society, the Columbus Zoo and Aquarium, and The Ohio State University.