Microsatellite analysis of maternity and the mating system in the Gulf pipefish Syngnathus scovelli, a species with male pregnancy and sex-role reversal

Highly variable microsatellite loci were employed to study the mating system of the sexually dimorphic Gulf pipefish Syngnathus scovelli . In this species, like others in the family Syngnathidae, 'pregnant' males provide all parental care. Gulf pipefish were collected from one locale in the northern Gulf of Mexico, and internally carried broods of 40 pregnant males were analysed genetically. By comparing multilocus microsatellite fingerprints for the inferred mothers against expected genotypic distributions from the population sample, it was determined that: (i) only one male had received eggs from more than a single female; and (ii) on two separate occasions, two different males had received eggs from the same female. Given the high power to detect multiple matings by males, the first finding indicates that only rarely are individual males impregnated by multiple females during the course of a pregnancy. Conversely, given the lower power to detect multiple matings by females due to sampling constraints, the second finding suggests a high frequency of multiple successful matings by females. Thus, this population of Gulf pipefish displays a polyandrous genetic mating system. The relevance of these genetic findings is discussed with regard to the evolution of secondary sex traits in this species, and in other syngnathids.     

Isolation of polymorphic tetranucleotide microsatellite markers for the bay pipefish Syngnathus leptorhynchus

We have isolated nine polymorphic microsatellite markers for the bay pipefish Syngnathus leptorhynchus from genomic libraries enriched for (AAGG)_n repetitive elements. The number of alleles ranged from two to 15 per locus with the observed heterozygosity ranging from 0.09 to 1.00. These markers will be useful for analyses of questions concerning population genetic structure.     

Patterns of multiple paternity and maternity in fishes

The characterization of patterns of multiple mating is a major facet of molecular ecology and is paramount to understanding the evolution of behaviours associated with parental care and mate choice. Over the last 15 years, fishes have been particularly well studied with respect to multiple maternity and paternity thanks to the widespread application of microsatellite markers. The present review focusses on the impressive literature on genetic parentage in fishes. In studies of natural populations, we find that multiple paternity is extremely common across fish species, whereas rates of multiple maternity are much more variable. In species with nest defence, for example, rates of multiple maternity are strongly bimodal, and the occurrence of multiple dams per brood is either rare or the rule. The sex of the care‐giving parent is correlated with the rate of multiple parentage: when males provide uniparental care, rates of multiple paternity are low compared to rates of multiple maternity; when females provide parental care, either alone or assisted by males, rates of multiple paternity are highly variable, whereas rates of multiple maternity are quite low. These patterns may reflect conflicts between the reproductive interests of males and females. We also find that fishes in which females brood the offspring internally display much higher rates of multiple paternity compared to mammals or birds, whereas reptiles are intermediate. Male‐nesting fish species, however, show rates of multiple paternity more similar to those found in other vertebrates. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 735–760.     

Northern pipefish,Syngnathus fuscus,occurrences over the Mid-Atlantic Bight continental shelf: evidence of seasonal migration

Synopsis Data from several areas in the northern Mid-Atlantic Bight indicate northern pipefish, Syngnathus fuscus, undergo seasonal, inshore-offshore migrations. Resident in estuaries during spring through fall, they move into nearshore continental shelf waters off Cape Cod in late September–October and off Long Island and New Jersey in November. Return to estuaries occurs in March–April. Most (> 90%) continental shelf otter trawl collections in fall (September–November) were at water temperatures of 10–15°C. Most (> 80%) spring (March–May) collections occurred at water temperatures of 3–6°C. The majority of offshore collections were within 20 km of the coast and over 90% were in depths between 10 and 20 m. Length frequency data reveal both young-of-year and older fish migrate, possibly to avoid colder estuarine water temperatures in winter.     

Feeding chronology,daily ration,and the effects of temperature upon gastric evacuation in the pipefish,Syngnathus fuscus

Synopsis Feeding chronology, daily ration, and the effects of temperature upon gastric evacuation were examined in the pipefish,Syngnathus fuscus, from field and laboratory data.S. fuscus displayed a pattern of diurnal feeding, characteristic of syngnathids. Daily ration calculations yielded estimates of 4.0 and 4.4% body weight per day, which are comparable to estimates for other teleosts. Evacuation rate was found to be temperature dependent. with more rapid evacuation with increasing temperature. In addition, evacuation rate was found to be positively correlated with gut content. Slowing of evacuation rate with decreasing gut content may allow for increased assimilation efficiency during periods of low food availability. Daily ration, although controlled by the temperature dependence of evacuation rate, may also be controlled by prey abundance; fish maximize food intake during periods of high prey availability, and maximize upon assimilation during periods of low prey availability.Contribution number 1035 of the Virginia Institute of Marine Science of the College of William and Mary.     

Evidence for multiple paternity in the school shark Galeorhinus galeus found in New Zealand waters

This study assessed the levels of relatedness of Galeorhinus galeus of progeny arrays using six microsatellite DNA markers. A parentage analysis from five families (mother and litter) from the North Island of New Zealand suggested the occurrence of genetic polyandry in G. galeus with two of the five litters showing multiple sires involved in the progeny arrays. This finding may be consistent with the reproductive characteristics of G. galeus, in which females can potentially store sperm for long periods of time after the mating season.     

Sexual size dimorphism predicts the frequency of multiple mating in the sex-role reversed pipefish Syngnathus typhle

The sex-role reversed pipefish Syngnathus typhle is a member of the Syngnathidae, a family of fishes in which males brood embryos on their body surface. As in most ectotherms, embryonic development is highly temperature dependent in syngnathids and male brooding periods are extended when water temperatures are reduced. The influence of temperature on reproduction is expected to effectively truncate the breeding season and reduce fecundity in cold waters, potentially enhancing the opportunity for both fecundity and sexual selection. We studied spatial variation in the morphology and reproductive biology of S. typhle in five European populations which vary in latitude and water temperature. Microsatellite analyses indicated that the average number of male mates per population ranged between 1.3 and 3.7. The frequency of multiple mating by males was negatively correlated with the degree of sexual size dimorphism in each population, suggesting that disproportionate increases in female fecundity may be able to compensate for increased male brood pouch capacity. Both sexes were larger and males had an increased brood size where water temperatures during the breeding season were lower. Morphological variation among populations may be mediated by differences in fecundity selection associated with different optimal reproductive strategies in cold and warm water environments.     

Power of exclusion for parentage verification and probability of match for identity in American Kennel Club breeds using 17 canine microsatellite markers

DNA analysis of microsatellite markers has become a common tool for verifying parentage in breed registries and identifying individual animals that are linked to a database or owner. Panels of markers have been developed in canines, but their utility across and within a wide range of breeds has not been reported. The American Kennel Club (AKC) authorized a study to determine the power to exclude non-parents and identify individuals using DNA genotypes of 17 microsatellite markers in two panels. Cheek swab samples were voluntarily collected at Parent Breed Club National Specialty dog shows and 9561 samples representing 108 breeds were collected, averaging 88.5 dogs per breed. The primary panel of 10 markers exceeded 99% power of exclusion for canine parentage verification of 61% of the breeds. In combination with the secondary panel of seven markers, 100% of the tested breeds exceeded 99% power of exclusion. The minimum probability match rate of the first panel was 3.6 x 10(-5) averaged across breeds, and with the addition of the second panel, the probability match rate was 3.2 x 10(-8); thus the probability of another random, unrelated dog with the same genotype is very low. The results of this analysis indicated that, on average, the primary panel meets the AKC's needs for routine parentage testing, but that a combination of 10-15 genetic markers from the two panels could yield a universal canine panel with enhanced processing efficiency, reliability and informativeness.     

Probability of random sire exclusion using microsatellite markers for parentage verification

Many microsatellite sequences have been described in the bovine genome. Being highly polymorphic these have been suggested as markers for parentage verification and individual identification in cattle. We have evaluated the use of five highly polymorphic microsatellite markers for parentage verification in 14 breeds of cattle in the UK. Three of the microsatellite loci occur within introns in genes: BoLA DRB3 , steroid 21-hydroxylase, and the beta subunit of the follicle-stimulating hormone. The other two are anonymous sites ETH131 and HEL6. Results were analysed by a statistical approach that takes in to account deviations from Hardy-Wienberg equilibrium and linkage disequilibrium for multiple loci. The method of determining the probability of random sire exclusion uses observed genotype frequencies instead of allele frequencies. Independently, the markers used have a probability of between 0.72 and 0.62 of identifying a parentage error, while used together the five markers give, on average across breeds, a probability of 0.99 of excluding an incorrect sire.     

A bayesian framework for parentage analysis: the value of genetic and other biological data

We develop fractional allocation models and confidence statistics for parentage analysis in mating systems. The models can be used, for example, to estimate the paternities of candidate males when the genetic mother is known or to calculate the parentage of candidate parent pairs when neither is known. The models do not require two implicit assumptions made by previous models, assumptions that are potentially erroneous. First, we provide formulas to calculate the expected parentage, as opposed to using a maximum likelihood algorithm to calculate the most likely parentage. The expected parentage is superior as it does not assume a symmetrical probability distribution of parentage and therefore, unlike the most likely parentage, will be unbiased. Second, we provide a mathematical framework for incorporating additional biological data to estimate the prior probability distribution of parentage. This additional biological data might include behavioral observations during mating or morphological measurements known to correlate with parentage. The value of multiple sources of information is increased accuracy of the estimates. We show that when the prior probability of parentage is known, and the expected parentage is calculated, fractional allocation provides unbiased estimates of the variance in reproductive success, thereby correcting a problem that has previously plagued parentage analyses. We also develop formulas to calculate the confidence interval in the parentage estimates, thus enabling the assessment of precision. These confidence statistics have not previously been available for fractional models. We demonstrate our models with several biological examples based on data from two fish species that we study, coho salmon (Oncorhychus kisutch) and bluegill sunfish (Lepomis macrochirus). In coho, multiple males compete to fertilize a single female's eggs. We show how behavioral observations taken during spawning can be combined with genetic data to provide an accurate calculation of each male's paternity. In bluegill, multiple males and multiple females may mate in a single nest. For a nest, we calculate the fertilization success and the 95% confidence interval of each candidate parent pair.     

Characterization of 13 polymorphic microsatellite loci for an evergreen tree,Myrica rubra

We developed 13 microsatellite markers from a genomic library enriched for dinucleotide (CT) repeats in a dioecious evergreen tree, Myrica rubra. We screened loci from 32 adult trees. The number of alleles ranged from two to 14, and the expected heterozygosity ranged from 0.324 to 0.884. The total paternity exclusionary power where the mother was known equalled 0.99965. These loci are of clear value in studying mating system and parentage analysis of this species.     

Using blocks of linked single nucleotide polymorphisms as highly polymorphic genetic markers for parentage analysis

Single nucleotide polymorphisms (SNPs) are plentiful in most genomes and amenable to high throughput genotyping, but they are not yet popular for parentage or paternity analysis. The markers are bi-allelic, so individually they contain little information about parentage, and in nonmodel organisms the process of identifying large numbers of unlinked SNPs can be daunting. We explore the possibility of using blocks of between three and 26 linked SNPs as highly polymorphic molecular markers for reconstructing male genotypes in polyandrous organisms with moderate (five offspring) to large (25 offspring) clutches of offspring. Haplotypes are inferred for each block of linked SNPs using the programs Haplore and Phase 2.1. Each multi-SNP haplotype is then treated as a separate allele, producing a highly polymorphic, 'microsatellite-like' marker. A simulation study is performed using haplotype frequencies derived from empirical data sets from Drosophila melanogaster and Mus musculus populations. We find that the markers produced are competitive with microsatellite loci in terms of single parent exclusion probabilities, particularly when using six or more linked SNPs to form a haplotype. These markers contain only modest rates of missing data and genotyping or phasing errors and thus should be seriously considered as molecular markers for parentage analysis, particularly when the study is interested in the functional significance of polymorphisms across the genome.     

Assignment of paternity groups without access to parental genotypes: multiple mating and developmental plasticity in squid

We present a novel approach to investigating sibling relationships and reconstructing parental genotypes from a progeny array. The Bayesian method we have employed is flexible and may be applicable to a variety of situations in addition to the one presented here. While mutation rates and breeding population allele frequencies can be taken into account, the model requires relatively few loci and makes few assumptions. Paternity of 270 veined squid (Loligo forbesi) hatchlings from three egg strings collected from one location was assigned using five microsatellite loci. Paternal and maternal genotypes reconstructed for each of the three strings were identical, strongly indicating that a single female produced the strings that were fertilized by the same four males. The proportion of eggs fertilized was not equal between males in all three strings, with male 1 siring most offspring (up to 68% in string 1), through to male 4 siring the least (as low as 2.4% in string 1). Although temperature had a profound effect on incubation time, paternity did not affect this trait at 12 degrees C or 8 degrees C.     

Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo‐Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co‐amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent–offspring relationships in natural populations, with over 99.6% accuracy in parent–offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28–96% of loci. The multiplex PCRs developed here provide a reliable and cost‐effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species.     

Exclusion probabilities of 22 bovine microsatellite markers in fluorescent multiplexes for semiautomated parentage testing

Six multiplexes developed for semiautomated fluorescence genotyping were evaluated for parentage testing. These multiplexes contained primer pairs for the amplification of 22 microsatellites on 17 bovine autosomes. Exclusion probabilities were determined from genotypes of 1022 Holstein cattle and 311 beef cattle belonging to five breeds. Two cases were considered: case 1, genotypes are known for an alleged parent and an offspring but genotypes of a confirmed parent are unknown; and case 2, genotypes are known for an alleged parent, a confirmed parent and an offspring. If the alleged parent is not the true parent, then the 22 markers will exclude the alleged parent with a probability of >0·9986 for case 1 and with a probability of >0·99999 for case 2. On the basis of these exclusion probabilities, the probability that an alleged parent will be falsely included as a parent is in the range of 1/716 to 1/2845 for case 1 and 1/1·2 million to 1/159753 for case 2. In addition to these results, a rapid and efficient non-organic method for extraction of DNA from semen is described.     

Strong male/male competition allows for nonchoosy females: high levels of polygynandry in a territorial frog with paternal care

Our knowledge about genetic mating systems and the underlying causes for and consequences of variation in reproductive success has substantially improved in recent years. When linked to longitudinal population studies, cross-generational pedigrees across wild populations can help answer a wide suite of questions in ecology and evolutionary biology. We used microsatellite markers and exhaustive sampling of two successive adult generations to obtain population-wide estimates of individual reproductive output of males and females in a natural population of the Neotropical frog Allobates femoralis (Aromobatidae), a pan-Amazonian species that features prolonged iteroparous breeding, male territoriality and male parental care. Parentage analysis revealed a polygynandrous mating system in which high proportions of males (35.5%) and females (56.0%) produced progeny that survived until adulthood. Despite contrasting reproductive strategies, successfully reproducing males and females had similar numbers of mating partners that sired the adult progeny (both sexes: median 2; range 1-6); the numbers of their offspring that reached adulthood were also similar (both sexes: median 2; range 1-8). Measures of reproductive skew indicate selection on males only for their opportunity to breed. Reproductive success was significantly higher in territorial than in nonterritorial males, but unrelated to territory size in males or to body size in both sexes. We hypothesize that female polyandry in this species has evolved because of enhanced offspring survival when paternal care is allocated to multiple partners.     

A single base transversion in the flanking region of an equine microsatellite locus affects amplification of one allele

The equine dinucleotide microsatellite HMS7 is part of a microsatellite panel utilized in a parentage verification programme at the Veterinary Genetics Laboratory (Davis, California, USA). Apparent non-Mendelian inheritance was noted when a Quarter Horse mare was excluded as the parent of two offspring based on analysis of the HMS7 locus. The mare's DNA type qualified her as a parent of the offspring at an additional 20 microsatellite loci. The three animals appeared homozygous for HMS7 with each possessing an allele different from that of the other two animals. Polymerase chain reaction primers designed to bind outside the published primer-binding sites amplified an additional shared allele in all three horses, which qualified the mare as the dam of the two offspring. Sequencing of this newly detected allele revealed a C to A transversion in one of the published primer-binding regions. Apparent non-Mendelian inheritance at the HMS7 locus has been encountered in an additional 26 Quarter Horse parentage cases. In all instances, the lack of amplification and resultant 'null' allele was shown to be caused by the same transversion.