Isolation and characterization of polymorphic microsatellite loci for the study of paternity and population structure in the little penguin Eudyptula minor

We isolated and characterized eight novel microsatellite loci in the little penguin Eudyptula minor, using nonradioactive polymerase chain reaction‐based techniques to screen GA and GAAA repeats from enriched genomic DNA libraries. All eight loci were polymorphic and seven were variable in our main study population (mean H_E = 0.613, mean N_A = 7.14). Cross‐amplification using a microsatellite primer developed in Spheniscus demersus (African penguin) yielded one additional polymorphic locus. This locus combined with six of the little penguin loci is suitable for paternity assignment in little penguins (exclusion probability for seven unlinked loci = 0.993).     

Identification of novel microsatellite loci in the sand martin, Riparia riparia, and cross-amplification of loci from other bird species

We isolated and characterised six novel microsatellite loci for paternity analysis in the sand martin Riparia riparia, by screening an enriched genomic library. In addition, we tested 16 already published microsatellite markers, five of which were also polymorphic in the sand martin. Only one of these 11 loci exhibited evidence of null alleles, and all were polymorphic (mean H _o = 0.68, range of number of alleles per locus = 4–24), making them suitable for individual heterozygosity quantification and paternity assessment in this species (exclusion probability of 11 unlinked loci = 0.999997).     

Isolation and characterization of microsatellite markers for paternity assessment in the golden whistler (Pachycephala pectoralis: Aves)

We isolated and characterized six novel microsatellite markers for paternity analysis in the golden whistler Pachycephala pectoralis, by screening an enriched genomic library using nonradioactive PCR techniques. The six loci exhibited little or no evidence of null alleles and showed high levels of polymorphism (mean H_E = 0.85, mean number of alleles = 15.2), making them suitable for paternity assessment in this species (exclusion probability of six unlinked loci = 0.9997).     

Novel polymorphic microsatellite markers for paternity analysis in the red‐capped robin (Petroica goodenovii: Aves)

Seven microsatellite loci were isolated and characterized from the red‐capped robin Petroica goodenovii, using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Five loci showed no evidence of null alleles and were variable [mean heterozygosity (H_E) = 0.440, mean number of alleles = 8]. Cross‐amplification using primers for microsatellites in Phylloscopus occipitalis and Emberiza schoeniclus yielded another two polymorphic loci. The combined set of five red‐capped robin and two cross‐amplified loci are suitable for paternity assignment (exclusion probability for seven unlinked loci = 0.9760).     

Polymorphic microsatellite loci for paternity analysis in the Madagascar paradise flycatcher (Terpsiphone mutata: Aves)

Nine polymorphic microsatellite loci from the Madagascar paradise flycatcher Terpsiphone mutata were isolated using nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic library. Seven polymorphic loci showed no evidence of null alleles and exhibited high levels of variation in 18 unrelated individuals (mean diversity = 0.80, mean number of alleles = 13.6). These loci are therefore suitable for analysis of population structure and paternity (exclusion probability for six unlinked loci = 0.9998).     

Development of EST‐derived microsatellite markers for Arabidopsis lyrata subspecies petraea (L.)

A set of expressed sequence tag–simple sequence repeat (EST‐SSR) loci has been developed for Arabidopsis lyrata ssp. petraea. From 768 root cDNA clones, 126 microsatellites, including di‐, tri‐, tetra‐ and pentanucleotide repeat motifs were identified and primers were designed to 24 EST‐SSRs. Eleven loci were subsequently screened on 150 individuals sampled from five natural populations, which revealed three to nine alleles per locus (mean 5.36) and expected heterozygosity (H_E) estimates ranging from 0.046 to 0.698. Significant deviations from random mating were observed at 10 EST‐SSR loci, likely due to inbreeding (global F_IS = 0.151) and population structure (global F_ST = 0.246).     

Isolation and characterization of novel microsatellite loci for parentage assessment in the lance‐tailed manakin (Chiroxiphia lanceolata)

The lance‐tailed manakin (Chiroxiphia lanceolata) is a lek‐breeding bird from Central America in which males court females with complex cooperative displays. To resolve detailed patterns of paternity in the wild, we isolated and characterized 12 novel microsatellite loci in this species. Eleven of these loci were polymorphic (five to 14 alleles), with observed heterozygosities ranging from 0.36 to 0.87 (N = 574 individuals). We tested for linkage disequilibrium using randomized subsamples of adults to control for known family structure among long‐lived and sedentary individuals. These loci will be valuable in resolving paternity among many candidate fathers in this species.     

Isolation and characterization of microsatellite markers in the southern emu‐wren (Stipiturus malachurus: Aves)

We isolated and characterized eight novel microsatellite loci in the southern emu‐wren (Stipiturus malachurus). We used nonradioactive polymerase chain reaction (PCR)‐based techniques to screen an enriched genomic DNA library. Based on genotypes from a single population, six loci showed no evidence of null alleles and were polymorphic (allele range = 2–9, mean heterozygosity = 0.57), and one locus was sex‐linked (N_A = 4). These loci were variable and had different allele size ranges in three other populations of southern emu‐wrens, and are therefore useful for determining levels of genetic diversity within and between populations of the species.     

Microsatellite markers for Vaccinium from EST and genomic libraries

We present 30 microsatellite loci isolated from expressed sequence tag (EST) and genomic libraries in Vaccinium corymbosum L. Allele number per locus in 11 tetraploid and one diploid V. corymbosum accessions ranged from two to 15 (mean = 8.16) in 24 single‐locus simple sequence repeats (SSRs). Cross‐species amplification in a panel of 12 species representing nine sections ranged from 30 to 100% (mean = 83%).     

Limited cross‐species microsatellite amplification and the isolation and characterization of new microsatellite markers for the greater stick‐nest rat (Leporillus conditor)

The greater stick‐nest rat (Leporillus conditor) is a conilurid rodent whose recent history provides an opportunity to examine genetic changes in reintroduced populations. We trialled 63 known microsatellite primers from Rattus rattus and Mus musculus in L. conditor. Three primer pairs produced polymorphic loci (number of alleles = 2–3, mean, H_E = 0.42). Subsequently, we isolated and characterized 12 novel polymorphic microsatellites (mean number of alleles = 5–16, mean H_E = 0.76) from L. conditor from genomic libraries for use in population genetic studies.     

A molecular analysis of African lion (Panthera leo) mating structure and extra‐group paternity in Etosha National Park

The recent incorporation of molecular methods into analyses of social and mating systems has provided evidence that mating patterns often differ from those predicted by group social organization. Based on field studies and paternity analyses at a limited number of sites, African lions are predicted to exhibit a strict within‐pride mating system. Extra‐group paternity has not been previously reported in African lions; however, observations of extra‐group associations among lions inhabiting Etosha National Park in Namibia suggest deviation from the predicted within‐pride mating pattern. We analysed variation in 14 microsatellite loci in a population of 164 African lions in Etosha National Park. Genetic analysis was coupled with demographic and observational data to examine pride structure, relatedness and extra‐group paternity (EGP). EGP was found to occur in 57% of prides where paternity was analysed (n = 7), and the overall rate of EGP in this population was 41% (n = 34). Group sex ratio had a significant effect on the occurrence of EGP (P < 0.05), indicating that variation in pride‐level social structure may explain intergroup variation in EGP. Prides with a lower male‐to‐female ratio were significantly more likely to experience EGP in this population. The results of this study challenge the current models of African lion mating systems and provide evidence that social structure may not reflect breeding structure in some social mammals.     

Sperm dispersal distances estimated by parentage analysis in a brooding scleractinian coral

Within populations of brooding sessile corals, sperm dispersal constitutes the mechanism by which gametes interact and mating occurs, and forms the first link in the network of processes that determine specieswide connectivity patterns. However, almost nothing is known about sperm dispersal for any internally fertilizing coral. In this study, we conducted a parentage analysis on coral larvae collected from an area of mapped colonies, to measure the distance sperm disperses for the first time in a reef‐building coral and estimated the mating system characteristics of a recently identified putative cryptic species within the Seriatopora hystrix complex (ShA; Warner et al. 2015). We defined consensus criteria among several replicated methods (colony 2.0, cervus 3.0, mltr v3.2) to maximize accuracy in paternity assignments. Thirteen progeny arrays indicated that this putative species produces exclusively sexually derived, primarily outcrossed larvae (mean t_m = 0.999) in multiple paternity broods (mean r_p = 0.119). Self‐fertilization was directly detected at low frequency for all broods combined (2.8%), but comprised 23% of matings in one brood. Although over 82% of mating occurred between colonies within 10 m of each other (mean sperm dispersal = 5.5 m ± 4.37 SD), we found no evidence of inbreeding in the established population. Restricted dispersal of sperm compared to slightly greater larval dispersal appears to limit inbreeding among close relatives in this cryptic species. Our findings establish a good basis for further work on sperm dispersal in brooding corals and provide the first information about the mating system of a newly identified and abundant cryptic species.     

Paternity testing of wild black rockfish <Emphasis Type="Italic">Sebastes inermis</Emphasis> (brownish type) from the Seto Inland Sea of Japan

We developed four microsatellite DNA loci to test for multiple paternity of black rockfish, Sebastes inermis, from the Seto Inland Sea of Japan. All loci showed a high degree of polymorphism (number of alleles per locus = 10–14, expected heterozygosity = 0.80) and discriminating power (probability of identity index = 3.71 × 10⁻⁶, exclusion probability = 0.999) in unrelated wild specimens (n = 32). Genotypic assignment of five dams (109–220 mm in total length) and 50 embryos from each dam (n = 50) indicated that four dams were mated with a single sire. Only for one dam and three of her embryos we could not exclude multiple paternity.     

Speciation genomics and a role for the Z chromosome in the early stages of divergence between Mexican ducks and mallards

Speciation is a continuous and dynamic process, and studying organisms during the early stages of this process can aid in identifying speciation mechanisms. The mallard (Anas platyrhynchos) and Mexican duck (A. [p.] diazi) are two recently diverged taxa with a history of hybridization and controversial taxonomy. To understand their evolutionary history, we conducted genomic scans to characterize patterns of genetic diversity and divergence across the mitochondrial DNA (mtDNA) control region, 3523 autosomal loci and 172 Z‐linked sex chromosome loci. Between the two taxa, Z‐linked loci (Φ_ST = 0.088) were 5.2 times more differentiated than autosomal DNA (Φ_ST = 0.017) but comparable to mtDNA (Φ_ST = 0.092). This elevated Z differentiation deviated from neutral expectations inferred from simulated data that incorporated demographic history and differences in effective population sizes between marker types. Furthermore, 3% of Z‐linked loci, compared to <0.1% of autosomal loci, were detected as outlier loci under divergent selection with elevated relative (Φ_ST) and absolute (d_XY) estimates of divergence. In contrast, the ratio of Z‐linked and autosomal differentiation among the seven Mexican duck sampling locations was close to 1:1 (Φ_ST = 0.018 for both markers). We conclude that between mallards and Mexican ducks, divergence at autosomal markers is largely neutral, whereas greater divergence on the Z chromosome (or some portions thereof) is likely the product of selection that has been important in speciation. Our results contribute to a growing body of literature indicating elevated divergence on the Z chromosome and its likely importance in avian speciation.     

Pollination by sexual deception promotes outcrossing and mate diversity in self‐compatible clonal orchids

The majority of flowering plants rely on animals as pollen vectors. Thus, plant mating systems and pollen dispersal are strongly influenced by pollinator behaviour. In Australian sexually deceptive orchids pollinated by male thynnine wasps, outcrossing and extensive pollen flow is predicted due to floral deception, which minimizes multiple flower visitations within patches, and the movement of pollinators under mate‐search rather than foraging behaviours. This hypothesis was tested using microsatellite markers to reconstruct and infer paternity in two clonal, self‐compatible orchids. Offspring from naturally pollinated Chiloglottis valida and C. aff. jeanesii were acquired through symbiotic culture of seeds collected over three seasons. In both species, outcrossing was extensive (t_m = 0.924–1.00) despite clone sizes up to 11 m wide. The median pollen flow distance based on paternity for both taxa combined was 14.5 m (n = 18, range 0–69 m), being larger than typically found by paternity analyses in other herbaceous plants. Unexpectedly for orchids, some capsules were sired by more than one father, with an average of 1.35 pollen donors per fruit. This is the first genetic confirmation of polyandry in orchid capsules. Further, we report a possible link between multiple paternity and increased seed fitness. Together, these results demonstrate that deceptive pollination by mate‐searching wasps enhances offspring fitness by promoting both outcrossing and within‐fruit paternal diversity.     

Isolation and characterization of eight microsatellite loci for the Neotropical freshwater catfish Pimelodella chagresi (Teleostei: Pimelodidae)

We report eight (CA)_10?35 unlinked microsatellite loci from the Neotropical freshwater catfish, Pimelodella chagresi (Siluriformes: Pimelodidae). These loci were characterized with 23 individuals collected in Panama. Number of alleles per locus varied from 7 to 23 (mean = 12.9) and observed heterozygosity ranged from 0.522 to 0.909 (mean = 0.732). These loci will be used to investigate the existence of cryptic species within the P. chagresi clade, and to study fine‐scale population structure.     

Isolation and characterization of microsatellite loci from mother‐of‐millions,Bryophyllum delagoense (Crassulaceae), and its hybrid with Bryophyllum daigremontianum, ‘Houghton's hybrid’

Nine microsatellite loci were developed, and are transferable, across the Madagascan succulents Bryophyllum daigremontianum, Bryophyllum delagoense (mother‐of‐millions) and their horticultural hybrid (Houghton's), from enriched libraries of the later two species. For B. delagoense, a tetraploid, three to 13 alleles per locus were found for native Madagascan (H_O = 0.4–1.0), and one to nine in invasive Australian (H_O = 0.0–1.0) samples. In addition for 91 Australian samples, only five multilocus genotypes were found (95% of individuals were of two genotypes), suggesting extensive clonality in its introduced range. These loci will be used to examine genetic diversity, hybrid origin and mating system in natural and introduced populations.     

Extensive seed and pollen dispersal and assortative mating in the rain forest tree Entandrophragma cylindricum (Meliaceae) inferred from indirect and direct analyses

Pollen and seed dispersal are key processes affecting the demographic and evolutionary dynamics of plant species and are also important considerations for the sustainable management of timber trees. Through direct and indirect genetic analyses, we studied the mating system and the extent of pollen and seed dispersal in an economically important timber species, Entandrophragma cylindricum (Meliaceae). We genotyped adult trees, seeds and saplings from a 400‐ha study plot in a natural forest from East Cameroon using eight nuclear microsatellite markers. The species is mainly outcrossed (t = 0.92), but seeds from the same fruit are often pollinated by the same father (correlated paternity, r_p = 0.77). An average of 4.76 effective pollen donors (N_ep) per seed tree contributes to the pollination. Seed dispersal was as extensive as pollen dispersal, with a mean dispersal distance in the study plot approaching 600 m, and immigration rates from outside the plot to the central part of the plot reaching 40% for both pollen and seeds. Extensive pollen‐ and seed‐mediated gene flow is further supported by the weak, fine‐scale spatial genetic structure (Sp statistic = 0.0058), corresponding to historical gene dispersal distances (σ_g) reaching approximately 1,500 m. Using an original approach, we showed that the relatedness between mating individuals (F_ij = 0.06) was higher than expected by chance, given the extent of pollen dispersal distances (expected F_ij = 0.02 according to simulations). This remarkable pattern of assortative mating could be a phenomenon of potentially consequential evolutionary and management significance that deserves to be studied in other plant populations.     

Isolation of microsatellites from two species of scleractinian coral

Microsatellites were isolated from two broadcast‐spawning species of scleractinian coral (Platygyra daedalea and Goniastrea favulus) from Australia's Great Barrier Reef. We found 27 microsatellites across both species, although only five loci were polymorphic in each species. Microsatellite loci displayed a wide range of diversity levels with four to 11 alleles per locus (H_O = 0.26–0.91) in P. daedalea and two to seven alleles per locus (H_O = 0.16–0.96) in G. favulus. Most loci showed departures from Hardy–Weinberg equilibrium which may reflect nonrandom mating but may also be related to difficulties associated with coral DNA.