Highly clustered mating networks in naturally fragmented riparian tree populations.

Understanding how spatial patterns of mating and gene flow respond to habitat loss and geographical isolation is a crucial aspect of forest fragmentation genetics. Naturally fragmented riparian tree populations exhibit unique characteristics that significantly influence these patterns. In this study, we investigate mating patterns, pollen-mediated gene flow, and genetic diversity in relict populations of Frangula alnus in southern Spain by testing specific hypotheses related to the riparian habitat. We employ a novel approach that combines paternity analysis, particularly suited for small and isolated populations, with complex network theory and Bayesian models to predict mating likelihood among tree pairs. Our findings reveal a prevalence of short-distance pollination, resulting in spatially driven local mating clusters with a distinct subset of trees being highly connected in the mating network. Additionally, we observe numerous pollination events over distances of hundreds of metres and considerable pollen immigration. Local neighbourhood density is the primary factor influencing within-population mating patterns and pollen dispersal; moreover, mating network properties reflect the population's size and spatial configuration. Conversely, among-population pollen dispersal is mainly determined by tree size, which influences floral display. Our results do not support a major role of directional pollen dispersal in longitudinal trends of genetic diversity. We provide evidence that long-term fragmented tree populations persist in unique environments that shape mating patterns and impose constraints to pollen-mediated gene flow. Nevertheless, even seemingly strongly isolated populations can maintain functional connectivity over extended periods, especially when animal-mediated mating networks promote genetic diversity, as in this riparian tree species.

Verification of a loss of heterozygosity at the D8S1179 locus in a paternity case by the MiSeq FGx system.

Capillary electrophoresis is widely used to study short tandem repeats (STRs) in forensic genetics. However, next-generation sequencing platforms have become a new strategy for forensic DNA typing. In this study, we report a false four-step STR mutation between an alleged father (AF) and child in a paternity case. A total of 23 autosomal STR loci were evaluated using the Huaxia™ Platinum and Goldeneye™ 20A kits, revealing a single mismatch in D8S1179 between the AF (10/10) and the male child (14/14). Additional Y-STR typing of the AF and child was performed, and the results were consistent with those based on 27 Y-STR loci. To further confirm the experimental results, we sequenced the individuals using the MiSeq FGx system and detected 10/15 unbalanced alleles in the D8S1179 locus of the AF and 14/15 unbalanced alleles in the D8S1179 locus of the child. Sanger sequencing revealed that both the AF and child had the C→G point mutation in the primer binding region of D8S1179 resulting in allelic dropout. Therefore, the verification of STR typing by different sequencing systems is helpful for the interpretation of results in cases of multistep STR mutations.

Multiple-brooding rockfishes (Sebastes spp.) can utilize stored sperm from individual sires to fertilize consecutive broods.

Viviparous rockfishes (Sebastes spp., family Scorpaenidae) mate and store sperm in the ovaries for several months prior to fertilization, as oocytes develop for the parturition season. Although multiple paternity has been documented in single-brooding rockfishes, paternity in consecutive broods of multiple-brooding species has not been studied. Analyses of multilocus microsatellite genotypes in both residual larvae left in the ovary from a previous parturition and upcoming fertilized broods in the same ovary demonstrated evidence of the same sires in consecutive broods in chilipepper (Sebastes goodei) and speckled (Sebastes ovalis) rockfishes. One S. goodei mother showed evidence of multiple paternity from the same two sires in both consecutive broods. The ability to retain sperm, even after a parturition event, for use in subsequent broods, confers an advantage to ensure fertilization and allows for extension of the parturition season. This life-history strategy provides a bet-hedging advantage in the California Current system, an environmentally dynamic ecosystem where larval survivorship and subsequent recruitment to adult populations can vary temporally by orders of magnitude.

Genetic variation in reproductive timing in a long-lived herbaceous perennial.

PREMISE: Reproductive fitness of individual plants depends on the timing of flowering, especially in mate-limited populations, such as those in fragmented habitats. When flowering time traits are associated with differential reproductive success, the narrow-sense heritability (h2 ) of traits will determine how rapidly trait means evolve in response to selection. Heritability of flowering time is documented in many annual plants. However, estimating h2 of flowering time in perennials presents additional methodological challenges, often including paternity assignment and trait expression over multiple years. METHODS: We evaluated the h2 of onset and duration of flowering using offspring-midparent regressions and restricted maximum likelihood methods in an experimental population of an iterocarpic, perennial, herbaceous plant, Echinacea angustifolia, growing in natural conditions. We assessed the flowering time of the parental cohort in 2005 and 2006; the offspring in 2014 through 2017. We also examined the effects of the paternity assignment from Cervus and MasterBayes on estimates of h2 . RESULTS: We found substantial h2 for onset and duration of flowering. We also observed variation in estimates among years. The most reliable estimates for both traits fell in the range of 0.1-0.17. We found evidence of a genotype by year interaction for onset of flowering and strong evidence that genotypes are consistent in their duration of flowering across years. CONCLUSIONS: Substantial heritabilities in this population imply the capacity for a response to natural selection, while also suggesting the potential for differential contributions to adaptive evolution among seasons.

Regional variation in multiple paternity in the brown smooth-hound shark Mustelus henlei from the northeastern Pacific.

Multiple paternity (MP) in the brown smooth-hound shark (Mustelus henlei) was assessed in 15 litters (15 mothers and 97 embryos) collected in the northern Gulf of California of which 86.7% were sired by more than one male (i.e., from 2 to 4 sires). When taken together with results from previous studies, this record indicates that there is regional variation in MP in M. henlei in the northeastern Pacific. This pattern is associated with variations in the reproductive traits of each population (e.g., female size and litter size). In the Gulf of California, the results of a generalized linear model (GLZ) indicated that the litters of larger females had a higher probability of MP compared to those of smaller females.

Fine-scale spatial genetic structure, mating, and gene dispersal patterns in Parkia biglobosa populations with different levels of habitat fragmentation.

PREMISE: A good understanding of genetic variation and gene dispersal in tree populations is crucial for their sustainable management, particularly in a context of rapid environmental changes. West African Sudanian savannahs are being fragmented and degraded, partly due to expansion of crop cultivation and monocultures that reduce tree density and may impact pollinators. The population dynamics of important indigenous trees could also be affected. We investigated the influence of habitat fragmentation on patterns of genetic diversity and gene dispersal of a key Sudanian agroforestry tree species, Parkia biglobosa. METHODS: Using 10 highly polymorphic nuclear microsatellites, we genotyped 2475 samples from reproductive trees, seedlings, and embryos in four tree populations presenting different levels of habitat fragmentation. RESULTS: Parkia biglobosa presented similar high genetic diversity across the four populations studied. Genetic diversity and inbreeding were similar between adults and embryo cohorts. In all four populations, the selfing rate was less than 1%. The effective number of pollen donors per tree was high (NEP ~ 18-22), as was the pollen immigration rate (from 34 to 74%). Pollen dispersal was characterized by a fat-tailed distribution with mean estimates exceeding 200 m. In three populations, stem diameter had a pronounced effect on male reproductive success. Here, the highest male reproductive success was observed in trees with a diameter at breast height between 60 and 75 cm. CONCLUSIONS: At the scale analyzed, fragmentation does not seem to pose limitations to gene flow in any of the sites investigated, regardless of the landscape configuration associated with the different tree stands. The study provides useful insights on the reproductive biology of an important tree species in the West African savannahs.

Male energy reserves, mate-searching activities, and reproductive success: alternative resource use strategies in a presumed capital breeder.

Sexual selection studies often focus on morphological traits that are important only in the later stages of mate acquisition. Comparatively little is known about traits that lead to mate acquisition, such as mate-searching activities. We experimentally manipulated body condition (i.e., the energy reserves) in male puff adders (Bitis arietans) prior to the mating season using supplemental feeding in the field, and used radio-telemetry and DNA paternity analyses to characterize the relationships between male energy reserves, mating activities, and reproductive success. We found that male mobility is a strongly sexually selected trait because males that travelled further in search of females have higher female encounter rates and reproductive success. However, supplemental feeding did not have a significant effect on mating activities or reproductive success, because control snakes compensated by foraging more often during the mating season. The time invested in foraging by control snakes did not come at the costs of decreased mating activities or opportunity compared to fed snakes, because the latter spent the spare time resting. Our experimental field research directly demonstrates the link between male mobility and reproductive success, identifying the ultimate mechanism leading to the evolution of prolonged male mate-searching activities in snakes, and indicates that male puff adders, presumed capital breeders, adjust their breeding tactics according to resource availability.

The first reproductive parameters and evidence of multiple paternity in one new spiny dogfish species, Squalus albicaudus (Squaliformes, Squalidae).

Understanding elasmobranch reproductive biology is necessary for species conservation. Multiple paternity (MP) has been reported for elasmobranchs, and this study investigates the reproductive aspects and mating system (paternity genetic analyses) for Squalus albicaudus. Thirteen pregnant females were analysed concerning reproductive parameters, and the mating system was assessed for nine females and their litters. The study found a mean fecundity of 2.84 pups per litter without correlation between total female length and the number of embryos per litter. One litter showed evidence of MP, indicating the presence of polyandrous behaviour of the species.

Mixed mating system and variable mating patterns in tropical woody bamboos.

BACKGROUND: So far, little is known in detail about mating systems of woody bamboos. Paternity analysis of offspring improved our understanding of these systems, and contributed to their germplasm conservation and genetic improvement. RESULTS: In this study, a paternity analysis of offspring from two consecutive mass or sporadically flowering events of Dendrocalamus membranaceus and D. sinicus were conducted to determine their mating system and pollen dispersal using the program COLONY based on simple sequence repeat (SSR) markers. Two sporadically flowering populations of D. sinicus (C1, C2) obtained relatively high paternity assignments rates (69.0-71.4%). Meanwhile, among three populations of D. membranaceus, the sporadically flowering population A also had much higher paternity assignments rates (56.4%) than mass flowering populations B1(28.6%) and B2 (42.5%). Both D. membranaceus and D. sinicus had mixed mating systems while their mating patterns were variable depending on pollination conditions. The maximum pollen dispersal distances were 90 m and 4378 m for D. membranaceus and D. sinicus populations, respectively, and the mating distances of these two species focused on ranges of ca. 0-50 m and 0-1500 m, respectively. CONCLUSIONS: These results revealed for the first time variable mating patterns in woody bamboos. This suggests half-sib seeds from the same bamboo clump may have different male parents and it is crucial to clarify genetic origin in woody bamboos' breeding programs. The results also indicate the importance of pollinators in the mating systems of tropical woody bamboos.

Pollen-mediated gene flow ensures connectivity among spatially discrete sub-populations of Phalaenopsis pulcherrima, a tropical food-deceptive orchid.

BACKGROUND: Gene flow in plants via pollen and seeds is asymmetrical at different geographic scales. Orchid seeds are adapted to long-distance wind dispersal but pollinium transfer is often influenced by pollinator behavior. We combined field studies with an analysis of genetic diversity among 155 physically mapped adults and 1105 F1 seedlings to evaluate the relative contribution of pollen and seed dispersal to overall gene flow among three sub-populations of the food-deceptive orchid Phalaenopsis pulcherrima on Hainan Island, China. RESULTS: Phalaenopsis pulcherrima is self-sterile and predominantly outcrossing, resulting in high population-level genetic diversity, but plants are clumped and exhibit fine-scale genetic structuring. Even so, we detected low differentiation among sub-populations, with polynomial regression analysis suggesting gene flow via seed to be more restricted than that via pollen. Paternity analysis confirmed capsules of P. pulcherrima to each be sired by a single pollen donor, probably in part facilitated by post-pollination stigma obfuscation, with a mean pollen flow distance of 272.7 m. Despite limited sampling, we detected no loss of genetic diversity from one generation to the next. CONCLUSIONS: Outcrossing mediated by deceptive pollination and self-sterility promote high genetic diversity in P. pulcherrima. Long-range pollinia transfer ensures connectivity among sub-populations, offsetting the risk of genetic erosion at local scales.

Primary pollinator exclusion has divergent consequences for pollen dispersal and mating in different populations of a bird-pollinated tree.

Pollination by nectarivorous birds is predicted to result in different patterns of pollen dispersal and plant mating compared to pollination by insects. We tested the prediction that paternal genetic diversity, outcrossing rate and realized pollen dispersal will be reduced when the primary pollinator group is excluded from bird-pollinated plants. Pollinator exclusion experiments in conjunction with paternity analysis of progeny were applied to Eucalyptus caesia Benth. (Myrtaceae), a predominantly honeyeater-pollinated tree that is visited by native insects and the introduced Apis mellifera (Apidae). Microsatellite genotyping at 14 loci of all adult E. caesia at two populations (n = 580 and 315), followed by paternity analysis of 705 progeny, revealed contrasting results between populations. Honeyeater exclusion did not significantly impact pollen dispersal or plant mating at Mount Caroline. In contrast, at the Chiddarcooping site, the exclusion of honeyeaters led to lower outcrossing rates, a threefold reduction in the average number of sires per fruit, a decrease in intermediate-distance mating and an increase in near-neighbour mating. The results from Chiddarcooping suggest that bird pollination may increase paternal genetic diversity, potentially leading to higher fitness of progeny and favouring the evolution of this strategy. However, further experimentation involving additional trees and study sites is required to test this hypothesis. Alternatively, insects may be effective pollinators in some populations of bird-adapted plants, but ineffective in others.

The future of parentage analysis: From microsatellites to SNPs and beyond.

Parentage analysis is a cornerstone of molecular ecology that has delivered fundamental insights into behaviour, ecology and evolution. Microsatellite markers have long been the king of parentage, their hypervariable nature conferring sufficient power to correctly assign offspring to parents. However, microsatellite markers have seen a sharp decline in use with the rise of next-generation sequencing technologies, especially in the study of population genetics and local adaptation. The time is ripe to review the current state of parentage analysis and see how it stands to be affected by the emergence of next-generation sequencing approaches. We find that single nucleotide polymorphisms (SNPs), the typical next-generation sequencing marker, remain underutilized in parentage analysis but are gaining momentum, with 58 SNP-based parentage analyses published thus far. Many of these papers, particularly the earlier ones, compare the power of SNPs and microsatellites in a parentage context. In virtually every case, SNPs are at least as powerful as microsatellite markers. As few as 100-500 SNPs are sufficient to resolve parentage completely in most situations. We also provide an overview of the analytical programs that are commonly used and compatible with SNP data. As the next-generation parentage enterprise grows, a reliance on likelihood and Bayesian approaches, as opposed to strict exclusion, will become increasingly important. We discuss some of the caveats surrounding the use of next-generation sequencing data for parentage analysis and conclude that the future is bright for this important realm of molecular ecology.

Skewed male reproductive success and pollen transfer in a small fragmented population of the heterodichogamous tree Machilus thunbergii.

Heterodichogamy is defined as the presence of two flower morphs that exhibit the male and female functions at different times among individuals within a population. Heterodichogamy is regarded as an adaptation to promote outcrossing through enhanced inter-morph mating, together with a 1:1 morph ratio. However, in highly fragmented populations, the morph ratio may be more likely to be biased by stochastic events. In such a situation, individuals of a minority morph within a population are expected to have higher reproductive success than those of a majority morph, which may suffer from pollen shortages of the minority morph. In this paper, we evaluated mating patterns and male reproductive success in a highly fragmented population of Machilus thunbergii, a putative heterodichogamous evergreen laurel tree. Results of paternity analysis indicated that the selfing rate was not clearly different between the two morphs. In contrast, the proportion of intra-morph mating was higher in the majority-morph (MM) mother trees than in the minority-morph (MF) mother trees. Bayesian estimated male reproductive success indicated that male reproductive success was higher in minority-morph (MF) than in majority-morph (MM) mother trees. These findings indicate that (1) the majority morph mothers, suffering a shortage of the opposite morph pollen, could partly compensate for the reduced reproductive success by intra-morph mating rather than by selfing, and (2) negative-frequency dependent selection may be involved in the maintenance of the two morphs.

Mating System Evolution under Strong Pollen Limitation: Evidence of Disruptive Selection through Male and Female Fitness in Clarkia xantiana.

Selection on floral traits in hermaphroditic plants is determined by both male and female reproductive success. However, predictions regarding floral trait and mating system evolution are often based solely on female fitness. Selection via male fitness has the potential to affect the outcomes of floral evolution. In this study, we used paternity analysis to assess individual selfing rates and selection on floral traits via male and female fitness in an experimental population of Clarkia xantiana where pollen limitation of seed set was strong. We detected selection through both female and male fitness with reinforcing or noninterfering patterns of selection through the two sex functions. For female fitness, selection favored reduced herkogamy and protandry, traits that promote increased autonomous selfing. For male fitness, selection on petal area was disruptive, with higher trait values conferring greater pollinator attraction and outcross siring success and smaller trait values leading to higher selfed siring success. Combining both female and male fitness, selection on petal area and protandry was disruptive because intermediate phenotypes were less successful as both males and females. Finally, functional relationships among male and female fertility components indicated that selfing resulted in seed discounting and pollen discounting. Under these functional relationships, the evolutionarily stable selfing rate can be intermediate or predominantly selfing or outcrossing, depending on the segregating load of deleterious mutations.

Euglossine bees mediate only limited long-distance gene flow in a tropical vine.

Euglossine bees (Apidae: Euglossini) have long been hypothesized to act as long-distance pollinators of many low-density tropical plants. We tested this hypothesis by the analysis of gene flow and genetic structure within and among populations of the euglossine bee-pollinated vine Dalechampia scandens. Using microsatellite markers, we assessed historical gene flow by the quantification of regional-scale genetic structure and isolation by distance among 18 populations, and contemporary gene flow by the estimation of recent migration rates among populations. To assess bee-mediated pollen dispersal on a smaller scale, we conducted paternity analyses within a focal population, and quantified within-population spatial genetic structure in four populations. Gene flow was limited to certain nearby populations within continuous forest blocks, whereas drift appeared to dominate on larger scales. Limited long-distance gene flow was supported by within-population patterns; gene flow was biased towards nearby plants, and significant small-scale spatial genetic structure was detected within populations. These findings suggest that, although female euglossine bees might be effective at moving pollen within populations, and perhaps within forest blocks, their contribution to gene flow on the regional scale seems too limited to counteract genetic drift in patchily distributed tropical plants. Among-population gene flow might have been reduced following habitat fragmentation.

Reproductive dynamics shapes genomotype composition in an allopolyploid complex.

Hybrid complexes are composed of organisms with multiple combinations of parental genomes (genomotypes) that interconnect through nets of crosses. Although several such complexes are well established without speciation or extinction, mechanisms shaping their dynamics remain poorly understood. In this study, we quantified the reproductive success of the allopolyploid Iberian fish Squalius alburnoides in experimental free-access and directional crosses involving the most common genomotypes. Specifically, we analysed the paternity of the offspring produced when females had free access to male genomotypes and quantified variations in egg allocation, fertilization rate, and offspring survival among crosses involving each male genomotype. The composition of the offspring produced from free-access crosses varied significantly from that expected from random mating, suggesting that offspring production and viability are not independent of parental male genomotype. Moreover, directional crosses producing the genomotype most commonly found in wild populations appeared to be the most successful, with females laying more eggs, and fertilization rate and offspring survival being the highest. These results suggest that reproductive dynamics plays a relevant role in structuring the genomotype composition of populations and opens a path to future research on the ecology and evolutionary biology of allopolyploids and their multiplicity of possible evolutionary pathways.

Estimating selection through male fitness: three complementary methods illuminate the nature and causes of selection on flowering time.

Our understanding of selection through male fitness is limited by the resource demands and indirect nature of the best available genetic techniques. Applying complementary, independent approaches to this problem can help clarify evolution through male function. We applied three methods to estimate selection on flowering time through male fitness in experimental populations of the annual plant Brassica rapa: (i) an analysis of mating opportunity based on flower production schedules, (ii) genetic paternity analysis, and (iii) a novel approach based on principles of experimental evolution. Selection differentials estimated by the first method disagreed with those estimated by the other two, indicating that mating opportunity was not the principal driver of selection on flowering time. The genetic and experimental evolution methods exhibited striking agreement overall, but a slight discrepancy between the two suggested that negative environmental covariance between age at flowering and male fitness may have contributed to phenotypic selection. Together, the three methods enriched our understanding of selection on flowering time, from mating opportunity to phenotypic selection to evolutionary response. The novel experimental evolution method may provide a means of examining selection through male fitness when genetic paternity analysis is not possible.

Unusually limited pollen dispersal and connectivity of Pedunculate oak (Quercus robur) refugial populations at the species' southern range margin.

Low-latitudinal range margins of temperate and boreal plant species typically consist of scattered populations that persist locally in microrefugia. It remains poorly understood how their refugial habitats affect patterns of gene flow and connectivity, key components for their long-term viability and evolution. We examine landscape-scale patterns of historical and contemporary gene flow in refugial populations of the widespread European forest tree Pedunculate oak (Quercus robur) at the species' southwestern range margin. We sampled all adult trees (n = 135) growing in a 20 km long valley and genotyped 724 acorns from 72 mother trees at 17 microsatellite loci. The ten oak stands that we identified were highly differentiated and formed four distinct genetic clusters, despite sporadic historical dispersal being detectable. By far most contemporary pollination occurred within stands, either between local mates (85.6%) or through selfing (6.8%). Pollen exchange between stands (2.6%) was remarkably rare given their relative proximity and was complemented by long-distance pollen immigration (4.4%) and hybridization with the locally abundant Quercus pyrenaica (0.6%). The frequency of between-stand mating events decreased with increasing size and spatial isolation of stands. Overall, our results reveal outstandingly little long-distance gene flow for a wind-pollinated tree species. We argue that the distinct landscape characteristics of oaks' refugial habitats, with a combination of a rugged topography, dense vegetation and humid microclimate, are likely to increase plant survival but to hamper effective long-distance pollen dispersal. Moreover, local mating might be favoured by high tree compatibility resulting from genetic purging in these long-term relict populations.

Spatial genetic structure, genetic diversity and pollen dispersal in a harvested population of Astrocaryum aculeatum in the Brazilian Amazon.

BACKGROUND: Astrocaryum aculeatum is a palm tree species native to the tropical regions of South America, exploited commercially by local farmers for the pulp extracted from its fruits. The objective of this research was to compare the genetic diversity between adult plants and seedlings from open-pollinated seeds, quantify the pollen flow and dispersal, the spatial genetic structure, and the effective size of a population that has been continuously harvested for its fruits. The study was carried out in a natural population of A. aculeatum distributed over approximately 8 ha in the State of Amazonas (Brazil), separated by 400 m from the closest neighboring population. In total, 112 potential pollen donors, 12 mother plants and 120 offspring were mapped and genotyped. RESULTS: Genetic diversity was high for parents and the offspring. The fixation indexes for adults (F = -0.035) and offspring (F = -0.060) were negative and not significant. A significant spatial genetic structure was detected for the adult plants (up to the distance of 45 m) indicating short-distance seed dispersal. Paternity analysis detected 9.2 % of pollen immigration and the average distance of pollination within the population was 81 m. The average effective pollination neighborhood area between plants was 1.51 ha. CONCLUSIONS: Our results indicate that substantial introduction of new alleles has occurred in the population through pollen immigration, contributing to the maintenance of genetic diversity. Conservation efforts aimed at maintaining the gene pool of the current population or establishing new populations should utilize offspring from mother plants selected to be spaced by at least 50 m to prevent collecting seeds from relatives.

Reciprocal herkogamy promotes disassortative mating in a distylous species with intramorph compatibility.

Mating patterns in heterostylous species with intramorph compatibility have the potential to deviate from symmetrical disassortative mating owing to ecological and reproductive factors influencing pollen dispersal. Here, we investigate potential and realized patterns of mating in distylous Luculia pinceana (Rubiaceae), a species with intramorph compatibility. Our analysis provides an opportunity to test Darwin's hypothesis that reciprocal herkogamy promotes disassortative pollen transfer. We combined measurements of sex-organ reciprocity and pollen production to predict potential pollen transfer and mating patterns in a population from SW China. Marker-based paternity analysis was then used to estimate realized patterns of disassortative and assortative mating at the individual and floral morph levels. Both potential and realized mating patterns indicated a significant component of disassortative mating, satisfying theoretical conditions for the maintenance of floral dimorphism. Levels of assortative mating (37.7%) were significantly lower than disassortative mating (62.3%), but numerous offspring resulting from intramorph mating were detected in the majority of maternal seed families in both floral morphs. Our results provide empirical support for Darwin's cross-promotion hypothesis on the function of reciprocal herkogamy, but indicate that in most heterostylous species strong diallelic incompatibility may be a general requirement for complete disassortative mating.