High Levels Of Inbreeding Research Articles

Conservation genomics of an Australian cycad Cycas calcicola, and the Absence of Key Genotypes in Botanic Gardens

Understanding the genetic diversity of wild populations is fundamental to conserving species in-situ and ex-situ. To aid conservation plans and to inform ex-situ conservation, we examined the genetic diversity of the cycad Cycas calcicola (Cycadaceae). Samples were collected from wild populations in the Litchfield National Park and Katherine regions in the Northern Territory, Australia. Additional samples were obtained from botanic garden plants that were originally collected in the Katherine region, Daly River and Spirit Hills in the Northern Territory, Australia. Using RADseq we recovered 2271 informative genome-wide SNPs, revealing low to moderate levels of gene diversity (uHe = 0.037 to 0.135), very low levels of gene flow, and significant levels of inbreeding (mean FIS = 0.491). Population structure and multivariate analysis showed that populations fall into two genetic groups (Katherine vs Litchfield + Daly River + Spirit Hills). Genetic differentiation was twice as high between populations of the Katherine and Litchfield regions (FST ~ 0.1) compared to within these two regions (FST ~ 0.05). Increasing population fragmentation together with high levels of inbreeding and very little gene flow are concerning for the future adaptability of this species. The results indicated that the ex-situ collections (1) had significantly lower genetic diversity than the wild populations, and (2) only partly capture the genetic diversity present, particularly because the Litchfield National Park populations are not represented. We recommend that ex-situ collections be expanded to incorporate the genetic diversity found in Litchfield National Park and to increase the number of representatives from Daly River/Spirit Hills, and that in-situ populations from the Katherine and Greater Litchfield regions be conserved as separate management units.

Analysis of Autozygosity Using Whole-Genome Sequence Data of Full-Sib Families in Pikeperch (Sander lucioperca).

Pikeperch (Sander lucioperca) has emerged as a high value species to the aquaculture industry. However, its farming techniques are at an early stage and its production is often performed without a selective breeding program, potentially leading to high levels of inbreeding. In this study, we identified and characterized autozygosity based on genome-wide runs of homozygosity (ROH) on a sample of parental and offspring individuals, determined effective population size (N e ), and assessed relatedness among parental individuals. A mean of 2,235 ± 526 and 1,841 ± 363 ROH segments per individual, resulting in a mean inbreeding coefficient of 0.33 ± 0.06 and 0.25 ± 0.06 were estimated for the progeny and parents, respectively. N e was about 12 until four generations ago and at most 106 for 63 generations in the past, with varying genetic relatedness amongst the parents. This study shows the importance of genomic information when family relationships are unknown and the need of selective breeding programs for reproductive management decisions in the aquaculture industry.

Whole-genome SNP markers reveal runs of homozygosity in indigenous cattle breeds of Pakistan

The runs of homozygosity (ROH) were identified in 14 Pakistani cattle breeds (n = 105) by genotyping with the Illumina 50 K SNP BeadChip. These breeds were categorized into Dairy, Dual, and Draft breeds based on their utility and production performance. We identified a total of 10,936 ROHs which mainly consisted of a high number of shorter segments (1–4 Mb). Dairy group exhibited the highest level of inbreeding (FROH: 0.078 ± 0.028) while the lowest (FROH: 0.002 ± 0.008) was observed in Dual group. In 48 genomic regions identified with a high frequency of ROH, 207 genes were detected in the three breed groups. A substantially higher number of ROH islands detected in dairy breeds indicated the impact of the positive selection pressure over the years. Important candidate genes and QTL were detected in the ROH islands associated with economic traits like milk production, reproduction, meat, carcass, and health traits in dairy cattle.

Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers

Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species’ genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies.

麋鹿沧桑命运与未来保护展望

麋鹿作为中国特有的鹿科动物，经历129年的保护努力，种群数量恢复到10000多头。因近亲繁殖严重，表面上看已无灭绝危险，但仍存在巨大隐忧。通过对麋鹿种群变迁、灭绝、重引入、恢复现状及四大种群（乌邦寺、南海子、大丰、石首野化种群）的发展趋势进行追踪分析，探究近千年来困扰麋鹿种群发展壮大的多重因素，提出种群保护与栖息地保护并重、强化疫病监测等保护对策，为麋鹿及其它大型陆生濒危哺乳动物突破种群发展瓶颈提供借鉴。;Père David's deer (Elaphurus davidianus), a unique species of deer in China, the population has recovered to more than 10000 in the world after more than 100 years of efforts. It seems that there is no danger of extinction due to the high levels of inbreeding in the population of Père David's deer, but there are still huge concerns. By analyzing the changes, extinction, and recovery of the population of Père David's deer and the development trend of four major populations (Woburn Abbey, Nanhaizi, Dafeng and Shishou), this paper explored the multiple factors that have been disturbing the development and expansion of the population of Père David's deer in the past thousand years. The protection measures, such as placing equal emphasis on population protection and habitat protection and strengthening disease monitoring, were put forward to provide reference for Père David's deer and other large terrestrial endangered mammals to break through the bottleneck of population development.

Genetic Diversity of the Mongolian Bactrian Camel Based on Mitochondrial Sequences

Current genetic diversity of 5 local breeds of Mongolian camels and 2 samples of Camelus ferus was studied and compared their levels of variability. DNA was extracted using Qiaamp® DNA kit (Qiagen) and amplified mtDNA D-loop control region from 36 blood samples of Mongolian Bactrian camels and 2 from Mongolian Camelus ferus. Three common haplotypes (H1, H2, H6) and 4 unique haplotypes (H3, H4, H5, H8) revealed in the population of domestic camels based on the mitochondrial DNA region. For the further study we carried out best result 26 sequences. We did not find high inbreeding levels in the different breeds. The phylogenetic analysis suggested that domestic and wild camels have two distinct lineages.

No Evidence of Low Genetic Diversity Despite High Levels of Inbreeding and Poor Genetic Connectivity AmongTetrastigma loheri(Vitaceae) Populations in Remaining Forest Areas in Cebu, Philippines

Abstract—Little is known about the effects of habitat fragmentation on the patterns of genetic diversity and genetic connectivity of species in the remaining tropical forests of Southeast Asia. This is particularly evident in Cebu, a Philippine island that has a long history of deforestation and has lost nearly all of its forest cover. To begin filling this gap, data from 13 microsatellite loci developed forTetrastigma loheri(Vitaceae), a common vine species in Philippine forests, were used to study patterns of genetic diversity and genetic connectivity for the four largest of the remaining forest areas in Cebu. Evidence of relatively high levels of inbreeding was found in all four areas, despite no evidence of low genetic diversity. The four areas are genetically differentiated, suggesting low genetic connectivity. The presence of inbreeding and low genetic connectivity in a commonly encountered species such asT. loheriin Cebu suggests that the impact of habitat fragmentation is likely greater on rare plant species with more restricted distributions in Cebu. Conservation recommendations for the remaining forest areas in Cebu include the establishment of steppingstone corridors between nearby areas to improve the movement of pollinators and seed dispersers among them.

Konik, Tarpan, European wild horse: An origin story with conservation implications

Horses are gaining importance in European nature conservation management, for which usually so-called primitive breeds are favored due to their claimed robustness. An increasingly popular breed, the Konik horse, is often said to be the direct descendent of the alleged European wild horse, the Tarpan. However, both the direct descent of the Konik from European wild horses and the existence of the Tarpan as a wild species are highly debated. In this review, we scrutinized both contemporary research and historical sources and suggest that the Tarpan and the Konik as its direct descendent are manmade myths that hinder effective conservation management. We did not find evidence that the Tarpan was a wild horse rather than a feral horse. We did not find any evidence either for a closer connection between the Konik and any extinct wild horse than between other domestic breeds and wild horses. We discuss three perspectives on why the myth has become widely accepted and survived to this day: a historical-political, a biological-ecological, and an emotional perspective. It seems that the origin story of the Konik and its connection to the Tarpan was shaped by personal and political interests, including nationalistic ideas. These as well as general human emotions towards horses have influenced researchers and laypeople to keep the myth alive, which has been possibly negatively impacting contemporary nature conservation. Indeed, today’s Koniks originated from a small founder population of only six male lines that were selected according to their phenotypic traits, with the aim to rebreed the ‘wild Tarpan’. Strict breeding practices have led to high inbreeding levels in recent Konik populations, which may undermine nature conservation purposes. Therefore, we suggest that mythologized origin stories should not be an argument for selecting breeds of grazers for nature conservation.

A preliminary analysis of the population genetic parameters indicates critically low genetic variation in a threatened killifish ( Anatolichthys transgrediens )

Levels of genetic variation can steer the future adaptability and correlate to reduced inbreeding of species. Therefore, for any conservation measure, determining the levels of genetic variations and its implications in endangered species is a priority. Here, we investigate allozyme genetic variation of a critically endangered killifish species, Anatolichthys transgrediens, one of the most threatened species in the world with extremely small distribution range. Our results clearly point out perilously low levels of genetic variation, with only three out of 15 loci examined were polymorphic, likely reflecting their small population size and genetically deteriorated population structure. Fixation indices estimated also indicate high levels of inbreeding within A. transgrediens which may result in inbreeding depression and further population declines. We discuss the possible drivers for this situation and call the urgency of an integrated action for conservation of this endangered killifish species.

Body measurements of Black Slavonian pig

The Black Slavonian pig is an autochthonous pig breed in Croatia, which has recorded a continuous growth of the population followed by the higher number of breeders in recent years. The increase in population has removed the Black Slavonian breed from the category of endangered local breeds. The consequences of such a significant increase in population size in local breeds are often a high level of inbreeding, but also a high variability of the external traits of breeding individuals. Given that the main goal of the Breeding Program for Black Slavonian pigs is to preserve its phenotypic traits and breed-specific features, the paper presents the results of external traits analysis at 10 different points on the body, on a sample of 102 animals, aged between 10 and 24 months. Estimated mean values for wither’s height were 65 cm and 64 cm in boars and sows, respectively. Average body length was 128 cm for boars, and 126 cm for sows. The heart girth was 114 cm for both categories, while the height at the sacrum was 72 cm (boars), and 71 cm (sows). These results indicate very small or insignificant differences between male and female individuals, and refer that the body measurements of Black Slavonian pigs have not changed significantly in relation to its formation and development over time, as well as in relation to other local breeds from the neighbouring regions. With this in mind, selection work should be focused on control and preservation. Furthermore, the paper gives an overview of the population throughout history, as well as breeding practices.

Population genetic structure of the Natterjack toad (Epidalea calamita) in Ireland: implications for conservation management

Molecular methods can play a crucial role in species management and conservation. Despite the usefulness of genetic approaches, they are often not explicitly included as part of species recovery plans and conservation practises. The Natterjack toad (Epidalea calamita) is regionally Red-Listed as Endangered in Ireland. The species is declining and is now present at just seven sites within a highly restricted range. This study used 13 highly polymorphic microsatellite markers to analyse the population genetic diversity and structure. Genetic diversity was high with expected heterozygosity between 0.55 and 0.61 and allelic richness between 4.77 and 5.92. Effective population sizes were small (Ne < 100 individuals), but not abnormal for pond breeding amphibians. However, there was no evidence of historical or contemporary genetic bottlenecks or high levels of inbreeding. We identified a positive relationship between Ne and breeding pond surface area, suggesting that environmental factors are a key determinant of population size. Significant genetic structuring was detected throughout the species’ range, and we identified four genetic entities that should be considered in the species’ conservation strategies. Management should focus on preventing further population declines and future loss of genetic diversity overall and within genetic entities while maintaining adequate local effective population size through site-specific protection, human-mediated translocations and head-start programs. The apparent high levels of genetic variation give hope for the conservation of Ireland’s rarest amphibian if appropriately protected and managed.

Not out of the woods yet: Signatures of the prolonged negative genetic consequences of a population bottleneck in a rapidly re-expanding wader, the black-faced spoonbill Platalea minor.

The long-term persistence of a population which has suffered a bottleneck partly depends on how historical demographic dynamics impacted its genetic diversity and the accumulation of deleterious mutations. Here we provide genomic evidence for the genetic effect of a recent population bottleneck in the endangered black-faced spoonbill (Platalea minor) after its rapid population recovery. Our data suggest that the bird's effective population size, Ne , had been relatively stable (7500-9000) since 22,000years ago; however, a recent brief yet severe bottleneck (Ne =20) which we here estimated to occur around the 1940s wiped out >99% of its historical Ne in roughly three generations. Despite a >15-fold population recovery since 1988, we found that black-faced spoonbill population has higher levels of inbreeding (7.4 times more runs of homozygosity) than its sister species, the royal spoonbill (P.regia), which is not thought to have undergone a marked population contraction. Although the two spoonbills have similar levels of genome-wide genetic diversity, our results suggest that selection on more genes was relaxed in the black-faced spoonbill; moreover individual black-faced spoonbills carry more putatively deleterious mutations (Grantham's score>50), and may therefore express more deleterious phenotypic effects than royal spoonbills. Here we demonstrate the value of using genomic indices to monitor levels of genetic erosion, inbreeding and mutation load in species with conservation concerns. To mitigate the prolonged negative genetic effect of a population bottleneck, we recommend that all possible measures should be employed to maintain population growth of a threatened species.

History or demography? Determining the drivers of genetic variation in North American plants.

Understanding the impact of historical and demographic processes on genetic variation is essential for devising conservation strategies and predicting responses to climate change. Recolonization after Pleistocene glaciations is expected to leave distinct genetic signatures, characterised by lower genetic diversity in previously glaciated regions. Populations’ positions within species ranges also shape genetic variation, following the central‐marginal paradigm dictating that peripheral populations are depauperate, sparse and isolated. However, the general applicability of these patterns and relative importance of historical and demographic factors remains unknown. Here, we analysed the distribution of genetic variation in 91 native species of North American plants by coupling microsatellite data and species distribution modelling. We tested the contributions of historical climatic shifts and the central‐marginal hypothesis on genetic diversity and structure on the whole data set and across subsets based on taxonomic groups and growth forms. Decreased diversity was found with increased distance from potential glacial refugia, coinciding with the expected make‐up of postglacially colonised localities. At the range periphery, lower genetic diversity, higher inbreeding levels and genetic differentiation were reported, following the assumptions of the central‐marginal hypothesis. History and demography were found to have approximately equal importance in shaping genetic variation.

Genome-wide structural variations in Brazilian Senepol cattle, a tropically adapted taurine breed

This study aimed to identify structural variations in the form of runs of homozygosity (ROH) and copy number variants (CNVs) in the genome of the Brazilian Senepol cattle and to better explore the potential biological functions of the genes located within such regions. A total of 140 animals were genotyped with the GeneSeek® Genomic Profiler™ Bovine 50 K. Autosomal ROH and CNVs were detected after appropriated quality control. A total of 5,531 ROH were identified, with an average number of 40.37 per animal and an average length of 5.09 Mb. BTA1 had the greatest number of ROH per chromosome (n = 458), while BTA20 displayed the greatest fraction of the chromosome covered with ROH (22.06%). A total of 181 CNVs were identified, with an average length size of 184.67 kb, with values ranging from 26.61 to 334.48 kb. A total of 16 CNV regions (CNVRs) were seen distributed among eleven autosomes, and the largest number of CNVRs (n = 4) was described on BTA12. Autozygosity islands were identified using an outlier approach, and none of them overlapped with CNVRs. Our study revealed that the limited genetic basis together with the narrow number of imported animals to disseminate the breed might have strongly contributed to the low effective population size and the high genomic autozygosity proportion described in this population. The functional enrichment analysis revealed several significant terms (p<0.05) within the autozygosity islands and CNVRs closely linked to molecular and immune response mechanisms. The average FROH of different lengths were low to in the studied population, however, the autozygotic proportion in the genome indicates moderate to high inbreeding levels. The results exposed the need of implementing mating programs to control the increase of inbreeding and coancestry in Brazilian Senepol cattle.In these sense, Senepol breeders should apply selection and mating strategies to minimize the occurrence of long ROH in the offspring, as well as import of new genetic material to avoid the loss of genetic diversity. This study revealed the existence of overlapped regions between CNVRs and ROH throughout the genome of the Senepol cattle, that contributes to a better understanding of the functional role of genomic structural variations in taurine cattle adapted to tropical regions.

Genomic signatures of inbreeding and mutation load in a threatened rattlesnake.

Theory predicts that threatened species living in small populations will experience high levels of inbreeding that will increase their genetic load, but recent work suggests that the impact of load may be minimized by purging resulting from long-term population bottlenecks. Empirical studies that examine this idea using genome-wide estimates of inbreeding and genetic load in threatened species are limited. Here we use individual genome resequencing data to compare levels of inbreeding, levels of genetic load (estimated as mutation load) and population history in threatened Eastern massasauga rattlesnakes (Sistrurus catenatus), which exist in small isolated populations, and closely related yet outbred Western massasauga rattlesnakes (Sistrurus tergeminus). In terms of inbreeding, S.catenatus genomes had a greater number of runs of homozygosity of varying sizes, indicating sustained inbreeding through repeated bottlenecks when compared to S.tergeminus. At the species level, outbred S.tergeminus had higher genome-wide levels of mutation load in the form of greater numbers of derived deleterious mutations compared to S.catenatus, presumably due to long-term purging of deleterious mutations in S.catenatus. In contrast, mutations that escaped species-level drift effects within S.catenatus populations were in general more frequent and more often found in homozygous genotypes than in S.tergeminus, suggesting a reduced efficiency of purifying selection in smaller S.catenatus populations for most mutations. Our results support an emerging idea that the historical demography of a threatened species has a significant impact on the type of genetic load present, which impacts implementation of conservation actions such as genetic rescue.

Correlation of performance traits of black-and-white first- calves

The long-term use of Holstein breeding bulls of foreign selection has led to the creation of a large quantity of Holsteinzed cattle in various climatic and ecological forage zones of the country, which also differs in their economically useful traits and biological features inherited from the breed resources of cattle in the breeding zone and the country of origin of the breeding bulls involved in crossing. In Sverdlovsk region the Holsteinzed black-and-white cattle of the Ural type are being bred, which are represented by large highly productive animals with a high genetic potential for milk productivity. The aim of the work is to study the correlation of productive qualities parameters among first-calves of black-and-white cattle. There is no difference between the breed lines in their productivity or they are so insignificant that they prove the uniformity of the bred livestock. This uniformity is achieved by strict selection and constant observance of the same type of selection applied for breeding bulls to serve in the herd. An insignificant difference in milk productivity parameters of cows of different Holstein lines is caused to the high level of inbreeding in the herd. The inbred animals account for up to 65 percent or more, depending on the age. A low positive correlation between the live weight of cows and their productive qualities is established, which practically has no dependence on the linear origin of cows.

Evaluation of genetic differentiation and genome-wide selection signatures in Polish local sheep breeds

Since their domestication, sheep breeds have been a subject of artificial selection directed towards production of wool, meat and milk as well as natural selection associated with regional climates and nutritional conditions. These selection pressures created a large diversity of sheep breeds and the opportunity to study their genetic differentiation. Despite the existence of several studies on genetic variation and selection signatures in sheep, information on the genetic background and variability of local sheep populations, especially on a genome wide-level, is still incomplete. This is mostly due to the large number of local sheep breeds and populations in existence. Several of these populations are under conservative breeding because of their largely primitive character, which is not suitable for the modern intensive animal production. In this study we analysed population parameters and quantified genetic differentiation among seven Polish conserved sheep breeds or local sheep populations, namely: Black-headed (BH; n=104), Polish Merino of coloured variety (MPC; n=59), ‘Old type’ Merino (MPOT; n=50), Polish Mountain Sheep (PMS; n=103), Świniarka (SW; n=100), Uhruska (UHR; n=69) and Wrzosówka (WRZ; n=90). The breeds represent mainly multipurpose production type with the emphasize on meat, wool and skin. Based on the data from OvineSNP50 BeadChip array (Illumina), we found that the genetic structures of the populations are not heavily affected, despite the bottlenecks in their recent breeding history. The only exception seems to be the SW breed in which we found very low effective population size (30.7) and high level of genomic inbreeding (17% as shown by FROH). Average MAF across breeds was high and ranged from 0.285 in MPC to 0.297 in PMS. The average observed heterozygosity per breed ranged from 0.378 in SW to 0.398 in UHR. The admixture patterns analysis showed that the analysed breeds have distinct genetic profiles with clear signs of common origin and admixture, similar to other worldwide sheep populations. By using FST statistics we also identified loci of the genome with the strongest divergence between the analysed breeds, presumably responsible for the fixed genetic differences among them which were created by artificial selection. The most pronounced of these results was selection signal at the locus of RXFP2 gene which is responsible for horn size/type in sheep. This analysis also allowed for genomic localization of genetic differences between the analysed breeds, providing a basis for further studies aimed at identification of sources of genetic variation in these sheep breeds.

Inbreeding reduces fitness of seed beetles under thermal stress.

Human‐induced environmental change can influence populations both at the global level through climatic warming and at the local level through habitat fragmentation. As populations become more isolated, they can suffer from high levels of inbreeding, which contributes to a reduction in fitness, termed inbreeding depression. However, it is still unclear if this increase in homozygosity also results in a corresponding increase in sensitivity to stressful conditions, which could intensify the already detrimental effects of environmental warming. Here, in a fully factorial design, we assessed the life‐long impact of increased inbreeding load and elevated temperature on key life history traits in the seed beetle, Callosobruchus maculatus. We found that beetles raised at higher temperatures had far reduced fitness and survival than beetles from control temperatures. Importantly, these negative effects were exacerbated in inbred beetles as a result of increased inbreeding load, with further detrimental effects manifesting on individual eclosion probability and lifetime reproductive success. These results reveal the harmful impact that increasing temperature and likelihood of habitat fragmentation due to anthropogenetic changes in environmental conditions could have on populations of organisms worldwide.

Development and validation of a SNP-based genotyping tool for pedigree establishment in Australian greenlip abalone Haliotis laevigata Donovan, 1808

To enable cumulative increases in aquaculture productivity, structured and efficient selective breeding programs are required. These are contingent on the management of genetic resources within the breeding population though the attainment of accurate pedigree information which can be provided by DNA-based parentage analysis. This study developed a SNP panel for greenlip abalone, Haliotis laevigata, from DArTSeq data produced from a genetic audit of greenlip abalone (n = 336) from five farms. The initial dataset consisted of 15,320 SNPs. Strict filtering on SNP quality control metrics was conducted to select the most informative 1,004 SNPs for the design of a DArTag™ genotyping panel for greenlip abalone. Sixteen broodstock and 1035 hatchery produced progeny were genotyped using the 1,004 SNP DArTag™ panel. The resulting genotypes were filtered, to produce 705 high performing SNPs. In silico parentage analysis using subsets of these SNPs (highest ranked by polymorphic information content) revealed that the inclusion of < 50 SNPs were satisfactory to resolve parentage for 10,000 simulated progeny to up to 200 candidate parents. The resolving power of the panel was also assessed under high levels of inbreeding (0–50 %) and relatedness (0–50 %) between candidate parents, and conditions of missing parental genotypes (10–50 %) in silico. Under all levels of inbreeding, relatedness and missing parental genotypes simulated, the panel was able to accurately assign parental pairs to the offspring. To validate these in silico results, parentage analysis of the hatchery produced progeny of the 16 candidate broodstock was conducted using both CERVUS and APIS. Complete parentage was assigned to all experimental progeny, with 100 % consensus between the two methods used. This study indicates this panel will serve as an efficient and cost-effective tool for accurate pedigree establishment for greenlip abalone.

Population Genetics of Species in the Genera Botrychium and Botrypus (Ophioglossaceae)

The population genetics of ferns, which results from initiation of individuals in a new location (often via long-distance dispersal) plus a wide range of mating systems, merit continued study. In the case of species in the subfamily Botrychioideae (specifically the genera Botrychium and Botrypus), previous work using allozyme and isozyme techniques revealed low genetic diversity and weak population genetic structure. This lack of genetic differentiation between populations is in spite of underground fertilization in the genus resulting in high levels of inbreeding and primarily fixed heterozygosity in tetraploids. In the present study, Amplified Fragment-Length Polymorphisms (AFLPs) were used to examine population genetics and structure of three species in the genus Botrychium and one species in the genus Botrypus. Measures of population genetic diversity were generally low, with the highest measures in the relatively common Botrypus virginianus. Across all species, measures of population differentiation were low and most genetic variation was contained within populations. Bayesian analysis of population structure using the program STRUCTURE corroborated these findings, with inferred genetic clusters that generally did not correspond to geographic collecting locations. These results agree with previous studies, with low genetic diversity within and among populations likely due to self-fertilization that limits outcrossing and long-distance spore dispersal that results in genetically similar populations.