Outcrossing Species Research Articles

Differential retention of transposable element-derived sequences in outcrossing Arabidopsis genomes

BackgroundTransposable elements (TEs) are genomic parasites with major impacts on host genome architecture and host adaptation. A proper evaluation of their evolutionary significance has been hampered by the paucity of short scale phylogenetic comparisons between closely related species. Here, we characterized the dynamics of TE accumulation at the micro-evolutionary scale by comparing two closely related plant species, Arabidopsis lyrata and A. halleri.ResultsJoint genome annotation in these two outcrossing species confirmed that both contain two distinct populations of TEs with either ‘recent’ or ‘old’ insertion histories. Identification of rare segregating insertions suggests that diverse TE families contribute to the ongoing dynamics of TE accumulation in the two species. Orthologous TE fragments (i.e. those that have been maintained in both species), tend to be located closer to genes than those that are retained in one species only. Compared to non-orthologous TE insertions, those that are orthologous tend to produce fewer short interfering RNAs, are less heavily methylated when found within or adjacent to genes and these tend to have lower expression levels. These findings suggest that long-term retention of TE insertions reflects their frequent acquisition of adaptive roles and/or the deleterious effects of removing nearly neutral TE insertions when they are close to genes.ConclusionOur results indicate a rapid evolutionary dynamics of the TE landscape in these two outcrossing species, with an important input of a diverse set of new insertions with variable propensity to resist deletion.

Effects of inbreeding depression on seed production in Scots pine (Pinus sylvestris)

Like other outcrossing species, Scots pine (Pinus sylvestris L.) is thought to carry a “genetic load” of deleterious recessive alleles. When these alleles occur as homozygotes in inbred progeny, their expression can give rise to “inbreeding depression”. Although this phenomenon has been studied in several conifer species through selfing, few studies have quantified inbreeding depression in crosses with lower levels of relatedness between parents. We report here on the generation of a set of F3 study materials in Scots pine in which 142 families arose from a mating design among 49 F2 parents, representing nine levels of expected inbreeding coefficients between 0.0 and 0.5, and repeated over two consecutive seasons. Whereas the numbers of extractable seeds were unaffected by inbreeding, the proportion with fully developed embryos was strongly affected. This was expressed as inbreeding depression in the yield of full seeds per cone but not in their mean mass. Levels of germination of these full seeds were affected by inbreeding, but the depression was rather small and only weakly significant. The roles of pollen competition and polyembryony in mitigating the impact of inbreeding depression are discussed. The materials have been outplanted for future assessment of inbreeding depression on growth and survival.

Reproductive ecology of Seselighafoorianum (Apiaceae) restricted to rocky cliffs in Iran

Endemic and relict species are of particular interest when evaluating the ecological intactness of a given area. In this paper, the reproductive ecology of Seseli ghafoorianum (Apiaceae) is investigated, aiming to contribute to its protection. The species is considered as a Tertiary relict species in Iran. Both dwarf form and cliff habitats are rare in the family, raising the question of how the plant has adapted to this environment. Population structure, plant architecture and flowering sequence, pollinators, pollen quantification, and seed set (bagging experiment) were investigated. Individuals produce about 20 flowering branches with ca. 120 umbels. Flowers and umbels are protandrous. The female stage of the terminal umbels usually overlaps the maleness of the 1st order umbels promoting geitonogamy. The pollen/ovule ratio is about 30,000:1, indicating outcrossing. The degree of andromonoecy (>70%) and pollen vitality (~90%) indicate that pollen availability is not a limiting factor. In contrast, fruit set is low (~7%). It depends on the presence of pollinators (Lasioglossum), which is low, most likely due to the use of pesticides. The population furthermore suffers from the low survival rate of seedlings (0.02%). S. ghafoorianum appears to be an outcrossing species suffering from low pollinator and survival rates. To protect the species, use of pesticides and grazing should be reduced.

Water influences how seed production responds to conspecific and heterospecific pollen.

Outcrossing species depend on pollen from conspecific individuals that may not be exposed to the same abiotic conditions as maternal plants. Additionally, many flowers receive heterospecific pollen, which can also influence seed production. Studies aimed to understand how abiotic conditions influence seed production tend to focus on maternal conditions and leave unexplored the effect of abiotic conditions experienced by pollen donors. We tested how water availability to pollen donors, both conspecific and heterospecific, influenced the seed production of recipient plants exposed to different water availability regimes. In a greenhouse setting we manipulated the water availability (low- or high-water treatment) to potted recipient plants (Phacelia parryi), to conspecific pollen donors, and to heterospecific pollen donors (Brassica nigra). We hand pollinated recipient plants with different pollen mixes that represented all combinations of conspecific pollen mixed with heterospecific pollen. From these hand pollinations we determined the amount of pollen that was transferred, pollen volume, pollen shape, and seed production. Higher water availability to conspecific pollen donors led to higher seed production. Under low water availability to heterospecific pollen donors, seed production was unaffected by recipient or conspecific pollen donor treatment. Under high water availability to heterospecific pollen donors, seed production was highest when conspecific pollen donors and pollen recipients also received the high-water treatment. Environmental conditions of pollen donors can influence the seed production of maternal plants. These results illustrate potential impacts of environmental heterogeneity on post-pollination events that lead to seed production and thus impact a pollinator's contribution to plant fitness.

Gene flow signature in the S-allele region of cultivated buckwheat

BackgroundBuckwheat (Fagopyrum esculentum Moench.) is an annual crop that originated in southern China. The nutritious seeds are used in cooking much like cereal grains. Buckwheat is an outcrossing species with heteromorphic self-incompatibility due to its dimorphic (i.e., short- and long-styled) flowers and intra-morph infertility. The floral morphology and intra-morph incompatibility are both determined by a single S locus. Plants with short-styled flowers are heterozygous (S/s) and plants with long-styled flowers are homozygous recessive (s/s) at this locus, and the S/S genotype is not found. Recently, we built a draft genome assembly of buckwheat and identified the 5.4-Mb-long S-allele region harbored by short-styled plants.In this study, the first report on the genome-wide diversity of buckwheat, we used a genotyping-by-sequencing (GBS) dataset to evaluate the genome-wide nucleotide diversity within cultivated buckwheat landraces worldwide. We also investigated the utility of the S-allele region for phylogenetic analysis of buckwheat.ResultsBuckwheat showed high nucleotide diversity (0.0065), comparable to that of other outcrossing plants, based on a genome-wide simple nucleotide polymorphism (SNP) analysis. Phylogenetic analyses based on genome-wide SNPs showed that cultivated buckwheat comprises two groups, Asian and European, and revealed lower nucleotide diversity in the European group (0.0055) and low differentiation between the Asian and European groups.The nucleotide diversity (0.0039) estimated from SNPs in the S-allele region is lower than that in genome-wide SNPs. Phylogenetic analysis based on this region detected three diverged groups, S-1, S-2, and S-3.ConclusionThe SNPs detected using the GBS dataset were effective for elucidating the evolutionary history of buckwheat, and led to the following conclusions: (1) the low nucleotide diversity of the entire genome in the European group and low differentiation between the Asian and European groups suggested genetic bottlenecks associated with dispersion from Asia to Europe, and/or recent intensified cultivation and selection in Europe; and (2) the high diversification in the S-allele region was indicative of gene flows from wild to cultivated buckwheat, suggesting that cultivated buckwheat may have multiple origins.

Genetic Diversity and Population Structure of Rhododendron canescens, a Native Azalea for Urban Landscaping

Rhododendron canescens is a deciduous azalea native to the southeastern United States that is used in landscaping due to its ornamental qualities. A genotyping-by-sequencing (GBS) approach was taken to characterize the genetic structure and diversity of a R. canescens germplasm collection. Single nucleotide polymorphisms (SNPs) were identified by two software platforms, STACKS and GBS-SNP-CROP. Three distinct R. canescens populations were detected by STRUCTURE analysis with GBS-SNP-CROP data, whereas two populations were distinguished using STACKS data. Principal component analysis (PCA) with data from both SNP pipelines supported the presence of three populations. Statistical results indicated that there was low genetic differentiation between the populations, but relatively high genetic diversity within populations. The inbreeding coefficient of the R. canescens accessions was low, which would be expected with an outcrossing species. These results suggest that there may be a significant level of gene flow between populations of R. canescens.

Reproductive biology and genetic population structure of two alien Lolium species inhabiting the sandy coasts of Japan

AbstractUnderstanding the characteristics of alien species is a prerequisite for any biological study or anti‐invasion management strategy. Lolium rigidum (Poaceae) is an alien species that has become naturalized on the sandy coasts of Japan; however, it exhibits extensive morphological variation, leading to speculation that several taxa of Lolium have become naturalized. Here, we compared the morphology, reproductive biology and genetic structure of this Lolium species by growing individuals from different locations in the same environment to clarify whether the observed morphological variation is genetically based or is caused by intraspecific variation as a result of environmental differences. Principle component analyses of 11 morphological traits separated the study species into two types. Bagging experiments showed that one type exhibited an outcrossing life history, whereas the other type selfed exclusively. Nuclear DNA microsatellite analyses supported this distinction between these morphological types, with no intermediate individuals being found. The outcrossing type exhibited high genetic diversity, whereas the selfing type exhibited almost no polymorphism, reflecting the differences in their breeding systems. Moreover, both types differed from the economically important outcrossing species Lolium multiflorum and Lolium perenne. These two types of Lolium are expected to have different introduction histories and invasive potential on the coasts of Japan. In conclusion, it is important to distinguish between these types through additional biological studies to design effective control measures.

Cryptic Lineages and a Population Dammed to Incipient Extinction? Insights into the Genetic Structure of a Mekong River Catfish.

An understanding of the genetic composition of populations across management boundaries is vital to developing successful strategies for sustaining biodiversity and food resources. This is especially important in ecosystems where habitat fragmentation has altered baseline patterns of gene flow, dividing natural populations into smaller subpopulations and increasing potential loss of genetic variation through genetic drift. River systems can be highly fragmented by dams built for flow regulation and hydropower. We used reduced-representation sequencing to examine genomic patterns in an exploited catfish, Hemibagrus spilopterus, in a hotspot of biodiversity and hydropower development-the Mekong River basin. Our results revealed the presence of 2 highly divergent coexisting genetic lineages which may be cryptic species. Within the lineage with the greatest sample sizes, pairwise FST values, principal component analysis, and a STRUCTURE analysis all suggest that long-distance migration is not common across the Lower Mekong Basin, even in areas where flood-pulse hydrology has limited genetic divergence. In tributaries, effective population size estimates were at least an order of magnitude lower than in the Mekong mainstream indicating these populations may be more vulnerable to perturbations such as human-induced fragmentation. Fish isolated upstream of several dams in one tributary exhibited particularly low genetic diversity, high amounts of relatedness, and a level of inbreeding (GIS = 0.51) that has been associated with inbreeding depression in other outcrossing species. Our results highlight the importance of assessing genetic structure and diversity in riverine fisheries populations across proposed dam development sites for the preservation of these critically important resources.

Transcriptomic analyses reveal groups of co-expressed, syntenic lncRNAs in four species of the genus Caenorhabditis

ABSTRACTLong non-coding RNAs (lncRNAs) are a heterogeneous class of genes that do not code for proteins. Since lncRNAs (or a fraction thereof) are expected to be functional, many efforts have been dedicated to catalog lncRNAs in numerous organisms, but our knowledge of lncRNAs in non vertebrate species remains very limited. Here, we annotated lncRNAs using transcriptomic data from the same larval stage of four Caenorhabditis species. The number of annotated lncRNAs in self-fertile nematodes was lower than in out-crossing species. We used a combination of approaches to identify putatively homologous lncRNAs: synteny, sequence conservation, and structural conservation. We classified a total of 1,532 out of 7,635 genes from the four species into families of lncRNAs with conserved synteny and expression at the larval stage, suggesting that a large fraction of the predicted lncRNAs may be species specific. Despite both sequence and local secondary structure seem to be poorly conserved, sequences within families frequently shared BLASTn hits and short sequence motifs, which were more likely to be unpaired in the predicted structures. We provide the first multi-species catalog of lncRNAs in nematodes and identify groups of lncRNAs with conserved synteny and expression, that share exposed motifs.

Outcrossing Rates and Gene Flow in Natural Population of the Endangered Endemic Aquatic Lycophyte Isoetes yunguiensis as Revealed by ISSR Markers

In this study, mating system, genetic diversity, and genetic structure of the endangered endemic aquatic Isoetes yunguiensis in China was investigated using ISSR markers. The results of ISSR analyses showed that the estimate of multilocus outcrossing rate (tm) was high at species level (tm = 0.955), indicating that diploid I. yunguiensis is a predominant outcrossing species. Nine selected ISSR primers used in the study amplified 66 reproducible bands, 41 of which were polymorphic among 37 individuals. High level of genetic diversity was detected at the species level (PPB = 62.12%), whereas, relatively low genetic diversity existed within populations (PPB = 39.39%). Analysis of molecular variance (AMOVA) revealed that 31.99% of the genetic variation was attributable to differences between populations and the rest (68.01%) to variability within populationsof I. yunguiensis. Value of Fst (0.320) indicated that genetic differentiation between populations also was significant. These results showed that I. yunguiensis predominantly favors crossing, and has a high level of genetic diversity and highly significant genetic variation between and within populations. Gene flow (Nm) among populations is equal to 1.177. High outcrossing rates may be responsible for the high levels of genetic diversity observed in the I. yunguiensis population. To maintain the current level of genetic diversity for this species, we recommend increasing in situ conservation sites.

Modeling a trait-dependent diversification process coupled with molecular evolution on a random species tree

Understanding the evolution of binary traits, which affects the birth and survival of species and also the rate of molecular evolution, remains challenging. In this work, we present a probabilistic modeling framework for binary trait, random species trees, in which the number of species and their traits are represented by an asymmetric, two-type, continuous time Markov branching process. The model involves a number of different parameters describing both character and molecular evolution on the so-called ‘reduced’ tree, consisting of only extant species at the time of observation. We expand our model by considering the impact of binary traits on dN/dS, the normalized ratio of nonsynonymous to synonymous substitutions. We also develop mechanisms which enable us to understand the substitution rates on a phylogenetic tree with regards to the observed traits. The properties obtained from the model are illustrated with a phylogeny of outcrossing and selfing plant species, which allows us to investigate not only the branching tree rates, but also the molecular rates and the intensity of selection.

Potential SNPs related to microspore culture inRaphanus sativusbased on a single-marker analysis

Radish (Raphanus sativus) is an economically important crop grown for its edible roots and leaves. It is a self-incompatible, outcrossing species, making the production of homozygous lines and the development of breeding populations difficult. However, this can be overcome with haploids production techniques using isolated microspores, providing the rapid production of homozygous lines for breeding. Thus, it would be useful to identify radishes with a high regeneration rate from microspore culture. In the current study, 96 radish cultivars or germplasms were evaluated for high regeneration rates. Also, a single-marker analysis (SMA) was applied to detect single nucleotide polymorphisms (SNPs) potentially associated with this trait using genotype-by-sequencing (GBS) technology. The regeneration rate from microspore culture of 96 lines showed a wide range, from 0% to 269.5%. From the SMA, 52 markers were detected at a p value of 0.001 and a total of 11 physically nearby genes with high levels of similarity in various species were identified as candidates for high regeneration rates. This result could be used for clarifying the genetic basis underlying these traits and developing molecular markers associated with regeneration rates and would be beneficial for generating homozygous inbred lines.

Rapid evolution in native plants cultivated for ecological restoration: not a general pattern

The growing number of restoration projects worldwide increases the demand for seed material of native species. To meet this demand, seeds are often produced through large-scale cultivation on specialised farms, using wild-collected seeds as the original sources. However, during cultivation, plants experience novel environmental conditions compared to those in natural populations, and there is a danger that the plants in cultivation are subject to unintended selection and lose their adaptation to natural habitats. Although the propagation methods are usually designed to maintain as much natural genetic diversity as possible, the effectiveness of these measures have never been tested. We obtained seed of five common grassland species from one of the largest native seed producers in Germany. For each species, the seeds were from multiple generations of seed production. We used AFLP markers and a common garden experiment to test for genetic and phenotypic changes during cultivation of these plants. The molecular markers detected significant evolutionary changes in three out of the five species and we found significant phenotypic changes in two species. The only species that showed substantial genetic and phenotypic changes was the short-lived and predominantly selfing Medicago lupulina, while in the other, mostly perennial and outcrossing species, the observed changes were mostly minor. Agricultural propagation of native seed material for restoration can cause evolutionary changes, at least in some species. We recommend caution, particularly in selfing and short-lived species, where evolution may be more rapid and effects may thus be more severe.

Andromonoecy in Gynandropsis gynandra (L.) Briq. (Cleomaceae) and effects on fruit and seed production

Spider plant (Gynandropsis gynandra) is a traditional leafy vegetable widespread in sub-Saharan Africa and tropical Asia that is also valued for its medicinal properties. Developing a breeding program for the species requires detailed knowledge of its phenology, floral morphology and pollination system. This study investigates the effects of floral morphology and pollination mechanisms on the reproductive success in G. gynandra. The experiments were conducted in two locations in Benin. A split-plot design was used with four randomized complete blocks. Three accessions were randomly assigned to the whole plots and five treatments including natural self-pollination, hand self-pollination, geitonogamy, open pollination and hand cross-pollination were randomized over the sub-plots. We observed that individual plant exhibited 70% of staminate (male) flowers and 30% of hermaphrodite flowers. G. gynandra was andromonoecious. Open pollination and hand cross-pollination led to higher fruit and seed set. Natural self-pollination and hand self-pollination resulted in lower fruit and seed production. G. gynandra is a self-compatible and predominantly out-crossing species. Cross-pollination resulted in a significant increase in fruit set. This study set the ground for the development of improved cultivars in G. gynandra.

Genetic diversity and population genetic structure of the endangered kazakh endemic Oxytropis almaatensis (fabaceae)

The central Asian narrow endemic species Oxytropis almaatensis is a highly endangered plant with a very restricted distribution in the Tian Shan Mountains. In this study, we present the basic conservation genetic characteristics of this species based on a DNA fingerprinting approach in order to provide yardsticks for official conservation agencies to develop an informed conservation strategy. The three currently known populations with two allopatric subpopulations at each site were sampled in the Trans-Ili Alatau Mountains (S Kazakhstan) and subject to AFLP analysis using four primer combinations. This was supplemented by flow cytometry of plants with remarkably different body sizes to check for possible ploidy differences. The presence or absence of AFLP bands was used in downstream analyses utilising various population genetic approaches. Genetic diversity of O. almaatensis was found to be on the upper end of the spectrum typical for other outcrossing species of similar life-history characteristics. Most of the genetic variation was attributable to within (sub)population variance, and we also found a remarkable gene flow between the populations. However, the geographically closer populations were found to be more close to each other genetically, and population differentiation showed the same pattern with a significant isolation by distance. Similar patterns were not found for subpopulations of the geographically more close populations, and the subpopulations living along the same river valley were found to be genetically more cohesive. Flow cytometry did not reveal any difference in DNA content between the small and large forms of the species. All these results suggest the presence of two separate populations at the three localities of this species. Conservation efforts should focus on these two populations, and, given the relatively high genetic diversity within each population, both ex situ and in situ conservation measures can be effectively carried out based on the currently known populations of this narrow endemic species.

The progress of genetic improvement in alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) is a perennial and outcrossing species, widely grown as a forage legume for hay, pasture and silage. The genetic engineering approaches involve the transfer of useful or novel gene(s) into alfalfa to improve desired traits. The recent development of genetic engineering is extensively applied to basic and applied research for alfalfa improvement, including improvement of herbicide resistance, reinforcement of the resistance to biotic and abiotic stresses, production of novel compounds, improved yield for industrial and/or pharmaceutical proteins and renewable energy sources. On the other hand, alfalfa forage needs to possess additional fermentable carbohydrates, proteins with a balanced amino acid profile that are gradually degraded in the rumen of domestic livestock, and zero anti-nutritional factors, which are the major concerns of recent interest in alfalfa. However, an advance of transgenic approach is contentious, requiring vigilant experimental methods and design to contest bio-safety challenges. More importantly, the technology of clustered regularly interspaced short palindromic repeats (CRISPR) is rapidly growing and might be a game player or changer in alfalfa. The present review can enable us to identify the proper direction, get familiar with new research methods and success of genetic engineering technology in alfalfa, to produce maximally improved cultivars.

Population Differences in Fecundity Components in the Hermaphroditic Freshwater SnailPlanorbella trivolvis

Fecundity in outcrossing species can be influenced by both maternal and paternal parents. To tease out these influences, we observed egg production in two populations of the hermaphroditic freshwater snail, Planorbella trivolvis (Say 1817). We carried out both intra- and inter-population matings between individuals from an inbred albino laboratory strain and individuals one generation removed from a natural population, and measured egg production for two 3-week periods - immediately after mating and 11 weeks later. In the female role, individuals from the albino laboratory population produced significantly more egg masses than the wild derived snails, regardless of whether they were mated to a partner of wild or laboratory origin, indicating that egg mass production is controlled by the maternal parent. Conversely, regardless of their own origin (laboratory or wild), snails mated to individuals from the wild derived population laid more eggs in each egg mass than those fertilized by sperm from the lab population. These results indicate that the number of egg masses a snail lays is determined at least in part by a maternal contribution, and that the number of eggs deposited in each mass may be influenced by the origin of the sperm donor. In addition, we confirmed a short-term trade-off between growth and reproduction as differences in egg production among mating types were mirrored by differences in body size. Further investigation into the nature of these differences will lead to better understanding of the reproductive biology of these hermaphrodite mollusks.

Mating system does not predict niche breath

AbstractAimSelf‐pollinating plants (‘selfers’) have larger geographical ranges and inhabit higher latitudes than their outcrossing relatives. This finding has led to the hypothesis that selfers also have broader climatic niches (‘niches’) because the increased likelihood of successful colonization into new areas and the initial purging of deleterious mutations could offset the inability of selfers to adapt to new environments owing to low heterozygosity. Here, we examine the niches of hundreds of closely related selfing and outcrossing species to determine whether selfers do indeed have larger niche breadths.LocationGlobal.Time periodCurrent.Major taxa studiedTwenty clades of flowering plants comprising 424 species.MethodsWe estimated and compared the climatic niches of 194 pairs of sister species across 15 families, which differed in mating system. We incorporated these results into models predicting niche breadth and its change over time to estimate the effects of mating system on niche breadth. Furthermore, we compared the degree of niche divergence between sister‐species pairs of various mating system combinations.ResultsSelfers did not have wider niche breadths than their outcrossing sister taxa. Sister pairs of selfers also exhibited greater niche overlap than outcrossing sisters, implying that niche expansion becomes limited after the transition to selfing. Furthermore, the niche breadth of selfers was predicted to decrease significantly faster than that of closely related outcrossers.Main conclusionsOur results demonstrate a decoupling in the range size and niche breadth of selfers. The larger geographical range and comparable niche breadths of many selfers is most likely to be a temporary phenomenon caused by a transiently expanded realized niche, and both will become constricted over time in comparison to outcrossers.

Mating System and Genetic Variability of the Endangered Endemic Aquatic Lycophyte,Isoëtes yunguiensis, in China Determined using AFLP Markers

In this study, mating system, genetic diversity, and genetic structure of the endangered endemic aquatic lycophyte Isoëtes yunguiensis in China was investigated using AFLP markers. The estimates of outcrossing rates (tm = 1.200) indicate that diploid I. yunguiensis is a predominantly outcrossing species. Six selected AFLP primer pairs used in the study amplified 195 reproducible bands, 160 of which were polymorphic (PPB = 82.05%), indicating significant genetic diversity at the species level. AMOVA revealed that 40.12% of the genetic variation was attributable to differences between populations and the rest (59.88%) to variability within populations. High outcrossing rates and the historical accumulation of genetic variation may be responsible for the high levels of genetic diversity observed in the I. yunguiensis population. To maintain the current level of genetic diversity for this species, we recommend increasing in situ conservation sites.

Estimation of Mating System in the Endangered Aquatic Fern Ceratopteris pteridoides in China Based on AFLP Molecular Marker and Selfing Test: Implications for Conservation

Mating system has important implications for the genetic structure and diversity of populations, especially threatened and endangered species. In this study, mating system of the endangered aquatic fern Ceratopteris pteridoides in China was investigated using AFLP markers and selfing test. The results of AFLP analyses showed that the estimate of multilocus outcrossing rate (tm) was high at species level (tm = 0.999), indicating that C. pteridoides is a predominant outcrossing species. The small differences between tm and ts (0.136 ± 0.003) at species level showed that there is a low tendency for mating between relatives. The selfing test showed that isolated gametophytes formed the rate of normal sporophytes in three populations (BLH, ZDH, WCH) ranging from 72.9% to 77.8% with a mean value of 75.2%, indicating that C. pteridoides has a high level of intragametophytic selfing potential. Our investigation found that some of the studied populations of C. pteridoides have a prolific capacity for vegetative reproduction (clonal growth). The results revealed that the diploid homosporous pteridophyte C. pteridoides predominantly favors gametophytic crossing with a certain level of inbreeding along with clonal growth. We suggest that mating system of C. pteridoides may be adjusted to promote selfing rate in response to factors including the characteristics of hermaphrodite, and colonization events and small extant populations. As a result of the high outcrossing rate of C. pteridoides, we recommend that ex situ plantings would be most suitable for enhancing possible intermingling of the different populations thus minimizing inbreeding.