Obligate Outcrossing Research Articles

Despite Extensive Pollinator Sharing, Invasive Blackberry has Negligible Impacts on Reproductive Success of a Rare Native Wildflower

Abstract Plant invasions have the potential to disrupt community dynamics and impair essential ecosystem services, including the pollination of native flowers. Invaders most likely to invoke pollinator competition are characterized by conspicuous, resource-rich blooms and long flowering periods, while the natives most likely to be impacted are obligate out-crossers that are sensitive to disturbance or locally rare. We investigated the effects of a widespread showy invader of the Pacific Northwest, Rubus armeniacus (Himalayan blackberry), on an imperiled endemic wildflower, Sidalcea hendersonii. We observed pollinators, quantified pollen deposition and conducted pollen-supplementation experiments on S. hendersonii plants growing at three distances (1 m, 15 m and 50 m) from well-established blackberry patches in five adjacent field plots. Individual R. armeniacus flowers received more than three times as many total visits as S. hendersonii inflorescences; however, there was minimal overlap between the polli...

Sexually antagonistic polymorphism in simultaneous hermaphrodites.

In hermaphrodites, pleiotropic genetic trade-offs between female and male reproductive functions can lead to sexually antagonistic (SA) selection, where individual alleles have conflicting fitness effects on each sex function. Although an extensive theory of SA selection exists for dioecious species, these results have not been generalized to hermaphrodites. We develop population genetic models of SA selection in simultaneous hermaphrodites, and evaluate effects of dominance, selection on each sex function, self-fertilization, and population size on the maintenance of polymorphism. Under obligate outcrossing, hermaphrodite model predictions converge exactly with those of dioecious populations. Self-fertilization in hermaphrodites generates three points of divergence with dioecious theory. First, opportunities for stable polymorphism decline sharply and become less sensitive to dominance with increased selfing. Second, selfing introduces an asymmetry in the relative importance of selection through male versus female reproductive functions, expands the parameter space favorable for the evolutionary invasion of female-beneficial alleles, and restricts invasion criteria for male-beneficial alleles. Finally, contrary to models of unconditionally beneficial alleles, selfing decreases genetic hitchhiking effects of invading SA alleles, and should therefore decrease these population genetic signals of SA polymorphisms. We discuss implications of SA selection in hermaphrodites, including its potential role in the evolution of "selfing syndromes."

Reproductive assurance drives transitions to self-fertilization in experimental Caenorhabditis elegans.

BackgroundEvolutionary transitions from outcrossing between individuals to selfing are partly responsible for the great diversity of animal and plant reproduction systems. The hypothesis of ‘reproductive assurance’ suggests that transitions to selfing occur because selfers that are able to reproduce on their own ensure the persistence of populations in environments where mates or pollination agents are unavailable. Here we test this hypothesis by performing experimental evolution in Caenorhabditis elegans.ResultsWe show that self-compatible hermaphrodites provide reproductive assurance to a male-female population facing a novel environment where outcrossing is limiting. Invasions of hermaphrodites in male-female populations, and subsequent experimental evolution in the novel environment, led to successful transitions to selfing and adaptation. Adaptation was not due to the loss of males during transitions, as shown by evolution experiments in exclusively hermaphroditic populations and in male-hermaphrodite populations. Instead, adaptation was due to the displacement of females by hermaphrodites. Genotyping of single-nucleotide polymorphisms further indicated that the observed evolution of selfing rates was not due to selection of standing genetic diversity. Finally, numerical modelling and evolution experiments in male-female populations demonstrate that the improvement of male fitness components may diminish the opportunity for reproductive assurance.ConclusionsOur findings support the hypothesis that reproductive assurance can drive the transition from outcrossing to selfing, and further suggest that the success of transitions to selfing hinges on adaptation of obligate outcrossing populations to the environment where outcrossing was once a limiting factor.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0093-1) contains supplementary material, which is available to authorized users.

A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.

BackgroundTuber melanosporum, also known in the gastronomic community as “truffle”, features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity.FindingsWe performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody (“truffle”), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes.ConclusionsThe datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles.

Reproductive ecology of Syzygium alternifolium (Myrtaceae), an endemic and endangered tropical tree species in the southern Eastern Ghats of India

Syzygium alternifolium is a semi-evergreen mass-flowering tree species of dry deciduous forest in the southern Eastern Ghats of India. It is a mass bloomer with flowering during dry season. The floral traits suggest a mixed pollination syndrome involving entomophily and anemophily together called as ambophily. Further, the floral traits suggest generalist pollination system adapted for a guild of pollinating insects. The plant is self-incompatible and obligate out-crosser. The flowers are many-ovuled but only a single ovule forms seed and hence, fruit and seed set rates are the same. Natural fruit set stands at 11%. Bud infestation by a moth, flower predation by the beetle, Popillia impressipyga and bud and flower mounds significantly limit fruit set rate. The ability of the plant to repopulate itself is limited by the collection of fruits by locals due to their edible nature, short viability of seeds, high seedling mortality due to water stress, nutrient deficiency and erratic rainfall or interval of drought within the rainy season. Therefore, S. alternifolium is struggling to populate itself under various intrinsic and extrinsic factors. Further studies should focus on how to assist the plant to increase its population size in its natural area taking into account the information provided in this paper.

Aspergillus: sex and recombination.

The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli.

English

Willow (Salix spp.) includes closely related dioecious polyploid species, which are obligate outcrossers. Natural populations of willows and their hybrids are represented by a mixture of highly heterozygous genotypes sharing a common gene pool. Random amplified polymorphism DNAs (RAPD) and microsatellites (simple sequence repeats, SSRs) are useful methods for studying genetic diversity of willow (Salix spp.). RAPD delivers a large number of data points from a single experiment and is a useful method for distinguishing between closely related individuals. SSR markers are very robust tools, and we have identified several markers that show high levels of polymorphism in willow. Genetic characterization of 94 genotypes (four female, ten male, and 80 half sibs) of Salix collected from Naganji Nursery of University of Horticulture and Forestry, Solan, Himachal Pradesh, India were analyzed using 10 SSRs and 15 RAPDs PCR-based molecular markers. RAPD analysis yielded 87 polymorphic fragments (98.9%), with an average of 5.8 polymorphic fragments per primer. Similarly, SSR analysis produced 33 bands, out of which 26 were polymorphic (78.8%) with an average of 2.6 polymorphic fragments per primer. The genetic diversity was high among the genotypes (Nei’s genetic diversity = 0.354 and Shannon’s information index = 0.536) as measured by combination of both RAPD and SSR markers. The mean coefficient of gene differentiation (Gst) was 0.037, indicating 96.6% of the genetic diversity resided within the genotypes. It was found that the genetic diversity among genotypes was broader, suggesting the importance and feasibility of introducing elite genotypes from different hybrids for willows germplasm conservation and breeding programs.   Key words: Salix sp., female, male, half sibs, molecular markers, genetic diversity, Random amplified polymorphism DNAs (RAPD), simple sequence repeats (SSRs).

Comparative Relationships and Chromosome Evolution in Switchgrass (Panicum virgatum) and Its Genomic Model, Foxtail Millet (Setaria italica)

A switchgrass (Panicum virgatum L.) consensus map was developed that combined data from two mapping populations and integrated recombination data from both parents of this largely obligate outcrossing species. The consensus map consisted of 1,321 loci and spanned 2,122 cM. An analysis of the distribution of genic and genomic markers across the length of a linkage group showed that genic markers were relatively overrepresented in distal regions, while genomic markers were relatively overrepresented in pericentromeric regions. Furthermore, genic markers located in distal and pericentromeric regions identified orthologs in the genome of the closely related species foxtail millet (Setaria italica (L.) P. Beauv.) at a similar rate, but fewer orthologs were found for genomic markers that mapped to pericentromeric regions compared to distal regions. Because cross-species sequence conservation is largely limited to genes, our data suggest that genomic markers that map to distal regions are more likely to be derived from genic regions than those that map to pericentromeric regions. A comparative analysis between loci on the switchgrass consensus map and their orthologs in the foxtail millet genome showed that the two species varied by at least nine inversions and one reciprocal translocation. Extending the comparative analysis to sorghum demonstrated that the majority of the rearrangements occurred in the foxtail millet genome in the past 13 million years. The reduction in chromosome number from 10 in the Andropogoneae to 9 in the Paniceae was achieved through a complex set of rearrangements involving three ancestral chromosomes orthologous to sorghum chromosomes 6, 8, and 9 and chromosomes III and VII in the lineage leading to switchgrass and foxtail millet. Upon insertion of ancestral chromosome 9 into ancestral chromosome 8, the centromere of the recipient chromosome was lost, and this was accompanied by a loss of repetitive DNA. The switchgrass–foxtail millet comparative map provides a guide of the rearrangements that need to be taken into account when using foxtail millet as a surrogate for switchgrass in genetic analyses.

Experimental coevolution: rapid local adaptation by parasites depends on host mating system.

Host-parasite interactions can drive rapid, reciprocal genetic changes (coevolution), provided both hosts and parasites have high heritabilities for resistance/infectivity. Similarly, the host's mating system should also affect the rate of coevolutionary change in host-parasite interactions. Using experimental coevolution, we determined the effect of obligate outcrossing verses partial self-fertilization (mixed mating) on the rate of evolutionary change in a nematode host (Caenorhabditis elegans) and its bacterial parasite (Serratia marcescens). Bacterial populations were derived from a common ancestor. We measured the effects of host mating system on host adaptation to the parasite. We then determined the extent of parasite adaptation to their local host populations. Obligately outcrossing hosts exhibited more rapid adaptation to parasites than did mixed mating hosts. In addition, most of the parasites became adapted to infecting their local hosts, but parasites from obligately outcrossing hosts showed a greater level of local adaptation. These results suggest that host populations evolved along separate trajectories and that outcrossing host populations diverged further than partially selfing populations. Finally, parasites tracking outcrossing host populations diverged further than parasites tracking the partially selfing host populations. These results show that the evolutionary trajectories of both hosts and parasites can be shaped by the host's mating system.

Selfing ability and dispersal are positively related, but not affected by range position: a multispecies study on southern African Asteraceae.

Dispersal and breeding system traits are thought to affect colonization success. As species have attained their present distribution ranges through colonization, these traits may vary geographically. Although several theories predict associations between dispersal ability, selfing ability and the relative position of a population within its geographic range, there is little theoretical or empirical consensus on exactly how these three variables are related. We investigated relationships between dispersal ability, selfing ability and range position across 28 populations of 13 annual, wind-dispersed Asteraceae species from the Namaqualand region of South Africa. Controlling for phylogeny, relative dispersal ability--assessed from vertical fall time of fruits--was positively related to an index of autofertility--determined from hand-pollination experiments. These findings support the existence of two discrete syndromes: high selfing ability associated with good dispersal and obligate outcrossing associated with lower dispersal ability. This is consistent with the hypothesis that selection for colonization success drives the evolution of an association between these traits. However, no general effect of range position on dispersal or breeding system traits was evident. This suggests selection on both breeding system and dispersal traits acts consistently across distribution ranges.

Characterization of Genetic Diversity in the Nematode Pristionchus pacificus from Population-Scale Resequencing Data

The hermaphroditic nematode Pristionchus pacificus is an established model system for comparative studies with Caenorhabditis elegans in developmental biology, ecology, and population genetics. In this study, we present whole-genome sequencing data of 104 P. pacificus strains and the draft assembly of the obligate outcrossing sister species P. exspectatus. We characterize genetic diversity within P. pacificus and investigate the population genetic processes shaping this diversity. P. pacificus is 10 times more diverse than C. elegans and exhibits substantial population structure that allows us to probe its evolution on multiple timescales. Consistent with reduced effective recombination in this self-fertilizing species, we find haplotype blocks that span several megabases. Using the P. exspectatus genome as an outgroup, we polarized variation in P. pacificus and found a site frequency spectrum (SFS) that decays more rapidly than expected in neutral models. The SFS at putatively neutral sites is U shaped, which is a characteristic feature of pervasive linked selection. Based on the additional findings (i) that the majority of nonsynonymous variation is eliminated over timescales on the order of the separation between clades, (ii) that diversity is reduced in gene-rich regions, and (iii) that highly differentiated clades show very similar patterns of diversity, we conclude that purifying selection on many mutations with weak effects is a major force shaping genetic diversity in P. pacificus.

Pollination and seed set of an obligatory outcrossing plant in an urban–peri-urban gradient

Abstract Urban land use strongly affects pollinators and plant pollination due to fragmentation and isolation of natural plant populations. On the other hand, urban land use can potentially be beneficial to pollinators through the presence of urban gardens and parks which are usually designed to ensure abundant flowering. However, little is known about the effects of urban land use on bees and the pollination services they provide. In an urban–peri-urban gradient around Leuven, Belgium we studied flower visitation rates to, and seed set of Trifolium repens (white clover) in public lawns. T. repens is an obligatory outcrossing plant and is therefore sensitive to reductions in pollinator services. We related our results to both local and regional variables using Boosted Regression Trees. The two variables that best explained the variability in visitation rates and seed set were the amount of green areas (gardens, parks, grasslands) in the surroundings and the abundance of T. repens in the lawns. Surprisingly, an increasing amount of green areas in the surroundings had a negative effect on both flower visitation rates to, and seed set of T. repens . Flower visitation rates by bumblebees responded positively to urban land use resulting in higher visitation rates and increased seed set in the more urban sites. This may point either to increased abundance of bumblebees in more urban sites or to a concentration effect of bumblebees in our urban study sites due to a lack of alternative forage resources. Responses will likely differ for other bee and plant species, but this shows that at least for T. repens , pollination is not compromised by urban land use.

Fertilization of Epichloë typhina stromata by mycophagous slugs

Epichloë typhina, a fungal endophyte of cool season grasses, is heterothallic and an obligate out-crosser. In areas of endemism, its spermatia are moved between stromata of the two opposite mating types through egg-laying activities of Botanophila flies. In western Oregon, where the fungus was inadvertently introduced into seed-production fields of Dactylis glomerata (= orchardgrass, cocksfoot), flies do not appear to be the sole vectors for E. typhina fertilization. Here we examined the role of the common agricultural slug pest Deroceras reticulatum and mycophagous slug species Prophysaon andersoni and Arion subfuscus in E. typhina spermatia transfer. Frass from P. andersoni, A. subfuscus and D. reticulatum fed stromata of one mating type was transferred to stromata of the opposite mating type, resulting in 100%, 93% and 25% stromata fertilization respectively. An experiment designed to mimic field conditions examined stromata fertilization on E. typhina-infected plants of opposite mating type in the presence of slugs. Treatments with P. andersoni and D. reticulatum had greater stromata fertilization compared to the no-slug control, but the slug treatments were not different. This appears to be the first report of mollusks vectoring viable spermatia leading to the cross fertilization of stromata of different mating types.

Hitchhiking of Deleterious Alleles and the Cost of Adaptation in Partially Selfing Species

Self-fertilization is generally seen to be disadvantageous in the long term. It increases genetic drift, which subsequently reduces polymorphism and the efficiency of selection, which also challenges adaptation. However, high selfing rates can increase the fixation probability of recessive beneficial mutations, but existing theory has generally not accounted for the effect of linked sites. Here, we analyze a model for the fixation probability of deleterious mutants that hitchhike with selective sweeps in diploid, partially selfing populations. Approximate analytical solutions show that, conditional on the sweep not being lost by drift, higher inbreeding rates increase the fixation probability of the deleterious allele, due to the resulting reduction in polymorphism and effective recombination. When extending the analysis to consider a distribution of deleterious alleles, as well as the average fitness increase after a sweep, we find that beneficial alleles generally need to be more recessive than the previously assumed dominance threshold (h < 1/2) for selfing to be beneficial from one-locus theory. Our results highlight that recombination aiding the efficiency of selection on multiple loci amplifies the fitness benefits of outcrossing over selfing, compared to results obtained from one-locus theory. This effect additionally increases the parameter range under which obligate outcrossing is beneficial over partial selfing.

The genetic basis and experimental evolution of inbreeding depression in Caenorhabditis elegans

Determining the genetic basis of inbreeding depression is important for understanding the role of selection in the evolution of mixed breeding systems. Here, we investigate how androdioecy (a breeding system characterized by partial selfing and outcrossing) and dioecy (characterized by obligatory outcrossing) influence the experimental evolution of inbreeding depression in Caenorhabditis elegans. We derived inbred lines from ancestral and evolved populations and found that the dioecious lineages underwent more extinction than androdioecious lineages. For both breeding systems, however, there was selection during inbreeding because the diversity patterns of 337 single-nucleotide polymorphisms (SNPs) among surviving inbred lines deviated from neutral expectations. In parallel, we also followed the evolution of embryo to adult viability, which revealed similar starting levels of inbreeding depression in both breeding systems, but also outbreeding depression. Under androdioecy, diversity at a neutral subset of 134 SNPs correlated well with the viability trajectories, showing that the population genetic structure imposed by partial selfing affected the opportunity for different forms of selection. Our findings suggest that the interplay between the disruptions of coevolved sets of loci by outcrossing, the efficient purging of deleterious recessive alleles with selfing and overdominant selection with outcrossing can help explain mixed breeding systems.

Low legitimate pollen flow in distylic Turnera hermannioides (Passifloraceae) and its consequences on fruit and seed set

Turnera hermannioides is a ruderal distylic subshrub, native to NE-Brazil. In the Catimbau National Park, situated within the semi-arid Caatinga region, we studied the pollination ecology of this species, emphasizing (1) effective pollinators; (2) characteristics of short- and long-styled flowers; (3) intra- and intermorph pollen flow; and (4) fruit and seed set. Short and long-styled morphs differ in pollen size and ornamentation, stigmatic surface, style and stamen length and nectar production. The flowers are obligate intermorphic outcrossers and depend on animals for pollination. The flowers of T. hermannioides attracted insect visitors of 25 species, among them butterflies, beetles, but mainly bees. Polylectic bees, such as Apis mellifera, stingless bees, and solitary Callonychium brasiliense were the most frequent visitors and the principal pollinators. The frequency of visits, however, was very low (on average 1.9 visits/flower/day), resulting in a low and unbalanced legitimate pollen flow, i.e. a much lower number of pollen grains from short-styled flowers reaching stigmas of long-styles than vice versa, which in turn compromised fruit and seed set. Surprisingly, Protomeliturga catimbaui and P. turnerae, both oligolectic on flowers of the Turneraceae-clade of the Passifloraceae s.l., were very rare flower visitors and did not contribute significantly to the pollination of Turnera hermannioides. We discuss how the low visitation rate and the inefficiency of pollinators (i.e. inefficiency in promoting legitimate pollination) compromise pollen flow and, consequently, fruit and seed set in this species.

MELITTOPHILY AND MALACOPHILY IN Ipomoea pes-caprae (CONVOLVULACEAE)

Ipomoea pes-caprae is a mangrove associate often found along shorelines. It flowers during August–February and is hermaphroditic and an obligate out-crosser due to self-incompatibility. It is both melittophilous and malacophilous. The natural fruit set rate is less than 10% probably due to the wastage of pollen in self-pollination, the ephemeral nature of flowers, predation, and nutrient requirements. Seeds are impermeable to water and germinate readily if they are covered and abraded by sand; those exposed to seawater rot and subsequently perish. The low natural fruit set rate, the mortality of seeds and seedlings due to rotting, burial by sand, space constraint, and shading by established plants appear to be regulating the population size of I. pes-caprae. The plant also propagates vegetatively through the expansion of branches that are broken into fragments by storm waves, transported by water, and then washed onshore into new locations.

Self-Incompatibility inByrsonima lucida(Malpighiaceae), a Threatened Pine Rockland Specialist

ABSTRACT Byrsonima lucida (Mill.) DC is the only member of the tropical plant family Malpighiaceae native to North America, and is considered a threatened species by the state of Florida. In order to better conserve the remaining natural populations of B. lucida, we studied the breeding system of this species in natural and garden settings. We carried out floral biology and breeding system experiments over three flowering seasons from 2009–2012. We found that stigmas were receptive mostly during the first 2 d of flowering, but not in the final day. Self-pollination yielded few fruits and pollinator exclusion yielded no fruit set, indicating self-incompatibility and pollinator dependence in this species. Obligate outcrossing and complete dependence on a specialist bee pollinator make B. lucida's small, fragmented populations vulnerable to extinction. Conservation measures should include protecting its specialized bee pollinator.

Plant mating system transitions drive the macroevolution of defense strategies

Understanding the factors that shape macroevolutionary patterns in functional traits is a central goal of evolutionary biology. Alternative strategies of sexual reproduction (inbreeding vs. outcrossing) have divergent effects on population genetic structure and could thereby broadly influence trait evolution. However, the broader evolutionary consequences of mating system transitions remain poorly understood, with the exception of traits related to reproduction itself (e.g., pollination). Across a phylogeny of 56 wild species of Solanaceae (nightshades), we show here that the repeated, unidirectional transition from ancestral self-incompatibility (obligate outcrossing) to self-compatibility (increased inbreeding) leads to the evolution of an inducible (vs. constitutive) strategy of plant resistance to herbivores. We demonstrate that inducible and constitutive defense strategies represent evolutionary alternatives and that the magnitude of the resulting macroevolutionary tradeoff is dependent on the mating system. Loss of self-incompatibility is also associated with the evolution of increased specificity in induced plant resistance. We conclude that the evolution of sexual reproductive variation may have profound effects on plant-herbivore interactions, suggesting a new hypothesis for the evolution of two primary strategies of plant defense.

Genetic structure of Linaria capraria Mill. (Plantaginaceae) and endemic species of the Tuscan Archipelago (central Mediterranean)

This study, based on inter-simple sequence repeats in 14 populations of Linaria capraria, shows that genetic diversity at the species level is 0.21 and represents 58% of total variance, whereas the level of intrapopulational genetic diversity is 0.07 and represents 42% of total variance. Gene flow is absent between populations from the same island. On the basis of analysis of molecular variance, three centers of genetic diversity were found: Elba and Pianosa, Montecristo, and Capraia; these correspond to the group of islands that remained isolated during the Würm marine regression. A mean proportion of species-level diversity resides among populations, so we hypothesize that the species has a mixed reproductive system that lies somewhere between obligate outcrossing and obligate selfing. The most genetically diverse populations live in natural and well-isolated rocky habitats that seem to have no particular intervention requirements for conservation but the entire system of populations of this endemic species should be preserved.