Complete chloroplast genomes of three copal trees (Bursera: Bullockia): comparative analysis and phylogenetic relationships.

BACKGROUND: Bursera trees are conspicuous elements of the tropical dry forests in the Neotropics that have significant cultural value due to their fragrant resins (incense), wood sources (handcrafts), and ecological benefits. Despite their relevance, genetic resources developed for the genus are scarce. METHODS AND RESULTS: We obtained the complete chloroplast (Cp) genome sequence, analyzed the genome structure, and performed functional annotation of three Bursera species of the Bullockia section: Bursera cuneata, B. palmeri, and B. bipinnata. The Cp genome sizes ranged from 159,824 to 159,872 bp in length, including a large single-copy (LSC) region from 87,668 to 87,656 bp, a small single-copy (SSC) from 18,581 to 18,571 bp, and two inverted repeats regions (IRa and IRb) of 26,814 bp each. The three Cp genomes consisted of 135 genes, of which 90 were protein-coding, 37 tRNAs, and 8 rRNAs. The Cp genomes were relatively conserved, with the LSC region exhibiting the greatest nucleotide divergence (psbJ, trnQ-UCC, trnG-UCC, and petL genes), whereas few changes were observed in the IR border regions. Between 589 and 591 simple sequence repeats were identified. Analysis of phylogenetic relationships using our data for each Cp region (LSC, SSC, IRa, and IRb) and of seven species within Burseraceae confirmed that Commiphora is the sister genus of Bursera. Only the phylogenetic trees based on the SSC and LSC regions resolved the close relationship between B. bipinnata and B. palmeri. CONCLUSION: Our work contributes to the development of Bursera's genomic resources for taxonomic, evolutionary, and ecological-genetic studies.

Do habitat fragmentation and degradation influence the strength of fine-scale spatial genetic structure in plants? A global meta-analysis.

As primarily sessile organisms, plants often show a non-random spatial distribution of genotypes over distance. This process known as fine-scale spatial genetic structure (FSGS) has been suggested through systematic reviews to depend on life form, mating system, and pollen and seed dispersal vectors, while there is no consensus on its behaviour due to external factors, such as anthropogenic habitat changes. By conducting a systematic review and global meta-analysis of empirical FSGS studies, we aimed to evaluate how anthropogenic habitat fragmentation and degradation influence the strength of FSGS in plant populations by means of the Sp statistic. Moreover, we tested how pollination and seed dispersal vectors contribute to the variation of the Sp statistic. We retrieved 243 FSGS studies from 1960 to 2020 of which only 65 were informative for the systematic review. Most empirical studies comprised outcrossers (84%) and trees (67%), with few herbs (23%) and scarce annual species (2%). In weighted meta-analyses for 116 plant populations (31 studies), we did not detect significant effects in the magnitude of effect sizes for the Sp statistic among undisturbed, degraded and fragmented habitats. Results showed significant effects for seed dispersal vectors, but not for pollination. Overall, we observed high variation among the effect sizes (not related to the goodness-of-fit of mixed models) of habitat status, pollination and seed dispersal categories, which precludes identifying biological trends on the Sp statistic. More empirical studies are needed that contrast multiple plant populations in disturbed versus undisturbed habitats, and by increasing the taxonomic groups, such as herbs and annual plants.

Geographic isolation and long-distance gene flow influence the genetic structure of the blue fan palm Brahea armata (Arecaceae).

The formation of the Baja California Peninsula (BCP) has impacted the microevolutionary dynamics of different species in ways that depend on biological traits such as dispersal capacity. Plants with relatively low levels of vagility have exhibited high genetic divergence between the BCP and Continental mainland. Brahea armata (Arecaceae) is a palm species inhabiting the northern part of the BCP and Sonora; its distribution occurs in isolated oases of vegetation. We aimed to evaluate the influence of the formation of the BCP on the genetic structure of B. armata using nuclear microsatellites and chloroplast markers (cpDNA) to compare patterns of genetic diversity and structure with previous published studies. Because gene flow through seeds is usually more limited compared to pollen flow, we expect to find stronger genetic structure at (cpDNA) than at nuclear markers. Moreover, larger genetic structure might also be explained by the smaller effective population size of cpDNA. We analyzed six microsatellite markers and two cpDNA regions. The main results indicated high levels of genetic differentiation among isolated populations located in the BCP, while low genetic differentiation was found between southern populations of the BCP and Sonora, suggesting long distance gene flow. In contrast, chloroplast markers indicated high levels of genetic structure between BCP and Sonora populations, suggesting asymmetrical gene flow between pollen (measured by nuclear microsatellites) and seed (cpDNA markers). This study provides valuable information on genetic diversity of B. armata that can be relevant for conservation and management; and develops microsatellites markers that can be transferred to other Brahea species.

Development of Genomic Resources in Mexican Bursera (Section: Bullockia: Burseraceae): Genome Assembly, Annotation, and Marker Discovery for Three Copal Species.

Bursera comprises ~100 tropical shrub and tree species, with the center of the species diversification in Mexico. The genomic resources developed for the genus are scarce, and this has limited the study of the gene flow, local adaptation, and hybridization dynamics. In this study, based on ~155 million Illumina paired-end reads per species, we performed a de novo genome assembly and annotation of three Bursera species of the Bullockia section: Bursera bipinnata, Bursera cuneata, and Bursera palmeri. The total lengths of the genome assemblies were 253, 237, and 229 Mb for B. cuneata, B. palmeri, and B. bipinnata, respectively. The assembly of B. palmeri retrieved the most complete and single-copy BUSCOs (87.3%) relative to B. cuneata (86.5%) and B. bipinnata (76.6%). The ab initio gene prediction recognized between 21,000 and 32,000 protein-coding genes. Other genomic features, such as simple sequence repeats (SSRs), were also detected. Using the de novo genome assemblies as a reference, we identified single-nucleotide polymorphisms (SNPs) for a set of 43 Bursera individuals. Moreover, we mapped the filtered reads of each Bursera species against the chloroplast genomes of five Burseraceae species, obtaining consensus sequences ranging from 156 to 160 kb in length. Our work contributes to the generation of genomic resources for an important but understudied genus of tropical-dry-forest species.

Molecular evidence and ecological niche modeling reveal an extensive hybrid zone among three Bursera species (section Bullockia).

The genus Bursera, includes ~100 shrub and trees species in tropical dry forests with its center of diversification and endemism in Mexico. Morphologically intermediate individuals have commonly been observed in Mexican Bursera in areas where closely related species coexist. These individuals are assumed to result from interspecific hybridization, but no molecular evidence has supported their hybrid origins. This study aimed to investigate the existence of interspecific hybridization among three Mexican Bursera species (Bullockia section: B. cuneata, B. palmeri and B. bipinnata) from nine populations based on DNA sequences (three nuclear and four chloroplast regions) and ecological niche modeling for three past and two future scenario projections. Results from the only two polymorphic nuclear regions (PEPC, ETS) supported the hybrid origin of morphologically intermediate individuals and revealed that B. cuneata and B. bipinnata are the parental species that are genetically closer to the putative hybrids. Ecological niche modeling accurately predicted the occurrence of putative hybrid populations and showed a potential hybrid zone extending in a larger area (74,000 km2) than previously thought. Paleo-reconstructions showed a potential hybrid zone existing from the Last Glacial Maximum (~ 21 kya) that has increased since the late Holocene to the present. Future ecological niche projections show an increment of suitability of the potential hybrid zone for 2050 and 2070 relative to the present. Hybrid zone changes responded mostly to an increase in elevational ranges. Our study provides the first insight of an extensive hybrid zone among three Mexican Bursera species based on molecular data and ecological niche modeling.

Influence of Pleistocene climatic oscillations on the phylogeography and demographic history of endemic vulnerable trees (section Magnolia) of the Tropical Montane Cloud Forest in Mexico.

The Tropical Montane Cloud Forest (TMCF) is a highly dynamic ecosystem that has undergone frequent spatial changes in response to the interglacial-glacial cycles of the Pleistocene. These climatic fluctuations between cold and warm cycles have led to species range shifts and contractions-expansions, resulting in complex patterns of genetic structure and lineage divergence in forest tree species. In this study, we sequenced four regions of the chloroplast DNA (trnT-trnL, trnK5-matk, rpl32-trnL, trnS-trnG) for 20 populations and 96 individuals to evaluate the phylogeography, historical demography, and paleodistributions of vulnerable endemic TMCF trees in Mexico: Magnolia pedrazae (north-region), M. schiedeana (central-region), and M. schiedeana population Oaxaca (south-region). Our data recovered 49 haplotypes that showed a significant phylogeographic structure in three regions: north, central, and south. Bayesian Phylogeographic and Ecological Clustering (BPEC) analysis also supported the divergence in three lineages and highlighted the role of environmental factors (temperature and precipitation) in genetic differentiation. Our historical demography analyses revealed demographic expansions predating the Last Interglacial (LIG, ~125,000 years ago), while Approximate Bayesian Computation (ABC) simulations equally supported two contrasting demographic scenarios. The BPEC and haplotype network analyses suggested that ancestral haplotypes were geographically found in central Veracruz. Our paleodistributions modeling showed evidence of range shifts and expansions-contractions from the LIG to the present, which suggested the complex evolutionary dynamics associated to the climatic oscillations of the Pleistocene. Habitat management of remnant forest fragments where large and genetically diverse populations occur in the three TMCF regions analyzed would be key for the conservation of these magnolia populations.

A comprehensive landscape genomics approach for seed sourcing strategies in landscapes under varying degrees of habitat disturbance.

As most ecosystems around the world are threatened by anthropogenic degradation and climate change, there is an increasing urgency to implement restoration strategies aiming at ensuring ecosystem self-sustainability and resilience. An initial step towards that goal relies on selecting the most suitable seed sources for a successful revegetation, which can be extremely challenging in highly degraded landscapes. The most common seed sourcing strategy is to select local seeds because it is assumed that plants experience strong adaptations to their natal sites. An alternative strategy is the selection of climate-adapted genotypes to future conditions. While considering future climatic projections is important to account for spatial shifts in climate to inform assisted gene flow and translocations, to restore highly degraded landscapes we need a comprehensive approach that first accounts for species adaptations to current at-site environmental conditions. In this issue of Molecular Ecology Resources, Carvalho et al. present a novel landscape genomics framework to identify the most appropriate seed sourcing strategy for moderately and highly degraded sites by integrating genotype, phenotype and environmental data in a spatially explicit context for two native plant species with potential to help restore iron-rich Amazonian savannas. This framework is amenable to be applicable and adapted to a broad range of restoration initiatives, as the dichotomy between focusing on the current or future climatic conditions should depend on the goals and environmental circumstances of each restoration site.

Morphotype divergence and genetic diversity of Hedeoma piperita Benth. in western Mexico.

Hedeoma piperita Benth. is a perennial herb from the Lamiaceae family, which is highly valued for its medicinal and culinary properties by the Purépecha ethnic group in Michoacán, Mexico. The species presents populations of two morphotypes (white and purple corollas) that have not been formally studied. In this work, we aimed to evaluate the morphological and genetic variation between the two morphotypes. We sampled individuals from 15 populations within the Purépecha Plateau in western Mexico to measure 33 quantitative and qualitative morphological variable characters (vegetative and reproductive) and to estimate genetic diversity and structure using six nuclear microsatellite markers. Principal Component Analysis showed a clear separation between populations of the two morphotypes, which differences were statistically significant for all vegetative (n = 11) and reproductive (n = 22) characters. Similarly, Bayesian and multivariate cluster analyses based on the microsatellite data supported the distinction of the two morphotypes, except for one population of the white corolla that was genetically closer to the purple corolla group. Genetic diversity was moderate to low across populations of the two morphotypes, and inbreeding (FIS) was significantly higher in populations of the purple corolla. Our morphological and genetic data support the presence of two divergent morphotypes in H. piperita. This species is of high importance within the Purépecha culture, but unfortunately is declining in the region due to its high extraction rates. Thus, our results are valuable to delineate germplasm zones for future breeding programs and for informing in situ conservation strategies.

Everything you always wanted to know about gene flow in tropical landscapes (but were afraid to ask).

The bulk of the world's biodiversity is found in tropical regions, which are increasingly threatened by the human-led degradation of natural habitats. Yet, little is known about tropical biodiversity responses to habitat loss and fragmentation. Here we review all available literature assessing landscape effects on gene flow in tropical species, aiming to help unravel the factors underpinning functional connectivity in the tropics. We map and classify studies by focus species, the molecular markers employed, statistical approaches to assess landscape effects on gene flow, and the evaluated landscape and environmental variables. We then compare qualitatively and quantitatively landscape effects on gene flow across species and units of analysis. We found 69 articles assessing landscape effects on gene flow in tropical organisms, most of which were published in the last five years, were concentrated in the Americas, and focused on amphibians or mammals. Most studies employed population-level approaches, microsatellites were the preferred type of markers, and Mantel and partial Mantel tests the most common statistical approaches used. While elevation, land cover and forest cover were the most common gene flow predictors assessed, habitat suitability was found to be a common predictor of gene flow. A third of all surveyed studies explicitly assessed the effect of habitat degradation, but only 14 of these detected a reduced gene flow with increasing habitat loss. Elevation was responsible for most significant microsatellite-based isolation by resistance effects and a single study reported significant isolation by non-forested areas in an ant. Our study reveals important knowledge gaps on the study of landscape effects on gene flow in tropical organisms, and provides useful guidelines on how to fill them.

Species delimitation and genetic structure of two endemic Magnolia species (section Magnolia; Magnoliaceae) in Mexico.

Magnolias are characteristic tree species of the Tropical Montane Cloud Forest (TMCF) in Mexico, an ecosystem that is highly threatened by habitat fragmentation and climate change. In this study, based on DNA sequences from five regions (chloroplast: trnT-trnL, trnK5-matK, trnS-trnG, rpl32-trnL, nuclear: ITS) and seven nuclear microsatellite markers, we aimed to delineate species boundaries between two-endemic species of the TMCF, Magnolia pedrazae and Magnolia schiedeana, and to estimate levels of genetic structure and diversity among populations. Phylogenetic and haplotype network analyses for the chloroplast and ITS regions did not support genetic differentiation as two distinctive species. Results from Bayesian and multivariate cluster analyses based on microsatellite loci showed high genetic differentiation across most populations, which was consistent with a strong and significant pattern of isolation by geographical distance. We found moderate to high levels of population genetic diversity, but it was lower in small populations relative to large populations. Our results suggest a contemporary decrease of genetic connectivity among populations, likely as a consequence of the current decline of suitable TMCF habitat. Managing landscape connectivity among remnant Magnolia populations within protected natural parks and surroundings, and with emphasis of small populations, would be key for the species conservation.

Landscape limits gene flow and drives population structure in Agassiz's desert tortoise (Gopherus agassizii).

Distance, environmental heterogeneity and local adaptation can strongly influence population structure and connectivity. Understanding how these factors shape the genomic landscape of threatened species is a major goal in conservation genomics and wildlife management. Herein, we use thousands (6,859) of single nucleotide polymorphism markers and spatial data from hundreds of individuals (n = 646) to re-evaluate the population structure of Agassiz's desert tortoise (Gopherus agassizii). Analyses resolve from 4 to 8 spatially well-defined clusters across the range. Western, central, and southern populations within the Western Mojave recovery unit are consistent throughout, while analyses sometimes merge other recovery units depending on the level of clustering. Causal modeling consistently associates genetic connectivity with least-cost distance, based on multiple landscape features associated with tortoise habitat, better than geographic distance. Some features include elevation, soil depth, rock volume, precipitation, and vegetation coverage, suggesting that physical, climatic, and biotic landscape features have played a strong evolutionary role restricting gene flow between populations. Further, 12 highly differentiated outlier loci have associated functions that may be involved with neurogenesis, wound healing, lipid metabolism, and possibly vitellogenesis. Together, these findings have important implications for recovery programs, such as translocations, population augmentation, reproduction in captivity and the identification of ecologically important genes, opening new venues for conservation genomics in desert tortoises.

Development of a genotype-by-sequencing immunogenetic assay as exemplified by screening for variation in red fox with and without endemic rabies exposure.

Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High-throughput sequencing (HTS) technologies, such as genotype-by-sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization-based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to "capture" specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300-kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on-target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer-read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune-related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems.

Spatial patterns of immunogenetic and neutral variation underscore the conservation value of small, isolated American badger populations.

Small and isolated populations often exhibit low genetic diversity due to drift and inbreeding, but may simultaneously harbour adaptive variation. We investigate spatial distributions of immunogenetic variation in American badger subspecies (Taxidea taxus), as a proxy for evaluating their evolutionary potential across the northern extent of the species' range. We compared genetic structure of 20 microsatellites and the major histocompatibility complex (MHC DRB exon 2) to evaluate whether small, isolated populations show low adaptive polymorphism relative to large and well-connected populations. Our results suggest that gene flow plays a prominent role in shaping MHC polymorphism across large spatial scales, while the interplay between gene flow and selection was stronger towards the northern peripheries. The similarity of MHC alleles within subspecies relative to their neutral genetic differentiation suggests that adaptive divergence among subspecies can be maintained despite ongoing gene flow along subspecies boundaries. Neutral genetic diversity was low in small relative to large populations, but MHC diversity within individuals was high in small populations. Despite reduced neutral genetic variation, small and isolated populations harbour functional variation that likely contribute to the species evolutionary potential at the northern range. Our findings suggest that conservation approaches should focus on managing adaptive variation across the species range rather than protecting subspecies per se.

Lack of Spatial Immunogenetic Structure among Wolverine (Gulo gulo) Populations Suggestive of Broad Scale Balancing Selection.

Elucidating the adaptive genetic potential of wildlife populations to environmental selective pressures is fundamental for species conservation. Genes of the major histocompatibility complex (MHC) are highly polymorphic, and play a key role in the adaptive immune response against pathogens. MHC polymorphism has been linked to balancing selection or heterogeneous selection promoting local adaptation. However, spatial patterns of MHC polymorphism are also influenced by gene flow and drift. Wolverines are highly vagile, inhabiting varied ecoregions that include boreal forest, taiga, tundra, and high alpine ecosystems. Here, we investigated the immunogenetic variation of wolverines in Canada as a surrogate for identifying local adaptation by contrasting the genetic structure at MHC relative to the structure at 11 neutral microsatellites to account for gene flow and drift. Evidence of historical positive selection was detected at MHC using maximum likelihood codon-based methods. Bayesian and multivariate cluster analyses revealed weaker population genetic differentiation at MHC relative to the increasing microsatellite genetic structure towards the eastern wolverine distribution. Mantel correlations of MHC against geographical distances showed no pattern of isolation by distance (IBD: r = -0.03, p = 0.9), whereas for microsatellites we found a relatively strong and significant IBD (r = 0.54, p = 0.01). Moreover, we found a significant correlation between microsatellite allelic richness and the mean number of MHC alleles, but we did not observe low MHC diversity in small populations. Overall these results suggest that MHC polymorphism has been influenced primarily by balancing selection and to a lesser extent by neutral processes such as genetic drift, with no clear evidence for local adaptation. This study contributes to our understanding of how vulnerable populations of wolverines may respond to selective pressures across their range.

Highly polymorphic microsatellite markers in Pulsatilla vulgaris (Ranunculaceae) using next-generation sequencing.

PREMISE OF THE STUDY: We developed novel microsatellite markers for the perennial plant Pulsatilla vulgaris (Ranunculaceae) to investigate the effects of fragmentation on gene flow in this imperiled species. METHODS AND RESULTS: We identified microsatellites and developed primers based on 454 shotgun sequences. We identified 14 markers that were polymorphic and produced clean bands. Of these, eight could be analyzed as diploids. Genotyping of 97 individuals across two populations revealed these markers to be highly polymorphic with seven to 17 alleles per locus and observed heterozygosity from 0.41 to 0.83. CONCLUSIONS: The markers are highly informative and will be used to test if the reintroduction of shepherding in southern Germany improves genetic connectivity among fragmented populations of P. vulgaris. The combination of diploid and tetraploid markers presented here will be useful in resolving the polyploidization history of this and related species.

Spatial patterns of neutral and functional genetic variations reveal patterns of local adaptation in raccoon (Procyon lotor) populations exposed to raccoon rabies.

Local adaptation is necessary for population survival and depends on the interplay between responses to selective forces and demographic processes that introduce or retain adaptive and maladaptive attributes. Host-parasite systems are dynamic, varying in space and time, where both host and parasites must adapt to their ever-changing environment in order to survive. We investigated patterns of local adaptation in raccoon populations with varying temporal exposure to the raccoon rabies virus (RRV). RRV infects approximately 85% of the population when epizootic and has been presumed to be completely lethal once contracted; however, disease challenge experiments and varying spatial patterns of RRV spread suggest some level of immunity may exist. We first assessed patterns of local adaptation in raccoon populations along the eastern seaboard of North America by contrasting spatial patterns of neutral (microsatellite loci) and functional, major histocompatibility complex (MHC) genetic diversity and structure. We explored variation of MHC allele frequencies in the light of temporal population exposure to RRV (0-60 years) and specific RRV strains in infected raccoons. Our results revealed high levels of MHC variation (66 DRB exon 2 alleles) and pronounced genetic structure relative to neutral microsatellite loci, indicative of local adaptation. We found a positive association linking MHC genetic diversity and temporal RRV exposure, but no association with susceptibility and resistance to RRV strains. These results have implications for landscape epidemiology studies seeking to predict the spread of RRV and present an example of how population demographics influence the degree to which populations adapt to local selective pressures.

Directed dispersal by rotational shepherding supports landscape genetic connectivity in a calcareous grassland plant.

Directed dispersal by animal vectors has been found to have large effects on the structure and dynamics of plant populations adapted to frugivory. Yet, empirical data are lacking on the potential of directed dispersal by rotational grazing of domestic animals to mediate gene flow across the landscape. Here, we investigated the potential effect of large-flock shepherding on landscape-scale genetic structure in the calcareous grassland plant Dianthus carthusianorum, whose seeds lack morphological adaptations to dispersal to animals or wind. We found a significant pattern of genetic structure differentiating population within grazed patches of three nonoverlapping shepherding systems and populations of ungrazed patches. Among ungrazed patches, we found a strong and significant effect of isolation by distance (r = 0.56). In contrast, genetic distance between grazed patches within the same herding system was unrelated to geographical distance but significantly related to distance along shepherding routes (r = 0.44). This latter effect of connectivity along shepherding routes suggests that gene flow is spatially restricted occurring mostly between adjacent populations. While this study used nuclear markers that integrate gene flow by pollen and seed, the significant difference in the genetic structure between ungrazed patches and patches connected by large-flock shepherding indicates the potential of directed seed dispersal by sheep across the landscape.

Effect of rotational shepherding on demographic and genetic connectivity of calcareous grassland plants.

Response to habitat fragmentation may not be generalized among species, in particular for plant communities with a variety of dispersal traits. Calcareous grasslands are one of the most species-rich habitats in Central Europe, but abandonment of traditional management has caused a dramatic decline of calcareous grassland species. In the Southern Franconian Alb in Germany, reintroduction of rotational shepherding in previously abandoned grasslands has restored species diversity, and it has been suggested that sheep support seed dispersal among grasslands. We tested the effect of rotational shepherding on demographic and genetic connectivity of calcareous grassland specialist plants and whether the response of plant populations to shepherding was limited to species dispersed by animals (zoochory). Specifically, we tested competing dispersal models and source and focal patch properties to explain landscape connectivity with patch-occupancy data of 31 species. We fitted the same connectivity models to patch occupancy and nuclear microsatellite data for the herb Dianthus carthusianorum (Carthusian pink). For 27 species, patch connectivity was explained by dispersal by rotational shepherding regardless of adaptations to zoochory, whereas population size (16% species) and patch area (0% species) of source patches were not important predictors of patch occupancy in most species. [Correction made after online publication, February 25, 2014: Population size and patch area percentages were mistakenly inverted, and have now been fixed.] Microsite diversity of focal patches significantly increased the model variance explained by patch occupancy in 90% of the species. For D. carthusianorum, patch connectivity through rotational shepherding explained both patch occupancy and population genetic diversity. Our results suggest shepherding provides dispersal for multiple plant species regardless of their dispersal adaptations and thus offers a useful approach to restore plant diversity in fragmented calcareous grasslands.