Spatial isolation and genetic differentiation in naturally fragmented plant populations of the Swiss Alps

Aims The effect of anthropogenic landscape fragmentation on the genetic diversity and adaptive potential of plant populations is a major issue in conservation biology. However, little is known about the partitioning of genetic diversity in alpine species, which occur in naturally fragmented habitats. Here, we investigate molecular patterns of three alpine plants (Epilobium fleischeri, Geum reptans and Campanula thyrsoides) across Switzerland and ask whether spatial isolation has led to high levels of population differentiation, increasing over distance, and a decrease of within-population variability. We further hypothesize that the contrasting potential for long-distance dispersal (LDD) of seed in these species will considerably influence and explain diversity partitioning.Methods For each study species, we sampled 20–23 individuals from each of 20–32 populations across entire Switzerland. We applied Random Amplified Polymorphic Dimorphism markers to assess genetic diversity within (Nei's expected heterozygosity, H e; percentage of polymorphic bands, P p) and among (analysis of molecular variance, Φ st) populations and correlated population size and altitude with within-population diversity. Spatial patterns of genetic relatedness were investigated using Mantel tests and standardized major axis regression as well as unweighted pair group method with arithmetic mean cluster analyses and Monmonier's algorithm. To avoid known biases, we standardized the numbers of populations, individuals and markers using multiple random reductions. We modelled LDD with a high alpine wind data set using the terminal velocity and height of seed release as key parameters. Additionally, we assessed a number of important life-history traits and factors that potentially influence genetic diversity partitioning (e.g. breeding system, longevity and population size).Important findings For all three species, we found a significant isolation-by-distance relationship but only a moderately high differentiation among populations (Φ st : 22.7, 14.8 and 16.8%, for E. fleischeri, G. reptans and C. thyrsoides, respectively). Within-population diversity (H e : 0.19–0.21, P p : 62–75%) was not reduced in comparison to known results from lowland species and even small populations with <50 reproductive individuals contained high levels of genetic diversity. We further found no indication that a high long-distance seed dispersal potential enhances genetic connectivity among populations. Gene flow seems to have a strong stochastic component causing large dissimilarity between population pairs irrespective of the spatial distance. Our results suggest that other life-history traits, especially the breeding system, may play an important role in genetic diversity partitioning. We conclude that spatial isolation in the alpine environment has a strong influence on population relatedness but that a number of factors can considerably influence the strength of this relationship.     

Niche shifts and range expansions along cordilleras drove diversification in a high‐elevation endemic plant genus in the tropical Andes

The tropical Andes represent one of the world's biodiversity hot spots, but the evolutionary drivers generating their striking species diversity still remain poorly understood. In the treeless high‐elevation Andean environments, Pleistocene glacial oscillations and niche differentiation are frequently hypothesized diversification mechanisms; however, sufficiently densely sampled population genetic data supporting this are still lacking. Here, we reconstruct the evolutionary history of Loricaria (Asteraceae), a plant genus endemic to the Andean treeless alpine zone, based on comprehensive population‐level sampling of 289 individuals from 67 populations across the entire distribution ranges of its northern Andean species. Partly incongruent AFLP and plastid DNA markers reveal that the distinct genetic structure was shaped by a complex interplay of biogeography (spread along and across the cordilleras), history (Pleistocene glacial oscillations) and local ecological conditions. While plastid variation documents an early split or colonization of the northern Andes by at least two lineages, one of which further diversified, a major split in the AFLP data correlate with altitudinal ecological differentiation. This suggests that niche shifts may be important drivers of Andean diversification not only in forest–alpine transitions, but also within the treeless alpine zone itself. The patterns of genetic differentiation at the intraspecific level reject the hypothesized separation in spatially isolated cordilleras and instead suggest extensive gene flow among populations from distinct mountain chains. Our study highlights that leveraging highly variable markers against extensive population‐level sampling is a promising approach to address mechanisms of rapid species diversifications.     

Population genetic diversity of the clonal plant Geum reptans (Rosaceae) in the Swiss Alps

In the alpine landscape most plant populations are spatially isolated due to extreme patchiness and strong natural fragmentation. We used RAPD-PCR (randomly amplified polymorphic DNA polymerase chain reaction) for a study of the genetic diversity within and among 20 populations of Geum reptans, an outcrossing clonal plant species in the Swiss Alps. Populations were sampled at different altitudes, in early-, medium- and late-successional habitats (population origin) using a spatially hierarchical design, with distances among populations ranging from 0.2 to 208 km. Seed and pollen dispersibility was estimated by direct measurements. Seed dispersibility by wind was low with only 0.015% of the seeds flying over 100 m. Observed pollen flow was even more restricted. Molecular diversity within populations was irrespective of population origin (H(e) = 0.22 ± 0.004) and similar to the average of other RAPD studies. Contrary to our expectation, populations were only moderately differentiated (G(st) = 0.14). However, there was a clear spatial genetic structure and a positive relationship between pairwise genetic and geographic distances. Our results indicate considerable gene flow among populations within the same regional area, and we found no indication for genetic depletion during succession or in peripheral habitats. We conclude that, despite the high natural fragmentation and the importance of vegetative reproduction in this alpine plant, gene flow and repeated seedling recruitment during succession might be more frequent than commonly suggested.     

Alpine species seed longevity in the soil in relation to seed size and shape – A 5-year burial experiment in the Central Alps

Long-term persistent seeds are generally expected to be small and spherically shaped. In contrast, transient seeds are often large, flattened and elongated. We hypothesized that alpine species follow these trends, and that most species of unstable alpine habitats have the potential to form long-term persistent seed banks. The latter would enable them to delay germination until favourable conditions are present in their environment thereby increasing the likelihood for establishment of seedlings. We selected nine alpine species of more or less unstable habitats (i.e. Achillea moschata, Artemisia genipi, Anthyllis vulneraria ssp. alpicola, Geum reptans, Linaria alpina, Oxyria digyna, Saxifraga aizoides, Saxifraga oppositifolia, and Trifolium pallescens) for this study. Seed longevity was estimated by a 5-year burial experiment in the field with seed excavations after 1 winter, 2 years and 5 years. Germination experiments in the growth chamber and viability tests were performed after each excavation. We detected longevity patterns ranging from transient to long-term persistent. Two species were classified as transient. All other species have the potential to form persistent seed banks, with almost half of the investigated species showing a trend for long-term persistence. Despite contradictory reports, long-term persistent seed banks are an important life history trait of species of unstable alpine habitats. We found that seed shape and hard-seededness are good predictors of seed persistence of alpine species. Seed size seems to be less important. The data from this study support the general hypotheses describing the relationship among seed size, seed shape and seed persistence in the soil.     

Phenotypic and genetic diversity in aposematic Malagasy poison frogs (genus Mantella)

Intraspecific color variation has long fascinated evolutionary biologists. In species with bright warning coloration, phenotypic diversity is particularly compelling because many factors, including natural and sexual selection, contribute to intraspecific variation. To better understand the causes of dramatic phenotypic variation in Malagasy poison frogs, we quantified genetic structure and color and pattern variation across three closely related species, Mantella aurantiaca, Mantella crocea, and Mantella milotympanum. Although our restriction site‐associated DNA (RAD) sequencing approach identified clear genetic clusters, they do not align with current species designations, which has important conservation implications for these imperiled frogs. Moreover, our results suggest that levels of intraspecific color variation within this group have been overestimated, while species diversity has been underestimated. Within major genetic clusters, we observed distinct patterns of variation including: populations that are phenotypically similar yet genetically distinct, populations where phenotypic and genetic breaks coincide, and populations that are genetically similar but have high levels of within‐population phenotypic variation. We also detected admixture between two of the major genetic clusters. Our study suggests that several mechanisms—including hybridization, selection, and drift—are contributing to phenotypic diversity. Ultimately, our work underscores the need for a reevaluation of how polymorphic and polytypic populations and species are classified, especially in aposematic organisms.     

Faunal breaks and species composition of Indo-Pacific corals: the role of plate tectonics,environment and habitat distribution

Species richness gradients are ubiquitous in nature, but the mechanisms that generate and maintain these patterns at macroecological scales remain unresolved. We use a new approach that focuses on overlapping geographical ranges of species to reveal that Indo-Pacific corals are assembled within 11 distinct faunal provinces. Province limits are characterized by co-occurrence of multiple species range boundaries. Unexpectedly, these faunal breaks are poorly predicted by contemporary environmental conditions and the present-day distribution of habitat. Instead, faunal breaks show striking concordance with geological features (tectonic plates and mantle plume tracks). The depth range over which a species occurs, its larval development rate and genus age are important determinants of the likelihood that species will straddle faunal breaks. Our findings indicate that historical processes, habitat heterogeneity and species colonization ability account for more of the present-day biogeographical patterns of corals than explanations based on the contemporary distribution of reefs or environmental conditions.     

Population structure of California coyotes corresponds to habitat-specific breaks and illuminates species history

Little is known about the relationship between animal movements and the emergent structure of populations, especially for species occupying large continuous distributions. Some such mammals disperse disproportionately into habitat similar to their natal habitat, a behavioural bias that might be expected to lead to habitat-conforming genetic structure. We hypothesized that coyotes (Canis latrans) would exhibit such natal-biased dispersal, and used 13 microsatellite loci to test, correspondingly, whether genetic structure conformed to major habitat breaks. First, we used a model-based approach to assign coyote genotypes to distinct genetic clusters irrespective of geographical location. Visualization on a geographical information system revealed a strong concordance between the locations of cluster assignments and habitat bioregions, not explainable in terms of physical dispersal barriers or intervening low-quality habitat. Next, we used a multiple Mantel test, which controlled for effects of geographical distance (itself, marginally significant; P = 0.06), to statistically determine that genetic distance was indeed higher between than within bioregions (P < 0.001). Whereas previously published examples of landscape effects on gene flow have typically been explainable in terms of species-wide habitat affinities or dispersal barriers, our finding that genetic subdivisions were associated with unobstructed boundaries between contiguous habitats suggests a role for intraspecific variability in habitat affinities as a factor underlying genetic structure. In addition, our data combined with previously published data suggest a pattern of genetic isolation-by-distance throughout western North America, consistent with independent evidence that the western half of the coyote range predates European settlement.     

Comparative phylogeography in a genus of coral reef fishes: biogeographic and genetic concordance in the Caribbean

Geographic barriers that limit the movement of individuals between populations may create or maintain phylogenetically discrete lineages. Such barriers are often inferred from geographic surveys of a single mitochondrial marker to identify phylogenetic splits. Mitochondrial DNA, however, has an effective population size one-fourth that of nuclear DNA, which can facilitate the rapid evolution of monophyletic mtDNA lineages in the absence of geographic barriers. The identification of geographic barriers will thus be more robust if barriers are proposed a priori, and tested with multiple independent genetic markers in multiple species. Here, we tested two proposed marine biogeographic breaks located at the Mona Passage in the Caribbean Sea and at the southern end of Exuma Sound in the Bahamas. We sequenced mitochondrial cytochrome b (400 bp) and nuclear rag1 (573 bp) for nine species and colour forms (183 individuals total) within the teleost genus Elacatinus (Gobiidae) that span the proposed breaks. Our results showed that Mona Passage separated mtcyb and rag1 lineages, with no genetic exchange between populations separated by just 23 km. However, the Central Bahamas barrier was only weakly supported by our data. Importantly, neither barrier coincided with deep genetic splits. This suggests that these two barriers did not initially isolate regional populations, but instead disrupt ongoing gene flow between regions. Our inferred relationships further suggested a division of the Caribbean region into northwestern and southeastern regions, a pattern reflected by some freshwater and terrestrial vertebrates. Our results, coupled with genetic and demographic data from other reef fishes and corals, provide robust support for the Mona Passage as a long-term biogeographic barrier for Caribbean animals.     

Phylogeny,phylogeography and geographical variation in the Crocidura monax (Soricidae) species complex from the montane islands of Tanzania,with descriptions of three new species

We assess morphological and multilocus genetic variation among 11 isolated montane populations of white‐toothed shrews from Tanzania that have been referred to either Crocidura monax Thomas or C. montis Thomas. The montane sites we sampled represent ‘sky‐islands’ from two geologically distinct archipelagos (Northern Highlands and the Eastern Arc Mountains) and are a significant component of the Eastern Afromontane Biodiversity Hotspot. We used multivariate analyses of morphometric traits and phylogenetic and species‐delimitation analyses of multilocus DNA sequence data to assess species‐level diversity. Our species delimitation analyses included a novel, pairwise validation approach that avoids potential biases associated with specifying a guide tree. These analyses reveal several distinct lineages, which we treat as six allopatric species. Each species is restricted to one, two or four mountains. We use available names to recognize C. monax, C. tansaniana Hutterer and C. usambarae Dippenaar, while naming and describing three new species. Our results demonstrate the effectiveness of combining morphological and genetic data to uncover and describe hidden diversity in a cryptic mammalian system. © 2015 The Linnean Society of London     

Evolutionary diversification of cryophilic <Emphasis Type="Italic">Grylloblatta</Emphasis>species (Grylloblattodea: Grylloblattidae) in alpine habitats of California

Background  

Climate in alpine habitats has undergone extreme variation during Pliocene and Pleistocene epochs, resulting in repeated expansion and contraction of alpine glaciers. Many cold-adapted alpine species have responded to these climatic changes with long-distance range shifts. These species typically exhibit shallow genetic differentiation over a large geographical area. In contrast, poorly dispersing organisms often form species complexes within mountain ranges, such as the California endemic ice-crawlers (Grylloblattodea: Grylloblattidae: Grylloblatta). The diversification pattern of poorly dispersing species might provide more information on the localized effects of historical climate change, the importance of particular climatic events, as well as the history of dispersal. Here we use multi-locus genetic data to examine the phylogenetic relationships and geographic pattern of diversification in California Grylloblatta.     

Understanding population history for conservation purposes: population genetics of Saxifraga aizoides (Saxifragaceae) in the lowlands and lower mountains north of the Alps

Several alpine species have outlying populations in the lowlands and lower mountains north of the Alps. These small, isolated populations are usually described as either (1) glacial relics, (2) descendants from populations living on forelands and moraines during the ice ages, or (3) populations founded by long-distance dispersal after glaciation. A floristic survey of the historic and present distributions and an allozyme investigation were performed on one of these relic species, Saxifraga aizoides. The species was historically more abundant and had more stations in more regions of northeastern Switzerland. The former population structures within regions, nowadays destroyed, were still reflected in distinct and high regional genetic diversity and variation. There was weak evidence of increased inbreeding in outlying populations, but populations did not deviate from Hardy-Weinberg equilibrium. No geographic pattern of genetic variation above the regional scale (>10 km) was found. Based on the spatial and genetic structures found, it was not possible to discriminate between the abovementioned hypotheses. Nevertheless, the study shows how a thorough evaluation of distribution and abundance data aids the interpretation of genetic data with respect to population history, biogeography, and conservation biology.     

Central Asian origin of and strong genetic differentiation among populations of the rare and disjunct Carex atrofusca (Cyperaceae) in the Alps

Aim Carex atrofusca has an arctic–alpine distribution in the Northern Hemisphere, with only a few, disjunct localities known in the European Alps. These alpine populations are declining in number and size. In contrast, C. atrofusca has a wide circumpolar distribution range and is abundant in large parts of the Arctic. The degree of genetic differentiation of the alpine populations and their importance for the conservation of the intraspecific genetic variation of the species is unknown. Location Eurasia and Greenland, with emphasis on the European Alps. Methods We applied amplified fragment length polymorphism (AFLP) fingerprinting and sequences of chloroplast DNA to determine the position of the alpine populations in a circumpolar phylogeography of C. atrofusca and to unravel the patterns of genetic diversity and differentiation within the Alps. Results Two distinct major groups were detected in a neighbour‐joining analysis of AFLP data and in parsimony analysis of chloroplast DNA sequences: one consisting of the populations from Siberia and Greenland and one consisting of all European populations as sister to the populations from Central Asia. Within Europe, the populations from the Tatra Mountains and those from Scotland and Scandinavia formed two well‐supported groups, whereas the alpine populations did not constitute a group of their own. The genetic variation in the Alps was almost completely partitioned among the populations, and the populations were almost invariable. Main conclusions The alpine populations possibly originated due to immigration from Central Asia. The strong differentiation among them suggests that genetic drift has been strongly acting on the populations, either as a consequence of founder events during colonization or due to subsequent reduction of population sizes during warm stages of the Holocene.     

Maintaining their genetic distance: Little evidence for introgression between widely hybridizing species of Geum with contrasting mating systems

Within the plant kingdom, many genera contain sister lineages with contrasting outcrossing and inbreeding mating systems that are known to hybridize. The evolutionary fate of these sister lineages is likely to be influenced by the extent to which they exchange genes. We measured gene flow between outcrossing Geum rivale and selfing Geum urbanum, sister species that hybridize in contemporary populations. We generated and used a draft genome of G. urbanum to develop dd‐RAD data scorable in both species. Coalescent analysis of RAD data from allopatric populations indicated that the species diverged 2–3 Mya, and that historical gene flow between them was extremely low (1 migrant every 25 generations). Comparison of genetic divergence between species in sympatry and allopatry, together with an analysis of allele frequencies in potential parental and hybrid populations, provided no evidence of contemporary introgression in sympatric populations. Cluster‐ and species‐specific marker analyses revealed that, apart from four early‐generation hybrids, individuals in sympatric populations fell into two genetically distinct groups that corresponded exactly to their morphological species classification with maximum individual admixture estimates of only 1–3%. However, we did observe joint segregation of four putatively introgressed SNPs across two scaffolds in the G. urbanum population that was associated with significant morphological variation, interpreted as tentative evidence for rare, recent interspecific gene flow. Overall, our results indicate that despite the presence of hybrids in contemporary populations, genetic exchange between G. rivale and G. urbanum has been extremely limited throughout their evolutionary history.     

Biogeography of the livebearing fish Poecilia gillii in Costa Rica: are phylogeographical breaks congruent with fish community boundaries?

One of the original goals of phylogeography was to use genetic data to identify historical events that might contribute to breaks among communities. In this study, we examine the phylogeography of a common livebearing fish ( Poecilia gillii ) from Costa Rica. Our goal was to determine if phylogeographical breaks in this species were congruent with previously defined boundaries among four fish community provinces. We hypothesized that if abiotic factors influence both community boundaries and genetic structuring in P. gillii then we might find four clades within our focal species that were geographically separated along community boundary lines. Similarly, we expected to find most of the genetic variation in P. gillii partitioned among these four geographical regions. We generated DNA sequence data (mitochondrial cytochrome b and nuclear S7 small ribosomal subunit) for 260 individuals from 42 populations distributed across Costa Rica. We analysed these data using phylogenetic (parsimony and likelihood) and coalescent approaches to estimate phylogenetic relationships among haplotypes, patterns of gene flow and effective population size . Contrary to our expectations, we did not find four monophyletic groups that mapped cleanly to our geographical community provinces. However, one of our clades was restricted to a single province, suggesting that common earth history events could be responsible for both genetic structuring in P. gillii and fish community composition in this area. However, our results show a complex pattern of gene flow throughout other regions in Costa Rica where genetic structuring is not predicted by community province boundaries.     

Phylogeography of Iris missouriensis (Iridaceae) based on nuclear and chloroplast markers

We investigated the phylogeography of Iris missouriensis (Iridaceae), which is widely distributed in western North America. We utilized transposon display and DNA sequencing to quantify nuclear and chloroplast genetic structure. Our objectives were (i) to characterize the geographic structure of genetic variation throughout the species range, (ii) to test whether both margins of the range show reduced genetic diversity as predicted by north-south expansion and contraction associated with climate change, and (iii) to determine whether the subspecies Iris missouriensis ssp. longipetala is genetically distinct. We found that genetic diversity was significantly lower in the northern part of the range but was not significantly different between the central and southern regions, indicating greater stability of the southern margin vs. the northern. Among-population differentiation was high (PhiPT=0.52). The largest divisions in each marker set were concordant and separated the southern Rocky Mountains and Basin and Range provinces from the remainder of the range. The boundaries of this phylogeographic break do not coincide with gaps in present-day distributions or phylogeographic breaks identified in other species, and may indicate a measure of reproductive isolation. Consistent with current treatments, we did not find support for the taxonomic placement of I. missourienis ssp. longipetala as a distinct species. Although transposon display has been used to investigate relationships among crop accessions and their wild relatives, to our knowledge, this is the first use of these markers for population-level phylogeography of a nonmodel species and further demonstrates their utility in species recalcitrant to amplified fragment length polymorphism protocols.     

Staying in situ or shifting range under ongoing climate change: A case of an endemic herb in the Himalaya-Hengduan Mountains across elevational gradients

Aim

How species respond to ongoing climate change has been a hot research topic, especially with the controversy in shifting range (movement) or persisting in local habitat (in situ) as the primary response. Assessing the relative roles of range shifts, phenotypic plasticity and genetic adaptation helps us predict the evolutionary fate of species. We aim to explore the evolutionary strategies of plants under climate change from a keystone herb in alpine ecosystems, Mirabilis himalaica, along its elevational gradient.

Location

Himalaya-Hengduan Mountains, China.

Methods

We combined evidence from population genomics and ecological data in both space and time to investigate the state of “staying” or “moving”. We identified migration events by assessing historical and contemporary gene flow and changes in species distribution. Morphological variation was compared by measuring five traits using specimen data. Moreover, we explored climate-driven genetic variation and local selection regimes acting on populations in the alpine landscape along an elevational gradient.

Results

Our results argue that staying in situ by morphological variation and local genetic evolution rather than range shifting plays an important role in M. himalaica response to climate change. We first found trace evidence of upward or climatic-driven shifting along an elevational gradient, although asymmetric gene flow was restricted within microenvironments of mid-elevational populations. Furthermore, morphological variation comparisons revealed clinal variation, as resource allocation showed a declining pattern in vegetative growth but increased reproductive growth with increasing elevation. Outlier tests and environment association analyses indicated adaptative loci primarily related to thermal-driven selection and continuous adaptations to high elevation in the Himalaya-Hengduan Mountains.

Main Conclusions

Our findings show M. himalaica may persist in local habitats rather than shifting range under climate change, exhibiting a low risk of genomic vulnerability in current habitats. This study has important implications in improving our understanding of the evolutionary response in alpine plants to climate change.     

The consequences of mating over a range of parental genetic similarity in a selfing allopolyploid plant species

In diploids, F(1) offspring performance is expected to increase with increasing genetic dissimilarity between the parents until an optimum is reached because outbreeding mitigates inbreeding depression and maximizes heterosis. However, many flowering plant species are derived through allopolyploidization, i.e. interspecific hybridization with genome doubling. This mode of plant speciation can be expected to considerably alter the consequences of inbreeding and outbreeding. We investigated the F1 fitness consequences of mating over a range of (genetic) distances in the allohexaploid plant species Geum urbanum. Offspring was raised under controlled conditions (632 plants). The performance of outcrossed progeny was not significantly better than that of their selfed half-siblings and did not increase with parental genetic dissimilarity (0-0.83). Our findings support low, if any, inbreeding depression and heterosis. We attribute this to the peculiar state of quasi-permanent heterozygosity in allopolyploids and frequent selfing.     

A population genetics perspective on the evolutionary histories of three clonal,endemic, and dominant grass species of the Qinghai–Tibet Plateau: Orinus (Poaceae)

We performed analyses of amplified fragment length polymorphism (AFLP) in order to characterize the evolutionary history of Orinus according to its population genetic structure, as well as to investigate putative hybrid origins of O. intermedius and to provide additional insights into relationships among species. The genus Orinus comprises three clonal grasses that are dominant species within xeric alpine grasslands of the Qinghai–Tibet Plateau (QTP). Here, we used eight selectively obtained primer pairs of EcoRI/MseI to perform amplifications in 231 individuals of Orinus representing 48 populations and all three species. We compared our resulting data to genetic models of hybridization using a Bayesian algorithm within NewHybrids software. We determined that genetic variation in Orinus was 56.65% within populations while the among‐species component was 30.04% using standard population genetics statistics. Nevertheless, we detected that species of Orinus were clustered into three highly distinct genetic groups corresponding to classic species identities. Our results suggest that there is some introgression among species. Thus, we tested explicit models of hybridization using a Bayesian approach within NewHybrids software. However, O. intermedius likely derives from a common ancestor with O. kokonoricus and is probably not the result of hybrid speciation between O. kokonoricus and O. thoroldii. We suspect that recent isolation of species of Orinus in allopatry via vicariance may explain the patterns in diversity that we observed, and this is corroborated by a Mantel test that showed significant positive correlation between geographic and genetic distance (r = 0.05, p < 0.05). Recent isolation may explain why Orinus differs from many other clonal species by exhibiting the highest diversity within populations rather than among them.     

Responses of biotic interactions of dominant and subordinate species to decadal warming and simulated rotational grazing in Tibetan alpine meadow

Warming increases competition among plant species in alpine communities by ameliorating harsh environmental conditions, such as low temperatures. Grazing, as the main human activity, may mitigate the effect of warming, as previously reported. However, it is critical to refine the effects of warming on biotic interactions among species, for example, by taking the competitive ability of species into consideration. Based on a 10-year warming and grazing experiment in a Tibetan alpine meadow, we evaluated interspecific biotic interactions of dominant and subordinate species, using the approach of interspecific spatial associations. Warming significantly increased competition between subordinate and dominant species as well as among subordinate species, but not among dominant species. Moreover, facilitation of dominant-subordinate species also increased under warming. Simulated rotational grazing had similar effects to warming, with increasing interspecific competition. Our results show that, when studying the effects of warming on biotic interactions among species, it is necessary to characterize different species pairs relative to their competitive ability, and that simulated rotational grazing does not mitigate the effects of warming in the long term. Our results also provide evidence that the spatial pattern of species is a critical mechanism in species coexistence.