Survival in nunatak and peripheral glacial refugia of three alpine plant species is partly predicted by altitudinal segregation.

Mountain biota survived the Quaternary cold stages most probably in peripheral refugia and/or ice-free peaks within ice-sheets (nunataks). While survival in peripheral refugia has been broadly demonstrated, evidence for nunatak refugia is still scarce. We generated RADseq data from three mountain plant species occurring at different elevations in the southeastern European Alps to investigate the role of different glacial refugia during the Last Glacial Maximum (LGM). We tested the following hypotheses. (i) The deep Piave Valley forms the deepest genetic split in the species distributed across it, delimiting two peripheral refugia. (ii) The montane to alpine species Campanula morettiana and Primula tyrolensis survived the LGM in peripheral refugia, while high-alpine to subnival Saxifraga facchinii likely survived in several nunatak refugia. (iii) The lower elevation species suffered a strong population decline during the LGM. By contrast, the higher elevation species shows long-term stability of population sizes due to survival on permanently ice-free peaks and small population sizes at present. We found peripheral refugia on both sides of the Piave Valley, which acted as a major genetic barrier. Demographic modelling confirmed nunatak survival not only for S. facchinii but also for montane to alpine C. morettiana. Altitudinal segregation influenced the species' demographic fluctuations, with the lower elevation species showing a significant population increase at the end of the LGM, and the higher elevation species either showing decrease towards the present or stable population sizes with a short bottleneck. Our results highlight the role of nunatak survival and species ecology in the demographic history of mountain species.

Dealing with disjunct populations of vascular plants: implications for assessing the effect of climate change.

Species distribution models are the most widely used tool to predict species distributions for species conservation and assessment of climate change impact. However, they usually do not consider intraspecific ecological variation exhibited by many species. Overlooking the potential differentiation among groups of populations may lead to misplacing any conservation actions. This issue may be particularly relevant in species in which few populations with potential local adaptation occur, as in species with disjunct populations. Here, we used ecological niche modeling to analyze how the projections of current and future climatically suitable areas of 12 plant species can be affected using the whole taxa occurrences compared to occurrences from geographically disjunct populations. Niche analyses suggest that usually the disjunct group of populations selects the climatic conditions as similar as possible to the other according to climate availability. Integrating intraspecific variability only slightly increases models' ability to predict species occurrences. However, it results in different predictions of the magnitude of range change. In some species, integrating or not integrating intraspecific variability may lead to opposite trend in projected range change. Our results suggest that integrating intraspecific variability does not strongly improve overall models' accuracy, but it can result in considerably different conclusions about future range change. Consequently, accounting for intraspecific differentiation may enable the detection of potential local adaptations to new climate and so to design targeted conservation strategies.

Integrative Taxonomy of Armeria arenaria (Plumbaginaceae), with a Special Focus on the Putative Subspecies Endemic to the Apennines.

Three subspecies of Armeria arenaria are reported from Italy, two of which are considered endemic to the Apennines. The taxonomic value of these two taxa (A. arenaria subsp. marginata and A. arenaria subsp. apennina) is unclear. Moreover, the relationships between A. arenaria subsp. praecox and Northern Italian populations-currently ascribed to A. arenaria subsp. arenaria-have never been addressed. Accordingly, we used an integrated taxonomic approach, including morphometry, seed morpho-colorimetry, karyology, molecular systematics (psbA-trnH, trnQ-rps16, trnF-trnL, trnL-rpl32, and ITS region), and comparative niche analysis. According to our results, French-Northern Italian populations are clearly distinct from Apennine populations. In the first group, there is evidence which allows the recognition of A. arenaria s.str. (not occurring in Italy) and A. arenaria subsp. praecox. In the second group, the two putative taxa endemic to the Northern Apennines cannot be separated, so a single subspecies is here recognized: A. arenaria subsp. marginata.

Niche Variation in Endemic Lilium pomponium on a Wide Altitudinal Gradient in the Maritime Alps.

The relationship between altitudinal and ecological gradients has long been a dominant theme in plant ecology; moreover, how species respond to climate change has renewed this interest. Mediterranean mountains are often hotspots of endemism, and some endemic species have local distributions that span different climatic belts; hence, local variations in topography and fine-scaled niche conditions may play crucial roles in their persistence along such gradients. Studies of the fine-scaled niche are, however, very rare; most studies involve broad-scale variations in climatic parameters. The Turban lily, Lilium pomponium L. is endemic to the Maritime and Ligurian Alps, where it occurs across a wide altitudinal gradient. Previous work has shown no link between climatic marginality and geographic range limits on morphological traits and genetic variability; however, possible variations of local topographic and ecological parameters have not yet been examined. The objective of this paper is to characterise local ecological niche conditions of L. pomponium populations in the different bioclimatic zones it occupies along the altitudinal gradient. The species occurs in four main types of microecological niches. One of these niche types, with a high mineral cover, is the most abundant­type 2: it was detected in 39% of sampled quadrats and occurs across the whole bioclimatic gradient. Other niche types are more limited to subsections of the gradient: type 3 (in 19% of sampled quadrats) is restricted to high-altitude sites (>1070 m.a.s.l.) and is characterised by high vegetation and litter cover; type 4 (26%) corresponds to more forested habitats on substrates with low water retention capacities, in more inland zones close to the centre of L. pomponium distribution and across a range of altitudes; and type 1 (16% of quadrat) only occurs in the Mediterranean part of the gradient, close to distribution limits in pockets of soil among large blocks of rocks, mainly found at mid-altitudes. Despite heterogeneity in the spatial locations of niche types, there is no correspondence between ecological gradients and the distribution limits of this species. Knowledge of the fine-scaled ecological conditions that determine niche types is thus essential for conservation management of the habitats of this species and for the exploration of its possible response to ongoing climate change.

Topography of the Dolomites modulates range dynamics of narrow endemic plants under climate change.

Climate change is expected to threaten endemic plants in the Alps. In this context, the factors that may modulate species responses are rarely investigated at a local scale. We analyzed eight alpine narrow endemics of the Dolomites (southeastern Alps) under different predicted climate change scenarios at fine spatial resolutions. We tested possible differences in elevation, topographic heterogeneity and velocity of climate change among areas of gained, lost, or stable climatic habitat. The negative impact of climate change ranged from moderate to severe, depending on scenario and species. Generally, range loss occurred at the lowest elevations, while gained and stable areas were located at highest elevations. For six of the species, climate change velocity had higher values in stable and gained areas than in lost ones. Our findings support the role of topographic heterogeneity in maintaining climatic microrefugia, however, the peculiar topography of the Dolomites, characterized by high altitude plateaus, resulted in high climate change velocity in areas of projected future climatic suitability. Our study supports the usefulness of multiple predictors of spatio-temporal range dynamics for regional climate-adapted management and eventual assisted colonization planning to not overlook or overestimate the potential impact of climate change locally.

When ecological marginality is not geographically peripheral: exploring genetic predictions of the centre-periphery hypothesis in the endemic plant Lilium pomponium.

BACKGROUND: Quantifying variation of genetic traits over the geographical range of species is crucial for understanding the factors driving their range dynamics. The center-periphery hypothesis postulates, and many studies support, the idea that genetic diversity decreases and genetic differentiation increases toward the geographical periphery due to population isolation. The effects of environmental marginality on genetic variation has however received much less attention. METHODS: We tested the concordance between geographical and environmental gradients and the genetic predictions of center-periphery hypothesis for endemic Lilium pomponium in the southern Alps. RESULTS: We found little evidence for concordance between genetic variation and both geographical and environmental gradients. Although the prediction of increased differentiation at range limits is met, genetic diversity does not decrease towards the geographical periphery. Increased differentiation among peripheral populations, that are not ecologically marginal, may be explained by a decrease in habitat availability that reduces population connectivity. In contrast, a decrease of genetic diversity along environmental but not geographical gradients may be due to the presence of low quality habitats in the different parts of the range of a species that reduce effective population size or increase environmental constraints. As a result, environmental factors may affect population dynamics irrespective of distance from the geographical center of the range. In such situations of discordance between geographical and environmental gradients, the predictions of decreasing genetic diversity and increasing differentiation toward the geographical periphery may not be respected.

Effects of environmental heterogeneity on phenotypic variation of the endemic plant Lilium pomponium in the Maritime and Ligurian Alps.

Geographical limits of species' distributions are assumed to be coincident with ecological margins, although this assumption might not always be true. Indeed, harsh environments such as Alpine and Mediterranean ecosystems may favour high phenotypic variability among populations, especially those in peripheral sites. Floral traits are often found to be less variable and less affected by environmental heterogeneity than vegetative traits because variation in the former may have negative effects on fitness. For this reason, it is important to quantify variation in floral traits and plant fecundity in study range limits. The objective of the study is to examine phenotypic variation and differences in reproduction in endemic Lilium pomponium in the Maritime and Ligurian Alps in relation to environmental variation across its distribution range. In this species, marginal climatic populations occur both in the peripheral and central geographical locations of the distribution range; hence, geographical and ecological gradients are not concordant. Floral trait variation is related to local environmental conditions with an array of interactions among resource availability, potential pollen limitation and population size that are differentially related to floral traits. Contrary to the general expectation, all central and peripheral populations had similar, moderate seed production with each group limited by different factors acting on different stages of the life-history strategy. Our results are in line with the idea that general expectations are confirmed only when its assumptions are met and that the differences in pollination environment along an environmental gradient may not be the main determinant of the distribution limit.

Range shifts of native and invasive trees exacerbate the impact of climate change on epiphyte distribution: The case of lung lichen and black locust in Italy.

While changing climatic conditions may directly impact species distribution ranges, indirect effects related to altered biotic interactions may exacerbate range shifts. This situation fully applies to epiphytic lichens that are sensitive to climatic factors and strongly depend on substrate occurrence and features for their dispersal and establishment. In this work, we modelled the climatic suitability across Italy under current and future climate of the forest species Lobaria pulmonaria, the lung lichen. Comparatively, we modelled the suitability of its main tree species in Italy, as well as that of the alien tree Robinia pseudoacacia, black locust, whose spread may cause the decline of many forest lichen species. Our results support the view that climate change may cause range shifts of epiphytes by altering the spatial pattern of their climatic suitability (direct effect) and simultaneously causing range shifts of their host-tree species (indirect effect). This phenomenon seems to be emphasized by the invasion of alien trees, as in the case of black locust, that may replace native host tree species. Results indicate that a reduction of the habitat suitability of the lung lichen across Italy should be expected in the face of climate change and that this is coupled with a loss of suitable substrate. This situation seems to be determined by two main processes that act simultaneously: 1) a partial reduction of the spatial overlap between the climatic niche of the lung lichen and that of its host tree species, and 2) the invasion of native woods by black locust. The case of lung lichen and black locust in Italy highlights that epiphytes are prone to both direct and indirect effects of climate change. The invasion of alien trees may have consequences that are still poorly evaluated for epiphytes.

Reproductive biology of the threatened Lilium pomponium (Liliaceae), a species endemic to Maritime and Ligurian Alps.

Pollination ecology and breeding system of Lilium pomponium L. were studied, and their effect on the reproductive outcome was assessed. This species has high conservation interest in Europe, because it is included in Annex V of the EU Habitat Directive and it is one out of the five Lilium species listed in IUCN Global Red List. To achieve our aim, the pollen vectors as well as the effect of bagging, emasculation and artificial pollination on reproductive output were studied. The most frequent visitor was the Lepidopteran Gonepteryx rhamnii. In general, reproductive outputs were close to zero for all the self-pollination treatments; however, geitonogamy and facilitated selfing seem slightly more efficient than autogamy, as also confirmed by self-compatibility and autofertility indices. Altogether, our results suggest a self-incompatible outcrossing breeding system, with a poor capacity for selfing. Nevertheless, climate change and anthropic threats might promote a shift toward self-fertilization, even maladaptive, favouring the few individuals able to produce selfed seeds.

The abundance and diversity of arbuscular mycorrhizal fungi are linked to the soil chemistry of screes and to slope in the Alpic paleo-endemic Berardia subacaulis.

Berardia subacaulis Vill. is a monospecific genus that is endemic to the South-western Alps, where it grows on alpine screes, which are extreme habitats characterized by soil disturbance and limiting growth conditions. Root colonization by arbuscular mycorrhizal fungi (AMF) is presumably of great importance in these environments, because of its positive effect on plant nutrition and stress tolerance, as well as on structuring the soil. However, there is currently a lack of information on this topic. In this paper, we tested which soil characteristics and biotic factors could contribute to determining the abundance and community composition of AMF in the roots of B. subacaulis, which had previously been found to be mycorrhizal. For such a reason, the influence of soil properties and environmental factors on AMF abundance and community composition in the roots of B. subacaulis, sampled on three different scree slopes, were analysed through microscopic and molecular analysis. The results have shown that the AMF community of Berardia roots was dominated by Glomeraceae, and included a core of AMF taxa, common to all three scree slopes. The vegetation coverage and dark septate endophytes were not related to the AMF colonization percentage and plant community did not influence the root AMF composition. The abundance of AMF in the roots was related to some chemical (available extractable calcium and potassium) and physical (cation exchange capacity, electrical conductivity and field capacity) properties of the soil, thus suggesting an effect of AMF on improving the soil quality. The non-metric multidimensional scaling (NMDS) ordination of the AMF community composition showed that the diversity of AMF in the various sites was influenced not only by the soil quality, but also by the slope. Therefore, the slope-induced physical disturbance of alpine screes may contribute to the selection of disturbance-tolerant AMF taxa, which in turn may lead to different plant-fungus assemblages.

Do floral and niche shifts favour the establishment and persistence of newly arisen polyploids? A case study in an Alpine primrose.

BACKGROUND AND AIMS: Polyploidization plays a key role in plant evolution. Despite the generally accepted 'minority-cytotype exclusion' theory, the specific mechanisms leading to successful establishment and persistence of new polyploids remain controversial. The majority of newly formed polyploids do not become established, because they are less common, have fewer potential mates or may not be able to compete successfully with co-occurring progenitors at lower ploidy levels. Changes in floral traits and ecological niches have been proposed as important mechanisms to overcome this initial frequency-dependent disadvantage. The aim of this study was to determine whether dodecaploids of the heterostylous P. marginata differ from their hexaploid progenitors in P. marginata and P. allionii for selected floral traits and ecological preferences that might be involved in establishment and persistence, providing a possible explanation for the origin of polyploidized populations. METHODS: Floral morphological traits and ecological niche preferences among dodecaploids and their hexaploid progenitors in P. marginata and P. allionii ,: all restricted to the south-western Alps, were quantified and compared KEY RESULTS: Differences in floral traits were detected between dodecaploids and their closest relatives, but such differences might be too weak to counter the strength of minority cytotype disadvantage and are unlikely to enable the coexistence of different cytotypes. Furthermore, the results suggest the preservation of full distyly and no transition to selfing in dodecaploids. Finally, dodecaploids occur almost exclusively in environments that are predicted to be suitable also for their closest hexaploid relatives. CONCLUSIONS: In light of the results, P. marginata dodecaploids have probably been able to establish and persist by occupying geographical areas not yet filled by their closest relatives without significant evolution in their climatic and pollination niches. Dispersal limitation and minority-cytotype exclusion probably maintain their current range disjunct from those of its close relatives.

Phylogeographic Insights into a Peripheral Refugium: The Importance of Cumulative Effect of Glaciation on the Genetic Structure of Two Endemic Plants.

Quaternary glaciations and mostly last glacial maximum have shaped the contemporary distribution of many species in the Alps. However, in the Maritime and Ligurian Alps a more complex picture is suggested by the presence of many Tertiary paleoendemisms and by the divergence time between lineages in one endemic species predating the Late Pleistocene glaciation. The low number of endemic species studied limits the understanding of the processes that took place within this region. We used species distribution models and phylogeographical methods to infer glacial refugia and to reconstruct the phylogeographical pattern of Silene cordifolia All. and Viola argenteria Moraldo & Forneris. The predicted suitable area for last glacial maximum roughly fitted current known distribution. Our results suggest that separation of the major clades predates the last glacial maximum and the following repeated glacial and interglacial periods probably drove differentiations. The complex phylogeographical pattern observed in the study species suggests that both populations and genotypes extinction was minimal during the last glacial maximum, probably due to the low impact of glaciations and to topographic complexity in this area. This study underlines the importance of cumulative effect of previous glacial cycles in shaping the genetic structure of plant species in Maritime and Ligurian Alps, as expected for a Mediterranean mountain region more than for an Alpine region.

Reproductive biology of an Alpic paleo-endemic in a changing climate.

Climate change is known to have a profound influence on plant reproduction, mainly because it affects plant/pollinator interactions, sometimes driving plants to extinction. Starting from the Neogene, the European climate was subjected to severe alterations. Nevertheless, several genera, including Berardia, survived these climatic changes. Despite the numerous studies performed about the relationship between climate change and plant reproductive biology, equivalent studies on ancient species are lacking, even though they may furnish crucial information on the strategies that allowed them to survive drastic climatic fluctuations. We investigated floral and reproductive features in Berardia subacaulis (Asteraceae), describing pollen vectors, capitulum and florets phenology, evaluating reproductive efficiency and defining the reproductive mode of the plant with bagging experiments and test of apomixis. B. subacaulis grows in habitats with low pollination services; it is self-compatible, but many typical features favouring cross-pollination are still present: florets are characterized by incomplete protandry, capitulum protogyny and high pollen-ovule ratio. The plant is not apomictic and self-fertilization is allowed within each capitulum. Similarly to other European Alpine endemics supposed to belong to the Mediterranean ancient tropical flora, the reproductive mode observed in the monospecific genus Berardia assured reproduction also under a pollinator decline. Differently from the other endemics, it took advantage of its spontaneous self-pollination and compatibility and its generalist pollination service, common both among high altitude plants and in the Asteraceae.

Pollination ecology in the narrow endemic winter-flowering Primula allionii (Primulaceae).

Reduction of pollen flow can affect plant abundance and population viability and cause selection on plant mating system and floral traits. Little is known on the effect of this phenomenon in species naturally restricted to small and isolated habitats, that may have developed strategies to cope with long-term isolation and small population size. We investigated the pollination ecology of the endemic distylous winter-flowering P. allionii to verify the possible limitation of female fitness due to reduced pollinator visits. We recorded a higher production of pollen grains in long-styled morph, and a higher seed set in short-styled morph. The high intra-morph variability of sexual organ position may explain the hybridization phenomena allowing and easier intra-morph pollination. The fruit set is constant, although its winter-flowering period might decrease pollen transfer. Nevertheless, the lower competition for pollinators with neighbouring plants and the long-lasting anthesis may offset its reproductive success. Even if our results show no evidence of imminent threats, changes in plant-pollinator interactions might increase inbreeding, resulting in an increased extinction risk.

Polyploid evolution and Pleistocene glacial cycles: A case study from the alpine primrose Primula marginata (Primulaceae).

BACKGROUND: Recent studies highlighted the role of Pleistocene climatic cycles in polyploid speciation and of southern Alpine refugia as reservoirs of diversity during glacial maxima. The polyploid Primula marginata, endemic to the southwestern Alps, includes both hexaploid and dodecaploid cytotypes that show no ecological or morphological differences. We used flow cytometry to determine variation and geographic distribution of cytotypes within and between populations and analyses of chloroplast (cp) and nuclear ribosomal (nr) DNA sequences from the Internal Transcribed Spacer (ITS) region to infer the evolutionary history of the two cytotypes and the auto- vs. allopolyploid origin of dodecaploid populations. RESULTS: We did not detect any intermediate cytotypes or variation of ploidy levels within populations. Hexaploids occur in the western and dodecaploids in the eastern part of the distributional range, respectively. The cpDNA and nrDNA topologies are in conflict, for the former supports shared ancestry between P. marginata and P. latifolia, while the latter implies common origins between at least some ITS clones of P. marginata and P. allionii. CONCLUSIONS: Our results suggest an initial episode of chloroplast capture involving ancestral lineages of P. latifolia and P. marginata, followed by polyploidization between P. marginata-like and P. allionii-like lineages in a southern refugium of the Maritime Alps. The higher proportion of ITS polymorphisms in dodecaploid than in hexaploid accessions of P. marginata and higher total nucleotide diversity of ITS clones in dodecaploid vs. hexaploid individuals sequences are congruent with the allopolyploid hypothesis of dodecaploid origin.

Molecular phylogeny of the Caryophyllaceae (Caryophyllales) inferred from chloroplast matK and nuclear rDNA ITS sequences.

Caryophyllaceae is a principally holarctic family including around 2200 species often classified into the three subfamilies Alsinoideae, Caryophylloideae, and Paronychioideae. Complex and possibly homoplasious morphological characters within the family make taxa difficult to delimit and diagnose. To explore part of the morphological evolution within the family, we investigated the phylogeny of the Caryophyllaceae by means of analyzing plastid and nuclear sequence data with parsimony and Bayesian methods. We describe a mode of tracing a stable phylogenetic signal in ITS sequences, and a significant common signal is shared with the plastid data. Parsimony and Bayesian analyses yield some differences in tree resolution. None of the subfamilies appear monophyletic, but the monophyly of the Caryophylloideae is not contradicted. Alsinoideae are paraphyletic, with Arenaria subg. Eremogone and Minuartia subg. Spergella more closely related to the Caryophylloideae. There is strong support for the inclusion of Spergula-Spergularia in an Alsinoideae-Caryophylloideae clade. Putative synapomorphies for these groupings are twice as many stamens as number of sepals and a caryophyllad-type of embryogeny. Paronychioideae form a basal grade, where tribe Corrigioleae are sister to the rest of the family. Free styles and capsules with simple teeth are possibly plesiomorphic for the family.