Fabien Arène

Laurence Affre

Aggeliki Doxa

Arne Saatkamp
email: arne.saatkamp@imbe.fr

Temperature but not moisture response of germination shows phylogenetic constraints while both interact with seed mass and lifespan

Keywords: base temperature, base water potential, germination traits, hydrotime, lifespan, phylogenetic signal, seed size, seed-plant, thermal time

Understanding how plant traits interact with climate to determine plant niches is decisive for predicting climate change impacts. While lifespan and seed size modify the importance of germination timing, germination traits such as base temperature and base water potential directly translate climatic conditions into germination timing, impacting performance in later life stages. Yet we do not know how base temperature, base water potential, seed mass, lifespan and climate are related. We tested the relationships between base temperature and base water potential for germination, seed size and lifespan while controlling for bioclimatic regions. We also quantified the phylogenetic signal in germination traits and seed size using Pagel's λ. We used a worldwide data set of germination responses to temperature and moisture, seed size and lifespan of 240 seed plants from 49 families. Both germination temperature and moisture are negatively related to seed size. Annual plants show a negative relation between seed size and base water potential, whereas perennials display a negative relation between base temperature and seed mass. Pagel's λ highlighted the slow evolution of base temperature for germination, comparable to seed mass while base water potential was revealed to be labile. In the future, base water potential and seed mass can be used when moisture niches of plants are to be predicted. Lifespan, seed size and base temperature should be taken into account when analysing thermal limits of species distributions.

Introduction

Understanding how changing climate influences earth ecosystems depends critically on how plants respond to gradients of temperature and moisture [START_REF] Parmesan | Empirical perspectives on species borders: from traditional biogeography to global change[END_REF][START_REF] Bykova | Temperature dependence of the reproduction niche and its relevance for plant species distributions[END_REF][START_REF] Silvertown | Hydrological niches in terrestrial plant communities: a review[END_REF]. This puts the question of which adaptive traits of seed plants correspond to climatic niches at the core of functional ecology [START_REF] Jump | Running to stand still: adaptation and the response of plants to rapid climate change[END_REF][START_REF] Mcgill | Rebuilding community ecology from functional traits[END_REF][START_REF] Chuine | Why does phenology drive species distribution?[END_REF][START_REF] Gallagher | Evidence for climatic niche and biome shifts between native and novel ranges in plant species introduced to Australia[END_REF]. If we want to predict plant responses to changing temperature and moisture, we need to know how the interaction of traits and the environment translates into realized niches [START_REF] García-Baquero | Dissecting the hydrological niche: soil moisture, space and lifespan[END_REF][START_REF] Silvertown | Hydrological niches in terrestrial plant communities: a review[END_REF]. The underlying trade-offs and the evolutionary constraints of traits related to climatic niches have been studied [START_REF] Wright | The worldwide leaf economics spectrum[END_REF][START_REF] Evans | Climate and lifehistory evolution in evening primroses (Oenothera, Onagraceae): a phylogenetic comparative analysis[END_REF][START_REF] Donoghue | Biome shifts and niche evolution in plants[END_REF], but the implication of regeneration traits in relation to climatic features is little explored despite its potentially decisive role in successful regeneration [START_REF] Holt | Variation in moisture duration as a driver of coexistence by the storage effect in desert annual plants[END_REF][START_REF] Huang | The contribution of germination functional traits to population dynamics of a desert plant community[END_REF].

Whereas traits of adult plants related to climate are studied on a worldwide scale [START_REF] Wright | The worldwide leaf economics spectrum[END_REF][START_REF] Chave | Towards a worldwide wood economics spectrum[END_REF][START_REF] Sack | How do leaf veins influence the worldwide leaf economic spectrum? Review and synthesis[END_REF], our understanding of the link between reproductive traits and climate is more limited -with the exception of seed size [START_REF] Moles | Global patterns in seed size[END_REF]. This contrasts with findings that show adaptations of seeds and seedlings determining range limits, community assembly, and population dynamics [START_REF] Morin | Process-based modeling of species' distributions: what limits temperature tree species' range boundaries?[END_REF][START_REF] Poorter | Are species adapted to their regeneration niche, adult niche, or both?[END_REF][START_REF] Donohue | Germination, postgermination adaptation, and species ecological ranges[END_REF][START_REF] Huang | The contribution of germination functional traits to population dynamics of a desert plant community[END_REF][START_REF] Jiménez-Alfaro | Seed germination traits can contribute better to plant community ecology[END_REF]. [START_REF] Harper | The behaviour of seeds in soil: I. The heterogeneity of soil surfaces and its role in determining the establishment of plants from seed[END_REF] and [START_REF] Grubb | Maintenance of species-richness in plant communities: the importance of the regeneration niche[END_REF] demonstrated how important 'regeneration niches' and 'safe sites' are by showing that biotic and abiotic environments of plants during germination, establishment of seedlings and onward growth determine position and success of adults. Whereas studies focusing on 'survival niche' of seedlings revealed the importance of climatic niches [START_REF] Leishman | The role of seed size in seedling establishment in dry soil conditions experimental evidence from semi-arid species[END_REF][START_REF] Bykova | Temperature dependence of the reproduction niche and its relevance for plant species distributions[END_REF]), the role of adaptations and trade-offs at the germination process with climate has received comparatively little attention [START_REF] Kruk | Light and thermal environments as modified by a wheat crop: effects on weed seed germination[END_REF][START_REF] Holt | Variation in moisture duration as a driver of coexistence by the storage effect in desert annual plants[END_REF][START_REF] Huang | The contribution of germination functional traits to population dynamics of a desert plant community[END_REF].

In contrast to dormancy, which gives larger seasonal timing of germination [START_REF] Donohue | Germination timing influences natural selection on life-history characters in Arabidopsis thaliana[END_REF][START_REF] Baskin | Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination[END_REF], germination is a short-term process transforming seeds into seedlings that are more vulnerable to environmental hazards [START_REF] Sarukhan | Studies on plant demography: Ranunculus repens L., R. bulbosus L[END_REF][START_REF] Harper | Population Biology of Plants[END_REF][START_REF] Mazer | Ecological, taxonomic, and life history correlates of seed mass among Indiana dune angiosperms[END_REF]. Germination timing influences the adaptation to environmental conditions that individuals will experience for long periods afterwards [START_REF] Donohue | Germination timing influences natural selection on life-history characters in Arabidopsis thaliana[END_REF]. Hence the regeneration niche determines the survival niche of seedlings and even trait contrasts in adults [START_REF] Poorter | Are species adapted to their regeneration niche, adult niche, or both?[END_REF]. This further underlines the importance of carrying out studies on the links between germination requirements, seed traits and the interaction with adult plant traits.

Germination of seeds depends primarily on climatic variables such as temperature and moisture conditions in the seed bed [START_REF] Kruk | Light and thermal environments as modified by a wheat crop: effects on weed seed germination[END_REF]Saatkamp et al., 2011a;[START_REF] Baskin | Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination[END_REF][START_REF] Huang | The contribution of germination functional traits to population dynamics of a desert plant community[END_REF]. The latter are particularly crucial germination cues as they also influence later growth [START_REF] Poorter | The role of biomass allocation in the growth response of plants to different levels of light, CO 2 , nutrients and water: a quantitative review[END_REF][START_REF] Rustad | A meta-analysis of the response of soil respiration, net nitrogen mineralization, and aboveground plant growth to experimental ecosystem warming[END_REF]. Germination timing of non-dormant seeds with the environment can be modelled effectively as a function of time passed above threshold values such as base temperature (T b ) and base water potential (Ψ b ) [START_REF] Steinmaus | Estimation of base temperatures for nine weed species[END_REF][START_REF] Bradford | Applications of hydrothermal time to quantifying and modeling seed germination and dormancy[END_REF][START_REF] Trudgill | Thermal time-concepts and utility[END_REF]. Hydrothermal time models (HTT models) rely on T b and Ψ b and have previously been used to model crop emergence and germination of agricultural weeds from soil seed banks [START_REF] Bradford | Applications of hydrothermal time to quantifying and modeling seed germination and dormancy[END_REF][START_REF] Kruk | Light and thermal environments as modified by a wheat crop: effects on weed seed germination[END_REF] and according to recent analysis also play an important role in persistence in the soil seed bank (Saatkamp et al., 2011b). Parameters of germination models are of growing interest in comparative germination ecology as they appear to be specific adaptations of plants to their environment [START_REF] Allen | Hydrothermal time as a tool in comparative germination studies[END_REF][START_REF] Huang | The contribution of germination functional traits to population dynamics of a desert plant community[END_REF].

Although the fundamental functional and comparative value of germination traits has been identified, little is known about the relationships between germination traits such as T b , Ψ b and other seed traits, such as seed size.

Seed size has notably been related to light and moisture conditions that seedlings experience during establishment [START_REF] Salisbury | The Reproductive Capacity of Plants[END_REF][START_REF] Foster | The relationship between seed size and establishment conditions in tropical woody plants[END_REF][START_REF] Metcalfe | Seed mass and light requirements for regeneration in Southeast Asian rain forest[END_REF]. Seed size is related to fitness of seedlings, as individuals producing larger seeds benefit from better seedling establishment and higher seedling survival in relation to smaller seeds that are more numerous [START_REF] Lloret | Seedling survival of Mediterranean shrubland species in relation to root:shoot ratio, seed size and water and nitrogen use[END_REF]Moles et al., 2004;Moles and Westoby, 2004). Beyond such advantages, greater seed size has been suggested as an adaptive trait in the reduction of between-year variation in mortality in the framework of bet-hedging when rainfall varies from year to year [START_REF] Venable | The selective inter-action of dispersal, dormancy and seed size as adaptations for reducing risks in variable environments[END_REF][START_REF] Leishman | The role of seed size in seedling establishment in dry soil conditions experimental evidence from semi-arid species[END_REF][START_REF] Metz | Plant survival in relation to seed size along environmental gradients: a long-term study from semi-arid and Mediterranean annual plant communities[END_REF]. Seed size has also been linked to moisture niches of plants [START_REF] Baker | Seed weight in relation to environmental conditions in California[END_REF], notably by conferring a higher rate of survival of large seedlings under drought [START_REF] Leishman | The role of seed size in seedling establishment in dry soil conditions experimental evidence from semi-arid species[END_REF][START_REF] Lloret | Seedling survival of Mediterranean shrubland species in relation to root:shoot ratio, seed size and water and nitrogen use[END_REF].

Taken together, this suggests that larger seeded plants can successfully reproduce in drier environments or environments with unpredictable drought compared with small seeds which would need more moisture. Thus large seeds should germinate at lower soil water potentials, and hence lower base water potentials (Ψ b ) than small seeds. In addition, if risk reduction is an important selective pressure for the evolution of seed size, then perennials should exhibit a stronger correlation between base water potential Ψ b and seed size than annual plants, because perennials can survive as adults for several reproductive seasons and produce seed in other years. Among the few studies that have explored the link between germination niche and seed traits, [START_REF] Daws | Germination responses to water potential in neotropical pioneers suggest large-seeded species take more risks[END_REF] illustrated for a guild of tropical pioneer trees that large seeds can germinate in drier conditions than small seeds by having lower base water potentials (Ψ b ), as a consequence of their higher drought resistance. However, for other bioclimatic zones, such as nemoral (temperate humid regions), Mediterranean and dry tropics, the relationships between germination traits such as base water potential and base temperature, seed size and lifespan have not yet been explored. Moreover, beyond the ecological significance, information on the evolution of thermal and hydric aspects of the germination niche remains scarce. Some authors [START_REF] Donohue | Environmental and genetic influences on the germination of Arabidopsis thaliana in the field[END_REF] have shown that a shift from autumn to spring germination due to different levels of dormancy can undergo rapid local adaptation. However, studies on dormancy classes highlighted that these traits display a strong phylogenetic signal [START_REF] Baskin | Taxonomy, anatomy and evolution of physical dormancy in seeds[END_REF][START_REF] Forbis | The evolution of embryo size in angiosperms and other seed plants: implications for the evolution of seed dormancy[END_REF][START_REF] Finch-Savage | Seed dormancy and the control of germination[END_REF][START_REF] Baskin | Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination[END_REF]. Across the entire seed plant phylogeny, climatic niche evolution shows inertia due to features inherited from ancestors [START_REF] Prinzing | The niche of higher plants: evidence for phylogenetic conservatism[END_REF][START_REF] Qian | Geographical distribution and ecological conservatism of disjunct genera of vascular plants in eastern Asia and eastern North America[END_REF][START_REF] Schnitzler | Climatic niche evolution and species diversification in the Cape flora, South Africa[END_REF], more commonly referred to as phylogenetic signal [START_REF] Losos | Phylogenetic niche conservatism, phylogenetic signal and the relationship between phylogenetic relatedness and ecological similarity among species[END_REF]. Unlike realized adult niche, the evolution of thermal and hydric aspects of germination niche has been less studied, with the notable exception of [START_REF] Rosbakh | Initial temperature of seed germination as related to species occurrence along a temperature gradient[END_REF] who found no clear phylogenetic signal for their initial temperature (i.e. the minimum temperature allowing 5% germination of a seed lot) which calls for a broader evaluation of phylogenetic conservatism for germination traits. In contrast, several analyses of the evolution of seed size [START_REF] Leishman | The evolutionary ecology of seed size[END_REF][START_REF] Moles | Factors that shape seed mass evolution[END_REF] revealed a strong phylogenetic signal.

Altogether, these contrasting findings raise the question whether germination traits show a phylogenetic signal and to what extent they are related to seed size and other fundamental plant traits.

In order to address the functional link between germination traits and seed mass, we asked four questions.

(i) Do germination traits show a strong phylogenetic signal, and how does it compare with the phylogenetic signal of seed mass? (ii) Are the germination base temperature and base water potential negatively related to seed mass?

(iii) What is the relationship between germination traits (T b and Ψ b ) and seed mass, and does this relationship differ between annual and perennial plants?

(iv) How do bioclimatic zones influence the relationships between germination traits, seed size and lifespan?

To answer these questions, we compiled a data set on base temperature T b and base water potential Ψ b , based on published germination experiments under controlled conditions of seed plants, as well as information on seed mass, lifespan and distribution in bioclimatic zones.

Materials and methods

Base temperatures, base water potentials and seed masses

We performed a literature search on studies reporting experimental data on hydrotime and thermal time models and found 82 publications, including the important database recently published by [START_REF] Dürr | Ranges of critical temperature and water potential values for the germination of species worldwide: contribution to a seed trait database[END_REF]. We searched for studies using the keyword 'germination', combined with one of 'temperature, thermal time, base temperature, water potential, moisture, base water potential, hydrotime, hydrothermal time, model'; we accessed Google Scholar and ScienceDirect databases until June 2015, and our search covers data until that date. This resulted in 201 species for T b and 102 species for Ψ b , and a total of 240 species were taken into account in our study. The number of species was necessarily limited because of the high number of experimental conditions and replicates needed for estimating T b and Ψ b and the need for having complete trait sets for comparison among traits. Nevertheless, our data covered 49 families and all major clades of seed plants, several species-rich biomes and plants with native ranges in contrasting rainfall and temperature conditions. There is, however, a lack of data for plants from arctic-alpine habitats, tropics, tropical mountains and extreme deserts. In most published work, authors studied thermal and hydric time models separately, which explains the difference in sample size between T b and Ψ b . The HTT model is a population-based model describing the germination course of a seed lot in terms of temperature and moisture. Among the possible parameters of HTT models, here we only use base temperatures (T b ) and base water potentials (Ψ b ), above which thermal time and hydric time accumulates until germination can occur. Although we present here the thermal time and hydrotime models separately, temperature and moisture parameters can be modelled together in HTT models [START_REF] Bradford | Applications of hydrothermal time to quantifying and modeling seed germination and dormancy[END_REF]. However, base water potential refers to a fraction of a seed lot, conventionally 50%, and Ψ b50 is the minimum water potential necessary to promote the germination of 50% of a seed lot. For convenience, in this paper we refer to the base water potential of the 50% fraction (Ψ b50 ) simply as the base water potential (Ψ b ). At sub-optimal temperatures, the Ψ b does not vary as a function of temperature; however, when temperatures rise above the optimal temperature the Ψ b increases with temperature and finally reaches a value of 0 MPa (free water) for all seed fractions, which prevents germination of seeds. Both T b and Ψ b can change according to dormancy status of the seed lot [START_REF] Batlla | A quantitative analysis of dormancy loss dynamics in Polygonum avicu lare L. seeds: development of a thermal time model based on changes in seed population thermal parameters[END_REF][START_REF] Bair | A hydrothermal after-ripening time model for seed dormancy loss in Bromus tectorum L[END_REF]. In both cases, dormancy breaking tends to lower base temperatures [START_REF] Batlla | A quantitative analysis of dormancy loss dynamics in Polygonum avicu lare L. seeds: development of a thermal time model based on changes in seed population thermal parameters[END_REF] and base water potentials [START_REF] Bair | A hydrothermal after-ripening time model for seed dormancy loss in Bromus tectorum L[END_REF] for germination. To quantify the influence of dormancy on our results, we therefore ran two analyses, one with mean and the other with the minimum base temperatures for germination when several different entries per species were available. However, Thompson (1970a,b) showed that fresh seed lots showed more contrasted germination niches in terms of minimum and maximum temperatures for germination compared with seed lots for which dormancy was alleviated by dry storage. We excluded from our analysis the data from studies that estimated base water potentials indirectly, by comparison of species under greenhouse or field conditions and/or by holding other thermal or hydrotime parameters constant, without using different levels of base water potentials, notably the important data in [START_REF] Köchy | Hydrothermal time model of germination: parameters for 36 Mediterranean annual species based on a simplified approach[END_REF]. We therefore expect that residual dormancy does not change the results in terms of phylogenetic signal or trait-trait relationships.

We extracted seed masses for the 240 species of our study from the Kew Garden database (Royal Botanic Gardens, Kew, 2014) and obtained missing values from the LEDA seed trait database [START_REF] Kleyer | The LEDA Traitbase: a database of life-history traits of the Northwest European flora[END_REF].

Finally, to assess the potential differences in the germination behaviour as a function of adult longevity, we distinguished perennial (108 and 46 for T b and Ψ b , respectively) from annual plants (93 and 56 for T b and Ψ b , respectively). We also attributed each plant to a major climatic type: nemoral, Mediterranean, dry tropic and wet tropic, according to their distribution given in regional floras of Europe [START_REF] Tutin | Flora Europaea[END_REF] and China (Flora of China: http://flora.huh.harvard.edu/china/).

We excluded from our analyses the only representative from the arctic-alpine biome, Dryas octopetala (Rosaceae).

Seed plant phylogeny

We compiled a phylogeny using the dated, ultrametric supertree for 4685 European vascular plants based on 518 recent molecular phylogenies by [START_REF] Durka | Daphne: a dated phylogeny of a large European flora for phylogenetically informed ecological analyses[END_REF]. We first completed this tree for the 89 species in our data set that were missing in the tree following the methods of [START_REF] Durka | Daphne: a dated phylogeny of a large European flora for phylogenetically informed ecological analyses[END_REF], based on molecular phylogenies. We then reduced this tree to the 240 species in our data set belonging to 49 families (Supplementary Figure S1). Phylogenies were pruned in each analysis to a more restricted species set using the comparative data algorithm implemented in the caper package [START_REF] Orme | The caper package: comparative analysis of phylogenetics and evolution in R. R package[END_REF].

Phylogenetic signal and evolution of climatic niche and seed mass

We used Pagel's λ and maximum likelihood [START_REF] Pagel | Inferring the historical patterns of biological evolution[END_REF][START_REF] Freckleton | Phylogenetic analysis and comparative data: a test and review of evidence[END_REF] to test whether more closely related species across the seed plant phylogeny are more likely to share similar features of base temperature, base water potential and seed mass. Pagel's λ uses an explicit evolutionary approach which indicates the influence of phylogeny in the between-species covariance for a given trait. The λ parameter varies between 0, indicating that the evolution of the trait is independent of phylogeny, without phylogenetic signal, and 1, trait evolution following Brownian motion. Any value of λ significantly higher than zero indicates phylogenetic signal approaching Brownian motion to a different degree.

Germination traits and seed mass links

We applied phylogenetic generalized least square (PGLS) models [START_REF] Felsenstein | Phylogenies and the comparative method[END_REF][START_REF] Grafen | The phylogenetic regression[END_REF] to study the relation between germination traits and seed mass when residual errors of the regressions were not independent between observations, as suggested by [START_REF] Revell | Phylogenetic signal and linear regression on species data[END_REF]; otherwise, we used an ordinary least square (OLS) model. We used PGLS to study the relationship between T b and seed mass in the whole data set and to distinguish annual from perennial plants.

We also used PGLS to study the relation between germination traits (T b and Ψ b ) and seed mass in the dry tropic and nemoral data subset. All other relations showing any conditions cited above were tested using OLS. We computed all statistical analyses using R Core Team software (2014).

Results

Germination traits and seed mass evolution

Our results showed a strong phylogenetic signal for seed mass with a λ significantly different from zero and equal to unity (λ = 1, P < 0.001). The germination traits T b and Ψ b displayed contrasting results. Whereas we found a strong phylogenetic signal for base temperature (λ = 0.96, P < 0.001), we detected no phylogenetic signal for base water potential (λ = 0.35, P > 0.1). We present values of seed mass, T b and Ψ b mapped on the phylogeny (Supplementary Figures S2, S3 andS4). The phylogenetic signal remained strong and significant when we used minimum values for base temperature (λ = 0.95, P < 0.001). When we replaced mean by minimum values for base water potential, still no phylogenetic signal was detected, with minimum instead of mean values (λ < 0.01, P > 0.1).

Seed mass, base temperature and base water potential for germination

Whereas we found a weak positive relation between base temperature and seed mass using ordinary least squares (n = 230, R 2 = 0.05, slope = 0.8, P < 0.001), this relationship was not significant in phylogenetic least squares regression (n = 192, R 2 < 0.01, slope = -0.11, P = 0.697, maximum-likelihood adjusted λ = 0.931).

In contrast, we found a weak but significant relationship between base water potential and seed mass (n = 93, R 2 = 0.039, slope = -0.14, P < 0.05).

No significant relationship was found between base temperature and seed mass in either the annual (Fig. 1A) or perennial data subset (Fig. 1B). We found a significant negative relationship between Ψ b and seed mass only in the annual data subset (Fig. 1C), while perennials did not show any relationship (Fig. 1D).

Separate analyses for the four biomes showed a negative relationship between T b and seed mass in the nemoral (n = 112, R 2 = 0.093, slope = -1.28, P < 0.01) and Mediterranean biomes (n = 50, R 2 = 0.22, slope = -2.18, P < 0.001) but not in dry or wet tropics. In contrast, we found a positive relationship between T b and seed mass for the dry tropic biome (n = 11, R 2 = 0.55, slope = 3.17, P < 0.01) and no relationship in the wet tropic data set. The regression analyses between Ψ b and seed mass revealed a significant negative relationship in nemoral (N = 36, R 2 = 0.114, slope = -0.201, P < 0.05) and Mediterranean biomes (N = 34, R 2 = 0.16, slope = -0.302, P < 0.05) but no significant relationship for data from dry or wet tropics.

When we separated annual from perennial plants within biomes, our analyses showed significant negative relationship between T b and seed mass only for perennial plants from the nemoral biome (N = 70, R 2 = 0.16, slope = -1.43, P < 0.001) and annual plants from the Mediterranean species (N = 35, R 2 = 0.26, slope = -2.06, P < 0.01) but a positive relationship for perennials from the dry tropics (N = 8, R 2 = 0.54, slope = 3.589, P < 0.05). The other combinations of lifespan x biome types had few data points and yielded no significant test. The regression analyses between Ψ b and seed mass revealed a significant negative relationship for Mediterranean annuals (n = 26, R 2 = 0.26, slope = -0.37, P < 0.01). There was no relationship for nemoral and tropical plants. None of the regression results related above changed when we used minimum instead of mean values for T b and Ψ b (see Supplementary Material).

Discussion

Evolution of base temperature and base water potential

Here, we provide first evidence of a phylogenetic signal in base temperature, a key temperaturerelated germination trait, in a worldwide data set for seed plants. This suggests that germination traits related to temperature are more phylogenetically conserved than base water potential and show a phylogenetic signal as strong as seed mass.

The strong phylogenetic signal of base temperature for germination may be the result of a multitude of different enzymatic processes involved in germination itself [START_REF] Holdsworth | Post-genomics dissection of seed dormancy and germination[END_REF], each based on many different proteins having their own thermal characteristics [START_REF] Daniel | A new understanding of how temperature affects the catalytic activity of enzymes[END_REF]. Thermal characteristics of enzymatic processes may receive contrasting selection pressures, since several of the involved proteins are also expressed in later life stages [START_REF] Schmid | A gene expression map of Arabidopsis thaliana development[END_REF] and are thus part of a plant's general adaptation to temperature. Our findings develop and extend data presented by [START_REF] Rosbakh | Initial temperature of seed germination as related to species occurrence along a temperature gradient[END_REF], who found no phylogenetic signal using Blomberg's K statistic for 50 species but found significant differences among some plant families for 'initial temperature', i.e. the lowest temperature at which they observed germination.

Our results go beyond previous findings since we used base temperature, T b , which is known to be a fundamental physiological trait synthesizing the response of germination to temperature [START_REF] Trudgill | Thermal time-concepts and utility[END_REF]. In addition, we used Pagel's λ to quantify phylogenetic signal, which is more efficient than Blomberg's K [START_REF] Münkemüller | How to measure and test phylogenetic signal[END_REF] in the detection of the phylogenetic signal. It has been shown that T b and Ψ b change according to dormancy status of the seed lot [START_REF] Batlla | A quantitative analysis of dormancy loss dynamics in Polygonum avicu lare L. seeds: development of a thermal time model based on changes in seed population thermal parameters[END_REF][START_REF] Bair | A hydrothermal after-ripening time model for seed dormancy loss in Bromus tectorum L[END_REF], and one might argue that this impedes their use in comparative studies. However, studies on germination in Silene (Thompson, 1970a,b) showed that fresh seed lots showed more contrasted germination niches in terms of minimum and maximum temperatures for germination compared with seed lots in which dormancy was alleviated by dry storage. Comparable analysis of traits brings important understanding of plant function although they are variable, e.g. specific leaf area also shows large variation in values according to water status of leaves, plant growing conditions, but which do not change the relative ranking of species [START_REF] Roche | Congruency analysis of species ranking based on leaf traits: which traits are the more reliable?[END_REF]. Moreover, the strong phylogenetic signal across a large data set indicates that base temperature for germination has a species-specific value that varies in a small range, and our data indicate that closely related species can be a predictor of that value.

The greater evolutionary lability of Ψ b can be understood as a consequence of more rapid local adaptation in response to a rapid shift in moisture conditions of the environment and its high spatial variability. Although there are no data on rapid evolution and local adaptation for Ψ b itself, studies on other drought resistance traits show their high heritability and rapid evolution [START_REF] Thomas | Osmotic adjustment in Lolium perenne; its heritability and the nature of solute accumulation[END_REF]. Moreover, [START_REF] Agrawal | A field experiment demonstrating plant life-history evolution and its eco-evolutionary feedback to seed predator populations[END_REF] have shown rapid evolution of life history traits in the evening primrose Oenothera biennis (Onagraceae), partly due to genotypic variation in seed germination.

Thus it seems possible that Ψ b varies greatly among populations of closely related species due to rapid evolution, overriding a long evolutionary history.

Base temperature and seed mass

Here, we provide evidence that the base temperature T b increases weakly with seed mass across all angiosperms, but this pattern appears to be related to larger seed mass of tropical compared with temperate clades and becomes non-significant when phylogeny is taken into account. When separating these data for different biomes, base temperature decreases with seed mass in seasonal climates, this relation being stronger for perennials than for annual plants in the nemoral biome, and stronger for annuals than for perennial plants in the Mediterranean biome. A plausible explanation for a stronger effect for perennials is that perennial plants cannot escape extreme temperatures such as frost since they usually must grow for at least two growing seasons before reproduction, spanning times of frost or drought, which annuals can overcome in the soil seed bank. A higher base water potential of perennial plants may contribute to later germination and this leaves time for potential dispersal to a wetter site. The necessary important short-distance dispersal in the seed bank has been quantified by [START_REF] Olano | Soil seed bank recovery occurs more rapidly than expected in semi-arid Mediterranean gypsum vegetation[END_REF]. This idea is also sustained by research showing that seedlings in their first year are highly vulnerable to climatic hazards [START_REF] Lodge | Establishment of warm-and cool-season native perennial grasses on the north-west slopes of New South Wales. I. Dormancy and germination[END_REF]. Germination timing should thus coincide with seasons that optimize survival of seedlings to following climatic hazards [START_REF] Baskin | Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination[END_REF]. This is in line with the observation that seedlings of perennials allocate most seed resources to survival in contrast to annuals, which allocate more to growth and reproduction [START_REF] Primack | Reproductive effort in annual and perennial species of Plantago (Plantaginaceae)[END_REF][START_REF] Garnier | Growth analysis of congeneric annual and perennial grass species[END_REF] and invest more in extensive root systems [START_REF] Roumet | Suites of root traits differ between annual and perennial species growing in the field[END_REF]. Since seedlings from larger seeds also survive frost better [START_REF] Aizen | Effects of acorn size on seedling survival and growth in Quercus rubra following simulated spring freeze[END_REF], it becomes plausible that adaptation to different temperatures is influenced by seed size.

In our data set for perennials, the lower base temperature of large compared with small seeds suggests that they can germinate at colder temperatures than small seeds. Germination at colder temperatures exposes seedlings necessarily to colder initial environments or even to higher risks of frost. However, some seeds of winter annuals can be very small (e.g. Carophyllaceae, Brassicaceae) and seedlings live and grow even under snow [START_REF] Baskin | Seeds: Ecology, Biogeography and Evolution of Dormancy and Germination[END_REF]. Larger seeds might withstand these adverse seasons better by allocating more seed resources to adaptations against cold temperatures [START_REF] Canham | Interspecific and intraspecific variation in tree seedling survival: effects of allocation to roots versus carbohydrate reserves[END_REF], such as larger root systems, higher tissue density, apoplastic proteins and increased concentration of soluble sugars [START_REF] Larcher | Physiological Plant Ecology: Ecophysiology and Stress Physiology of Functional Groups[END_REF].

Annuals also show a larger and less predictable realized climatic niche compared with perennials, resulting in lower vulnerability to climatic changes [START_REF] Broennimann | Do geographic distribution, niche property and life form explain plants' vulnerability to global change?[END_REF][START_REF] Hanspach | Predictive performance of plant species distribution models depends on species traits[END_REF][START_REF] Boulangeat | Niche breadth, rarity and ecological characteristics within a regional flora spanning large environmental gradients[END_REF], again increasing the range of optimal temperature conditions at germination initiation. Altogether, this suggests that seedlings and juveniles from perennial plants are subject to stronger selective pressures with regard to temperature conditions than annuals, leading to a narrower range of temperatures for germination, and hence their optimal temperature for germination is influenced by seed size.

In Mediterranean climates, small-seeded species displayed higher base temperature values than large-seeded species. For Mediterranean annual species, [START_REF] Marañón | Physiological basis and ecological significance of the seed size and relative growth rate relationship in Mediterranean annuals[END_REF] showed that small-seeded species had higher relative growth rate (RGR) than large-seeded species. Therefore, small-seeded species could delay germination with higher value of T b and accomplish the life cycle before drought. On the other hand, to complete their life cycle, large-seeded species with smaller RGR might germinate earlier by lower values of T b than small-seeded species.

In tropical climates, larger seeds are more weakly associated with higher germination temperatures than small seeds, which leads to slower germination for larger seeds compared with small seeds. Also, larger seeds have been shown to germinate more slowly than small-seeded species [START_REF] Norden | The relationship between seed mass and mean time to germination for 1037 tree species across five tropical forests[END_REF]. Thus larger seeds slow germination by increasing the germination temperatures.

Whereas the evolutionary constraints for slower germination of larger tropical tree seeds still need to be studied, previous work suggests a positive relationship between small seeds and high drought sensitivity [START_REF] Daws | Germination responses to water potential in neotropical pioneers suggest large-seeded species take more risks[END_REF]. This suggests that smaller seeds select for faster development in order to escape unfavourable conditions.

Base water potential and seed mass

The base water potentials (Ψ b ) of seed plants, and especially those from Mediterranean climate zones, show that Ψ b decreases with seed mass. However, our analyses also highlight that lifespan critically influences this relationship, since only annual plants show a base water potential-seed mass relation in our data set.

A lower base water potential enables seed populations to germinate earlier and faster under dry conditions, as suggested by hydrotime models [START_REF] Bradford | Applications of hydrothermal time to quantifying and modeling seed germination and dormancy[END_REF]. By germinating under drier conditions, seedlings from seeds with low base water potential are exposed to higher risks, since subsequent dry conditions or droughts might kill them [START_REF] Daws | Germination responses to water potential in neotropical pioneers suggest large-seeded species take more risks[END_REF][START_REF] Mollard | Germination sensitivities to water potential among coexisting C3 and C4 grasses of cool semi-arid prairie grasslands[END_REF]. However, earlier germinating seedlings might also have higher fitness [START_REF] Luis | Early to rise makes a plant healthy, wealthy, and wise[END_REF], because they produce more offspring and are larger or have higher survival rates at the end of the first growing season. Larger seeds are known to generate seedlings with higher survival rates in dry environments [START_REF] Leishman | The role of seed size in seedling establishment in dry soil conditions experimental evidence from semi-arid species[END_REF] and are even known to be associated with drier environment in local moisture gradients [START_REF] Baker | Seed weight in relation to environmental conditions in California[END_REF]. This makes it clear that lower base water potentials enable large-seeded plants to exploit the advantages of the drought adaptation larger seeds confer on them. This is clear for the Mediterranean subset of our data, but probably applies to all climates with a marked dry season. The higher drought resistance of seedlings from larger seeds may be explained by their capacity to store more water or develop a more extensive root system, exploring larger volumes of soil to counterbalance lack of water [START_REF] Leishman | The role of seed size in seedling establishment in dry soil conditions experimental evidence from semi-arid species[END_REF][START_REF] Saverimuttu | Seedling longevity under deep shade in relation to seed size[END_REF]. Models of evolution of seed banks suggest that large seeds act as a risk-reducing trait to withstand unpredictable drought and diminish the importance of other risk-reducing traits such as seed banks or dispersal [START_REF] Venable | The selective inter-action of dispersal, dormancy and seed size as adaptations for reducing risks in variable environments[END_REF][START_REF] Philippi | Hedging one's evolutionary bets, revisited[END_REF]). High base water potential might also be a risk-reducing trait: it will lead to germination in much moister conditions and in this way prevent seedlings from being exposed to dry conditions in the beginning. In this sense base water potential and seed size can be seen as alternative risk-reducing traits. In contrast to annual plants, perennials rely on a longer lifespan and reproduction at greater age due to adaptations permitting their persistence through adverse seasons, such as storing organs, extensive root systems, dormant buds or extreme drought resistance [START_REF] Larcher | Physiological Plant Ecology: Ecophysiology and Stress Physiology of Functional Groups[END_REF]. These adaptations might further decrease the importance of adaptations at the seed stage due to selective interactions [START_REF] Venable | The selective inter-action of dispersal, dormancy and seed size as adaptations for reducing risks in variable environments[END_REF] and hence might lead to the observed picture of no relation between seed size and base water potential in perennials compared with a negative relation for annuals. Finally, since water conditions show a much higher inter-annual and spatial variability than temperature conditions, the resulting contrasting selective pressures for Ψ b may also contribute to the absence of phylogenetic signal in Ψ b . Moreover, our data suggest that the adaptive value of Ψ b differs between annuals and perennials. Since the shift from perennials to annuals is a frequent convergent feature in many seed plant lineages, this additionally contributes to the evolutionary lability of Ψ b .

When we separate different biomes, the importance of the seed-size water-potential relationship varied among biomes and our analyses highlight its importance for the Mediterranean and nemoral climate. Moreover, data for tropical pioneer trees also suggest that high base water potential for germination avoids germination in dry environments even for these perennial plants, for which small seeds have higher base water potential [START_REF] Daws | Germination responses to water potential in neotropical pioneers suggest large-seeded species take more risks[END_REF]. The negative relationship between seed size and base water potential for tropical pioneer trees is blurred in our data set by data from tropical plants from other habitats than pioneer trees, and we were thus not able to retrieve the same relationship.

Thermal aspects of germination niche, in contrast, have been proposed to be a close covariate of a plant climatic niche [START_REF] Rosbakh | Initial temperature of seed germination as related to species occurrence along a temperature gradient[END_REF], which is known to be highly phylogenetically conserved [START_REF] Prinzing | The niche of higher plants: evidence for phylogenetic conservatism[END_REF][START_REF] Qian | Geographical distribution and ecological conservatism of disjunct genera of vascular plants in eastern Asia and eastern North America[END_REF][START_REF] Schnitzler | Climatic niche evolution and species diversification in the Cape flora, South Africa[END_REF], as the result of historical-geographical constraints on niche evolution. Clearly, this reflects in our data set into

Figure 1 .

 1 Figure 1. Phylogenetic least square regression of the base temperature (T b ) on seed mass (log 10 transformed); (A) across the annual data subset (n = 82; not significant, n.s.) and (B) across perennial data subset (n = 121; n.s.). Ordinary least square regression of the base water potential (Ψ b ) on seed mass (log 10 transformed); (C) across the annual data subset (n = 52 ; R 2 = 0.23 ; slope = 0.36 ; P < 0.001) and (D) across perennial data subset (n = 41; n.s.).

Acknowledgements

We thank Michael Paul for revising the English of our manuscript. A.S., L.A. and F.A. were supported by the Région Provence-Alpes Côtes d'Azur, Gévoclé program, and Aix Marseille Université; A.S. and A.D. are funded by the European Union ENPI CBC-MED GREAT-MED program. The authors declare no competing interests.

higher T b for tropical compared with temperate species and between tropical and temperate seed plant clades (Supplementary Fig. S3). This gives support to the idea that T b evolved in response to similar selection pressures as the plant's climatic niches when comparing tropical with temperate species.

Base temperature turned out to be as phylogenetically conserved as seed size, implying that thermal aspects of regeneration by seeds might show slow adaptation and therefore be associated with high species replacement along gradients. Future studies might evaluate if changing the time for germination by modifying the level of dormancy can compensate and make seeds germinate in seasons or burial depths in accordance with their thermal germination requirements [START_REF] Donohue | Germination, postgermination adaptation, and species ecological ranges[END_REF]Saatkamp et al., 2011a). Water relations turned out to be more labile and seem to compensate for the evolutionary conservation of seed sizes; if this compensation mechanisms holds true, base water potential should increase in environments with decreasing moisture or increasing rainfall variability and be one of the traits increasing soil seed banks as a risk dispersion mechanism [START_REF] Venable | Bet hedging in a guild of desert annuals[END_REF]Saatkamp et al., 2011b;[START_REF] Saatkamp | The functional role of soil seed banks in natural communities[END_REF][START_REF] Huang | The contribution of germination functional traits to population dynamics of a desert plant community[END_REF].

Supplementary material

To view supplementary material for this article, please visit: https://doi.org/10.1017/S0960258517000083


Cesar Esar Ruiz

Guilherme Muricy

Anaira Ira Lage

Celso Domingos

Sandrine Chenesseau

Thierry Perez

Thierry P Erez

Descriptions of new sponge species and genus, including aspiculate Plakinidae, overturn the Homoscleromorpha classification

Keywords:

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Sponges often dominate in terms of both diversity and biomass in semi-dark and dark rocky marine habitats [START_REF] Hart | The fauna of Atlantic marine caves: evidence of dispersal by sea floor spreading while maintaining ties to deep waters[END_REF][START_REF] Vacelet | Sponges (Porifera) in submarine caves[END_REF]. In the last four decades, studies of submarine caves have revealed many new species which often exhibit original biological (adaptive) traits [START_REF] Hart | The fauna of Atlantic marine caves: evidence of dispersal by sea floor spreading while maintaining ties to deep waters[END_REF][START_REF] Vacelet | Hexactinellid Cave, a unique deep-sea habitat in the scuba zone[END_REF][START_REF] Vacelet | Sponges (Porifera) in submarine caves[END_REF]. These studies were mainly conducted in the Mediterranean Sea and focused on habitat, community and species descriptions [START_REF] Pouliquen | Les spongiaires des grottes sous-marines de la r egion de Marseille: ecologie et syst ematique[END_REF][START_REF] Harmelin | Les grottes sous-marines obscures: un milieu extrême et un remarquable biotope refuge[END_REF][START_REF] Fichez | Decrease in allochthonous organic inputs in dark submarine caves, connection with lowering in benthic community richness[END_REF][START_REF] Harmelin | Biodiversit e des habitats cryptiques marins du parc national de Port-Cros (M editerran ee, France). Assemblages de bryozoaires d 0 une grotte sous-marine et des faces inf erieures de pierres[END_REF][START_REF] Bitar | Two new lithistids (Porifera: Demospongiae) from a shallow eastern Mediterranean cave (Lebanon)[END_REF][START_REF] Gerovasileiou | Census of biodiversity in marine caves of eastern Mediterranean Sea[END_REF]. Among submarine cavedwellers, homoscleromorph sponges represent an abundant and diverse taxonomic group; 82% of the Mediterranean homoscleromorph species can be found in submarine caves, and 41% of them are cave-exclusive [START_REF] Ereskovsky | Overview on the Homoscleromorpha sponges diversity in the Mediterranean[END_REF][START_REF] Gerovasileiou | Marine caves of the Mediterranean Sea: a sponge biodiversity reservoir within a biodiversity hotspot[END_REF]. It is likely that surveys of as yet unexplored submarine caves throughout the oceans will lead to the discovery of *Corresponding author. E-mail: thierry.perez@imbe.fr new representatives of this poorly known group of sponges.

Homoscleromorpha was recently elevated to the rank of class [START_REF] Gazave | No longer Demospongiae: Homoscleromorpha formal nomination as a fourth class of Porifera[END_REF], and is currently represented by one order, Homosclerophorida [START_REF] Dendy | Report on the sponges collected by Professor Herdman at Celyon in 1902[END_REF], and two families, Plakinidae Schulze, 1880and Oscarellidae Lendenfeld, 1887. Plakinidae comprises the following six genera: Corticium Schmidt, 1862; Plakina Schulze, 1880; Plakortis Schulze, 1880; Plakinastrella [START_REF] Schulze | Untersuchungen € uber den Bau und die Entwicklung der Spongien. Neuten Mittheilung. Die Plakiniden[END_REF]Placinolopha Topsent, 1897;Tetralophophora R€ utzler et al., 2014. Plakinidae is currently defined as 'Homosclerophorida with siliceous skeletal components represented by calthrops, diods and triods' (Gazave et al., 2010: 11). This family is also characterized by a sylleibid-like or leuconoid aquiferous system harbouring eurypylous or aphodal choanocyte chambers [START_REF] Gazave | Molecular phylogeny restores the supra-generic subdivision of homoscleromorph sponges (Porifera, Homoscleromorpha)[END_REF]. Oscarellidae is defined as 'Homosclerophorida without spicules, with a sylleibid-like or leuconoid aquiferous system made up of spherical, eurypylous or diplodal choanocyte chambers' (Gazave et al., 2010: 11). Within Oscarellidae, only two genera are recognized as valid in the World Porifera Database [START_REF] Van Soest | World Porifera database[END_REF]: Oscarella Vosmaer, 1884 and Pseudocorticium Boury-Esnault, [START_REF] Boury-Esnault | Sponges without skeleton: a new Mediterranean genus of Homoscleromorpha (Porifera, Demospongiae)[END_REF] The lack of diagnostic morphological characters combined with phenotypic plasticity has resulted in much debate regarding the systematics of Homoscleromorpha, and its challenging taxonomy has led to several erroneous identifications, cases of alleged cosmopolitanism and unresolved species-complexes [START_REF] Boury-Esnault | The integrative taxonomic approach applied to Porifera: a case study of the Homoscleromorpha[END_REF]. Clearly there was a need for the exploration of other characters and datasets which may have the potential to shed light on the taxonomy and systematics of this group. Allozymes were first used to distinguish sibling species in Oscarella and Corticium (Boury-Esnault, Sol e- Cava & Thorpe, 1992;[START_REF] Sol E-Cava | Biochemical genetic divergence and systematics in sponges of the genera Corticium and Oscarella (Demospongiae: Homoscleromorpha) in the Mediterranean Sea[END_REF]. Cytological investigations also provided complementary characters for species delimitation among Oscarellidae [START_REF] Boury-Esnault | Genetic and cytological divergence between colour morphs of the Mediterranean sponge Oscarella lobularis Schmidt (Porifera, Demospongiae, Oscarellidae)[END_REF][START_REF] Muricy | Cytological evidence for cryptic speciation in Mediterranean Oscarella species (Porifera, Homoscleromorpha)[END_REF][START_REF] Muricy | An evaluation of morphological and cytological data sets for the phylogeny of Homosclerophorida (Porifera: Demospongiae)[END_REF] and to a lesser extent in the genus Plakina [START_REF] Muricy | Anatomy, cytology and symbiotic bacteria of four Mediterranean species of Plakina Schulze, 1880 (Demospongiae, Homosclerophorida)[END_REF]. More recently, metabolomics was presented as a promising method to be included in an integrative approach to solve the new challenges in sponge systematics [START_REF] Boury-Esnault | Sponge systematics facing new challenges[END_REF]. This integrative approach led to new insights into Homoscleromorpha interspecific relationships [START_REF] Ivani Sevi C | Metabolic fingerprinting as an indicator of biodiversity: towards understanding inter-specific relationships among Homoscleromorpha sponges[END_REF][START_REF] Boury-Esnault | The integrative taxonomic approach applied to Porifera: a case study of the Homoscleromorpha[END_REF][START_REF] Ruiz | Integrative taxonomic description of Plakina kanaky, a new polychromatic sponge species from New Caledonia (Porifera: Homoscleromorpha)[END_REF] but maintained the distinction between the aspiculate family Oscarellidae and the spiculate Plakinidae.

In this paper, we use morphological, cytological and molecular datasets to describe one new genus and two new species of Plakinidae from submarine caves in the Caribbean Sea (Lesser Antilles, eastern Caribbean). We also present original molecular data which indicate that Oscarella nathaliae [START_REF] Ereskovsky | Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis[END_REF] should be reassigned to the genus Plakina, and consequently to the family Plakinidae. Together, these new data demonstrate that the present definitions of the two Homosclerophorida families are too simplistic and must be revised, especially because our new species descriptions provide new evidence that characters (synapomorphies) can be lost during evolution. Although this is not new for the Porifera, it was a matter of debate for Homoscleromorpha. 1). The specimens were all found on vertical walls, under horizontal rocks or on the ceiling of submarine caves at 7-25 m depth. Underwater photos and general information (size, shape, colour and consistency) were registered for each individual. Each sample was fixed in 95% ethanol and in 2.5% glutaraldehyde for molecular and morphological analyses, respectively.

MATERIAL AND METHODS

SAMPLING

SKELETAL ANALYSIS

A piece of sponge was embedded in Araldite, at least three sections of about 5 mm thick were cut with a low-speed saw and wet-ground with abrasive paper or polishing discs to obtain thinner sections (< 1 mm thick), which were then mounted on glass slides and observed under a light microscope. For spicule preparations, a small fragment of each specimen was boiled with 5 mL nitric acid for organic degradation, and then washed several times with distilled water. The resulting solution was placed on slides for light microscopy. A minimum of 20 measurements were carried out for each spicule type. Spicule measurements are expressed as minimal and maximal length in micrometres. For scanning electron microscopy (SEM), slides containing spicules were coated with gold-palladium and observed under a Hitachi S-570 microscope.

CYTOLOGY

A small fragment of each specimen was fixed in 2.5% glutaraldehyde in 2 M phosphate buffer and filtered seawater (1:4:5, by vol.), then post-fixed in 2% OsO 4 in seawater [START_REF] Boury-Esnault | Comparative study of the choanosome of Porifera. 1: the Homoscleromorpha[END_REF]. Siliceous spicules were dissolved with 5% hydrofluoric acid for 2 h. For semi-thin and ultrathin sections, each fragment was embedded in Araldite. Semi-thin sections were stained with toluidine blue and observed under a Leica DMBL light microscope. Ultra-thin sections were made using an RMC ultramicrotome PTXL. The cuts were placed on a copper grid (3.05 mm in diameter, 300 meshes) and stained with 2% uranyl acetate for 15 min. Observations were carried out with a JEOL JEM-1400 (Peabody, MA, USA) transmission electron microscope. The cytological and prokaryotic composition of each sample was analysed, taking into account the cells' morphology, dimensions and cytoplasm characteristics.

DNA ANALYSIS

For our molecular systematics study, we chose cytochrome oxidase 1 (CO1) markers because of their known value in differentiating closely related species among Homoscleromorpha (see, for instance, [START_REF] Sevi C | Oscarella balibaloi, a new sponge species (Homoscleromorpha: Plakinidae) from the Western Mediterranean Sea: cytological description, reproductive cycle and ecology[END_REF][START_REF] Boury-Esnault | The integrative taxonomic approach applied to Porifera: a case study of the Homoscleromorpha[END_REF][START_REF] Ruiz | Integrative taxonomic description of Plakina kanaky, a new polychromatic sponge species from New Caledonia (Porifera: Homoscleromorpha)[END_REF]. DNA extractions were performed from small sponge fragments (2 cm 3 ) using a QIAamp DNA Mini Kit (Qiagen, Courtaboeuf, France). Two sets of primers were used: the universal primers LCO1490 and HCO2198 to amplify a 584-bp portion of the CO1 mitochondrial gene [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF], and C1-Npor2760 and C1-J2165 to amplify a 507-bp fragment (I3-M11) of the CO1 mitochondrial gene [START_REF] Misof | Mitochondrial gene fragments suggest paraphyly of the genus Panorpa (Mecoptera, Panorpidae)[END_REF][START_REF] Erpenbeck | Unraveling host and symbiont phylogenies of halichondrid sponges (Demospongiae, Porifera) using a mitochondrial marker[END_REF]. Amplifications were done in a 40lL total reaction volume with: 4 lL of each primer (10 lM), 6.4 lL dNTPs (10 mM), 8 lL polymerase buffer, 5 lL MgCl 2 (25 mM), 0.2 lL Taq polymerase (5 U lL À1 ), 2.4 lL extracted DNA and 10 lL of ultrapure (Milli-Q) water. PCRs were performed on a Mastercycler gradient PCR-S Eppendorf thermocycler with an initial step of 5 min at 94 °C followed by 40 amplification cycles (denaturation at 94 °C for 1 min; annealing at 42 °C for 1 min; and extension at 72 °C for 1 min), and a final extension step at 72 °C for 5 min. PCR products were directly sequenced in each primer direction by Eurofins laboratory. For the phylogenetic tree using the Folmer fragment, 14 sequences were downloaded from GenBank and compared with five sequences obtained during this study. For the I3-M11 fragment, we used 14 sequences downloaded from GenBank with eight new sequences from the present study. A concatenated tree was also constructed using all new and downloaded sequences obtained from both fragments (total of 1078 bp). At least one species of each homoscleromorph genus was included in the analysis with the exception of Placinolopha.

Sequences were aligned using BIOEDIT 7.0.5.3 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT[END_REF]. Phylogenetic trees were constructed using both the neighbour-joining (NJ) method (1000 bootstrap replicates) with CLUSTAL X 2.0 [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF] and maximum-likelihood (ML) method, under the GTR + I + G model (previously tested using Jmodeltest software; [START_REF] Darriba | jMo-delTest 2: more models, new heuristic and parallel computing[END_REF] with a non-parametric bootstrap resampling of 100 replicates using PhyML algorithms [START_REF] Guindon | New algorithms and methods to estimate Maximum-Likelihood phylogenies: assessing the performance of PhyML 3.0[END_REF]. The concatenated tree was made with RAxML [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF] on XSEDE with the online platform CIPRES (http://www.phylo.org). In all analyses, the Demospongiae Xestospongia muta [START_REF] Schmidt | Grundz € uge einer Spongien-Fauna des Atlantischen gebietes[END_REF] was used as the out-group. [START_REF] Muricy | Order Homosclerophorida Dendy, 1905, Family Plakinidae, Schulze[END_REF]. Etymology: Plakina arletensis refers to the little village 'Les Anses d'Arlet', close to the cave where this sponge was first found, which is one of the most beautiful places in La Martinique. This is also a special dedication to the kind and warm inhabitants of this village.

RESULTS

SYSTEMATICS

PLAKINA ARLETENSIS SP. NOV.
Diagnosis: Thin encrusting and white Plakina, with a rugose folded surface, a skeleton made of monolophose, trilophose and tetralophose calthrops, actines with and without terminal spines. Diods and triods are absent. Well-developed mesohyl with a high abundance of prokaryotic symbionts.

Description

Small crust, about 2-15 cm in size, 0.2-0.5 cm thick. White colour in vivo, becoming slightly cream or light brown in alcohol (Fig. 2A,B). The consistency is cartilaginous and the surface is rugose and irregularly folded. Oscules are circular, 1-2 mm in diameter, with a slightly elevated rim.

Skeleton:

The skeleton is dense, especially at the surface which is pierced principally by trilophose calthrops. The choanosomal skeleton has an alveolar arrangement formed by all spicule types (Fig. 2C).

Spicules: Diods and triods are absent. Calthrops are irregular, abundant, their actines (20-30 lm long) being with or without spines. Spines (1-4) are short and conical, usually at the basis of each actine (Fig. 2D,E).

Monolophose calthrops are abundant (13-28 lm actines long). The lophose actine is distally ramified by 2-4 short conical rays with sharp endings (Fig. 2F).

Trilophose calthrops are common; lophose actines ramify close the base in 2-3 rays, distally in 2-4 smaller rays with terminal spines (ramification pattern 1 proximal, 2 distal, terminal spines; for the terminology of ramification patterns, see [START_REF] Muricy | Order Homosclerophorida Dendy, 1905, Family Plakinidae, Schulze[END_REF]. The non-lophose actines are 10-30 lm long (Fig. 2G).

Tetralophose calthrops are heterolophose and the less abundant spicule type. The three apical actines ramify distally in 3-4 rays, then distally in 2-5 short rays with terminal spines. The basal actine ramifies distally in two rays, and then distally again in 2-3 short rays with terminal spines (ramification pattern 1 distal, 2 distal, terminal spines). The actine length is 5 lm on average (Fig. 2H; Table 1).

Tissue general organization:

The ectosome is 15-30 lm thick, separated from the choanosome by subectosomal cavities (15-40 lm) with a welldeveloped system of inhalant/exhalant canals (25-140 lm wide). The aquiferous system is leuconoid (Fig. 3A,B). Choanocyte chambers, 47-78 lm in diameter, are spherical and diplodal (Fig. 3B,C).

Cytology: Choanocytes are 3-7 lm wide and 4-6 lm high, their collar, 40-50 lm in diameter, is composed of about 50 microvilli. The choanocyte nucleus is spherical and located on the apical area of the cell (4-5 lm in diameter). Their cytoplasm is dense, often with one to five phagosomes of about 1.0-1.5 lm (Fig. 3B,C). The exo-and endopinacocytes are flat or ovoid (15-27 lm long and 7-11 lm large), with several vacuoles observed in their cytoplasm (Fig. 3B-D). Their nucleus is also flattened (4-5 lm in diameter). Choanocytes and pinacocytes are flagellated, underlined by a basement membrane (Fig. 3C,D). Few archaeocytes were observed. They have an irregular form with a spherical nucleus 2 lm in diameter (Fig. 3D,E).

Symbiotic prokaryotes: Plakina arletensis sp. nov. can be considered as a high microbial abundance (HMA) sponge. Three main morphotypes were easily detected, all three being extracellular and randomly dispersed in most of the mesohyl (Fig. 3E,F). The first morphotype corresponds to an abundant ovoid cell (1.5-1.8 lm long; 0.4-1 lm high), with a more or less dense periplasm. We consider most of the small spherical forms as a perpendicular view of morphotype 1 (Fig. 3E,F). The second morphotype, less abundant, has a rod-like to irregular shape (2-3 9 0.3-0.8 lm) with several translucent vacuoles. The third morphotype, less abundant, has the same ovoid form and dense periplasm as the first morphotype, but is bigger (3-5 9 1-1.7 lm) (Fig. 3E,F).

Ecology 2). As with most Plakina species, the new species does not have vacuolar cells in the mesohyl, but prokaryote symbionts are abundant [START_REF] Muricy | Anatomy, cytology and symbiotic bacteria of four Mediterranean species of Plakina Schulze, 1880 (Demospongiae, Homosclerophorida)[END_REF]. In addition to the similarities in morphological traits, the assignment of the new species to Plakina is also highly supported by phylogenetic analyses. The CO1 sequences for our new species show it clustering with other Plakina species and also show it to be a distinct species (see below). Cave-dwelling thin and translucent aspiculate Plakinidae. Colour milky beige, sometimes pale orange, forming pendulous thin layers with a perforated surface and a clearer membrane in the periphery. Consistency soft, slimy and very fragile. Two mesohylar cells with inclusions: large, abundant vacuolar cells and small, rare granular cells. [START_REF] Ereskovsky | Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis[END_REF]: Caribbean: Greater Antilles, Jamaica, and Lesser Antilles, Saint Martin, La Guadeloupe, La Martinique, Les Grenadines (Bequia); south-western Caribbean: Colombia (Santa Marta). This species is always found in semi-dark caves, from 10 to 50 m depth.

Distribution (modified from

Taxonomic remarks

Eight specimens of P. nathaliae were identified by comparing morphological, cytological and/or genetic characters. All individuals share the features of the species described by [START_REF] Ereskovsky | Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis[END_REF]: cavedwelling species, thin layer yellow to beige in colour (Fig. 4A-D), thin ectosome (3-50 lm high) with syllebeid aquiferous system and spherical choanocyte chambers (Fig. 4E). The two mesohyl cells originally described were also found in the studied specimens.

ASPICULOPHORA GEN. NOV. ASPICULOPHORA MADININA SP. NOV.

FIGS 5, 6

Diagnosis: Plakinidae without spicules, welldeveloped mesohyl with a thick collagen layer and without subectosomal cavities. Generally brown, but also cream to white. Smooth and 'grooved' surface. Jelly-like consistency. Leuconoid aquiferous system and aphodal choanocyte chambers. High abundance of prokaryotic symbionts.

Holotype: MNHN DJV180, Rocher du Diamant at 12 m depth, La Martinique (14°26.5 0 N, 61°03.083 0 W). Collector: T. P erez, 13 June 2011. 150530-GU6-TP7, Grotte Am edien at 12 m depth, La Guadeloupe (16°30.033 0 N, 061°28.774 0 W). Collector: T. P erez, 30 May 2015.

Etymology: The genus name reflects the absence of skeleton: from Latin a (= without) spiculum (= spearhead, arrowhead) phora (= bearing). The species name, madinina, refers to the amerindian name, 'Flower Island', of La Martinique Island, which is the first island of the Caribbean Sea where this aspiculate Plakinidae was found.

Description: Aspiculophora madinina has mainly a cushion shape, measuring 2-15 cm in diameter by 2-5 cm high, but sometimes it can be found hanging down, with prominent oscules surrounded by a thin membranous collar (Fig. 5A). The colour in vivo is mainly brown, occasionally yellow to cream (Fig. 5A). Brown specimens produce a dark exudate in contact with alcohol. The consistency is soft, almost gelatinous. The surface is smooth and grooved. Oscules are circular, 1-2 mm in diameter with an elevated rim. Soft tissue organization: The ectosome is 6-15 lm.

The aquiferous system is leuconoid with inhalant/ exhalant canals about 30-100 lm wide. A dense collagen fibril layer, about 8 lm in thickness, surrounds the exhalant canals. Some specimens also exhibit a thicker collagenous layer, about 1 mm, between the ectosome and the basal part of the sponge, where very few cells or prokaryotes are found (Fig. 5B). Choanocyte chambers (18-36 lm in diameter) are spherical and aphodal.

Cytology: The choanocytes are cylindrical to spherical, 4-7 lm wide and 6-7 lm high. Their nucleus, 2 lm in diameter, is spherical and in apical position (Fig. 6A,B). Their cytoplasm is not dense, often with one to three phagosomes and microgranular inclusions of about 1.5 lm in diameter. These inclusions are also observed in endopinacocytes (Fig. 6C). The exo-and endopinacocytes are flattened, 15-20 lm long and 7-11 lm wide. Only one type of vacuolar cell is present and restricted to the ectosomal region, sometimes in aggregates, always close to exhalant canals (Fig. 6C). This spherical cell, 9-13 lm in diameter, has a nucleus of 3-4 lm and harbours between two and ten vacuoles. Few archaeocytes were observed, with an irregular form and a nucleus of about 2 lm in diameter.

Symbiotic prokaryotes:

This sponge can be considered as an HMA sponge. Five morphotypes were distinguished based on their morphological traits. All prokaryotes are randomly dispersed and occupy most of the mesohyl (Fig. 6B-D). The first morphotype corresponds to an abundant ovoid cell (2-3 lm long; 1-1.5 lm wide), with a clear membrane about 0.1 lm wide. The second morphotype, also abundant in the sponge mesohyl, has a rod form (1.5 lm long; 0.5 lm wide). The third morphotype has an irregular ovoid form (1.5-2.0 lm long; 1 lm wide), with a dense cytoplasm and one to three external vacuoles in contact with the cells' outer membrane. The fourth morphotype is a spherical cell, with a dense periplasm, of about 1 lm in diameter. The fifth morphotype is a small spherical cell of about 0.1 lm in diameter. This morphotype was sometimes observed in groups of three to five cells in a row (Fig. 6D).

Ecology: Aspiculophora madinina sp. nov. is a sciaphilous species, living on vertical walls of semidark and dark caves or under overhangs. It was recorded between 5 and 50 m depth.

Taxonomic remarks

Aspiculophora madinina sp. nov. is a homoscleromorph sponge without skeleton and with a high content of collagen, providing a jelly-like consistency. As yet, massive forms such as this have not been reported in the aspiculate Oscarellidae. On the other hand, this species could be a Plakortis without spicules because most of its representatives occur as massive or globular forms. There is one report of such a Plakortis without spicules in the Caribbean Sponge Guide [START_REF] Zea | The sponge guide: a picture guide to Caribbean sponges, 3rd edn[END_REF], but the taxonomic affiliation to Plakortis was based only on the external morphology and the pungent smell of some specimens. It is difficult to compare the cytological characteristics of a putative new species with other Plakinidae, because of the lack of data at this level of biological organization. Indeed, traditional taxonomy has placed most attention on the skeletal description of this family, even if skeletal differences are poorly detectable in many species. Some other characters such as a leuconoid aquiferous system, eurypylous choanocyte chambers, well-developed mesohyl and subectosomal cavities are not sufficiently diagnostic, as they can be present or absent among Plakina, Plakortis and Plakinastrella species.

We thus believe that more thorough cytological investigations of Plakortis, Plakinastrella and Corticium might help in resolving a good number of taxonomic questions in this family.

The new genus is proposed because of the singularity of its CO1 sequences positioning the new species among the Plakinidae, near the base of this clade, but outside all previously known genera.

Moreover, we believe that its internal organization, with a thick collagen layer surrounding the inhalant canals and within the mesohyl, is unique among Homoscleromorpha.

DNA ANALYSIS OF STUDIED SPECIES

GenBank accession numbers for all sequences in this study are presented in Table 3. The two families of Homoscleromorpha are well supported in the phylogenetic reconstructions . With the Folmer partition (Fig. 7), Corticium appears as a monophyletic taxon of Plakinidae. Plakortis seems to be paraphyletic, a first clade being composed of Plakortis angulospiculatus [START_REF] Carter | Contributions to our Knowledge of the Spongida[END_REF] and the type species P. simplex Schulze, 1880, while a second clade clusters P. albicans Cruz-Barraza & Carballo, 2005 with two Plakinastrella species. In this Table 3. Accession numbers of the specimens sequenced in the present study using the Folmer [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF] and the I3-M11 fragment [START_REF] Misof | Mitochondrial gene fragments suggest paraphyly of the genus Panorpa (Mecoptera, Panorpidae)[END_REF][START_REF] Erpenbeck | Unraveling host and symbiont phylogenies of halichondrid sponges (Demospongiae, Porifera) using a mitochondrial marker[END_REF] The second phylogenetic tree using the I3-M11 CO1 fragment (Fig. 8) supports three monophyletic genera among Plakinidae, Aspiculophora, Plakinastrella and Plakortis. In this analysis Plakina is paraphyletic, P. monolopha (type species) forming a highly supported clade with C. candelabrum Schmidt, 1862, a second group containing P. arletensis sp. nov. and P. jani, and a third with P. crypta and P. trilopha.

The third phylogenetic tree was made by concatenating all available sequences (Fig. 9). Because of missing data for one of the two fragments, the alignment contains several gaps and the concatenated tree appears less robust than the two-first representations of the inter-specific relationships among Homoscleromorpha. However, the topology of this third tree is quite congruent with the previous ones. In this representation, Aspiculophora forms a separate clade within the Plakinidae. Also, Plakina seems paraphyletic with two different clades, the first one grouping P. arletensis sp. nov. with P. kanaky, P. jani, P. trilopha and P. nathaliae, whereas the second one, containing the type species P. monolopha, P. muryciae and P. crypta, is related to Corticium, which seems monophyletic. Plakortis also appears paraphyletic, a first clade grouping the type species P. simplex, P. halichondroides and P. angulospiculatus, and a second clade containing P. albicans, Plakinastrella onkodes and an undetermined Plakinastrella. Finally, a separate clade containing five specimens of Plakinastrella also makes this genus paraphyletic in this phylogenetic representation.

DISCUSSION

Eighty species of Plakinidae are recognized as valid in the World Porifera Database [START_REF] Van Soest | World Porifera database[END_REF], to which the present study adds another three. Plakinidae is found throughout the world's oceans except in polar regions, and appears to be often found in shady rocky habitats. In the Caribbean Sea, several new species and a new genus have recently been described [START_REF] Ereskovsky | Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis[END_REF][START_REF] R€ Utzler | Diversity of the sponges (Porifera) from cryptic habitats on the Belize barrier reef near Carrie Bow Cay[END_REF], although it is likely that the diversity of this family remains underestimated, mainly because of the lack of exploration of cryptic habitats and submarine caves.

Twenty-seven species of Plakina are now described, mainly on the basis of the composition of their spicules. The presence of lophose calthrops is considered a diagnostic character that separates Plakina species from other Plakinidae. Different genetic markers have been used to shed light on Homoscleromorpha systematics, and the phylogenetic trees using 18S and 28S [START_REF] Gazave | Molecular phylogeny restores the supra-generic subdivision of homoscleromorph sponges (Porifera, Homoscleromorpha)[END_REF][START_REF] Gazave | Systematics and molecular phylogeny of the family Oscarellidae (Homoscleromorpha) with description of two new Oscarella species[END_REF], cytochrome oxidase b and CO1 [START_REF] Ereskovsky | Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis[END_REF][START_REF] Cruz-Barraza | Taxonomy of family Plakinidae (Porifera: Homoscleromorpha) from eastern Pacific coral reefs, through morphology and cox1 and cobmtDNA data[END_REF][START_REF] Ruiz | Integrative taxonomic description of Plakina kanaky, a new polychromatic sponge species from New Caledonia (Porifera: Homoscleromorpha)[END_REF] are largely congruent. In the present study, CO1 has been used in an integrative systematics approach, combining the analyses of morphological and cytological characters, to place our specimens among the different genera and families of Homoscleromorpha. Our phylogenetic analysis supports the allocation of Aspiculophora madinina gen. nov., sp. nov. to Plakinidae, and clearly differentiates this new representative of the family from all other taxa. Taking into account these results and an original internal bodyplan, we propose the creation of the first genus of aspiculate Homoscleromorpha outside of Oscarellidae.

Previous classifications of Homoscleromorpha recognized the strength and weakness of the present genus composition in both families, but until now no one has clearly established the phylogenetic interspecific relationships among the different genera. Plakina arletensis sp. nov. and P. jani form a well-supported clade (bootstrap values 967/76), which is in accordance with their morphological traits. We conclude that P. arlentensis is a new species and must be included in clade B3 proposed by [START_REF] Gazave | Molecular phylogeny restores the supra-generic subdivision of homoscleromorph sponges (Porifera, Homoscleromorpha)[END_REF] grouping P. trilopha, P. jani and P. kanaky. We add new data showing the nonmonophyly of Plakina, but before proposing any changes to the status of this genus, we consider it necessary to obtain more DNA sequences and a revision of the morphological descriptions of the type species Plakina monolopha and Corticium candelabrum. Indeed, the I3-M11 sequences of these two species form a single, well-supported clade. We are convinced that the addition of other datasets such as metabolomic fingerprints or meta-barcoding of the symbiotic prokaryote communities will help to better understand the evolutionary mechanisms at play in the origin of this group of sponges.

We decided to rename Oscarella nathaliae as analyses of the CO1 sequences derived from the first specimen collected in La Martinique in 2011 showed it was clearly affiliated with Plakinidae. Due to technical difficulties, this specimen remained the only one that could be sequenced. In the meantime, [START_REF] Ereskovsky | Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis[END_REF] described this new species without any molecular data. Our genetic analysis of the CO1 of numerous new records of this species, from the south-western Caribbean (Colombia) to the northernmost part of the eastern Caribbean, showed clear affiliations with Plakinidae. Considering its position among most representatives of the Plakina (Fig. 7), even given the sometimes low bootstrap support and the recognized non-monophyletic status of the genus, we considered the renaming of this species as Plakina nathaliae as the most parsimonious option. This sponge was originally described as having a unique external morphology within Oscarella: 'a thin and leaf-like sponge, with a surface perforated by abundant pores' (Ereskovsky et al., 2013: 289). Another characteristic noted by authors was the 'irregular and punctual attachment to the substratum' (Ereskovsky et al., 2013: 302). These morphological traits are actually shared with Plakina crypta, a sciaphilous, exclusively Mediterranean species. Plakina nathaliae also has an internal organization that is similar to Plakina crypta and P. endoumensis. These three species share the absence of subectosomal cavities, a similar proportion mesohyl/choanocyte chambers, their leuconoid aquiferous system and their eurypylous choanocyte chambers. Finally, all three Plakina species can be considered as LMA sponges (Table 2), another anatomical trait which separates them from several other Plakina species such as P. jani or P. kanaky [START_REF] Muricy | Anatomy, cytology and symbiotic bacteria of four Mediterranean species of Plakina Schulze, 1880 (Demospongiae, Homosclerophorida)[END_REF][START_REF] Ruiz | Integrative taxonomic description of Plakina kanaky, a new polychromatic sponge species from New Caledonia (Porifera: Homoscleromorpha)[END_REF]. Meanwhile the report of two species without skeleton within Plakinidae again demonstrates that spicules can be secondarily lost in sponges and the heavy reliance on a classification based on spicule composition can often lead to homoplasious results. Recent sampling cruises that focused on submarine caves from the Pacific Ocean and the Caribbean Sea identified numerous new representatives of Homoscleromorpha which have as yet not been described. It is likely that the inclusion of new taxa in combination with additional complementary molecular and cytological investigations will permit a thorough revision of Plakina. For now, on the basis of these new descriptions and revision, we propose to update the taxonomic keys for Homoscleromorpha taking into account that Plakinidae contains 'Homosclerophorida with inorganic (spicular) skeletal complements', which can be lost in some cases. The absence of spicules is no longer exclusive to Oscarellidae.

  Specimens were collected by SCUBA diving in June 2011, March 2012, December 2013 and March 2014 from various sites around La Martinique Island, in December 2014 from the Caribbean coast of Colombia and then during the PACOTILLES cruise across the Lesser Antilles in May and June 2015 (Fig.

Figure 1 .

 1 Figure 1. Known distribution in the Caribbean Sea of Plakina arletensis sp. nov. (white stars), Plakina nathaliae comb. nov. (grey stars) and Aspiculophora madinina gen. nov., sp. nov. (black stars).

FIGS

  FIGS 2, 3

Figure 2 .

 2 Figure 2. Plakina arletensis sp. nov. A, in situ photo. B, same specimen after fixation in alcohol. C, transversal cut of skeleton. D, calthrops without spines. E, calthrops with spines. F, monolophose calthrops. G, trilophose calthrops. H, tetralophose calthrops.

Figure 3 .

 3 Figure 3. A, general soft tissue organization of Plakina arletensis sp. nov. B, detailed view of the mesohyl, showing the aquiferous system. C, transmission electron micrograph of a choanocyte chamber. D transmission electron micrograph with archeocytes and pinacocytes. E, transmission electron micrograph showing the abundance of prokaryotic cells inside the mesohyl and the three main morphotypes (1-3). F, transmission electron micrograph with a detailed view of the prokaryote symbionts (1-3). Ar, archeocytes; cc, choanocyte chamber; Ch, choanocyte; en, endosome; EC, exhalant canal; Mb, basement membrane; M, mesohyl; ph, phagosome; Pi, pinacocytes; Pk, prokaryotes.

  RBINS POR 90, 22 m depth. Le Diamant, Grotte du Fer a Cheval, La Martinique (14°28.067 0 N, 61°00.983 0 W). Collector: P. Willenz and A. Ereskovsky. 7 June 2003. Paratype: MNHN DJV179, 17 m depth. Rocher du Diamant, Grotte de Zeb, La Martinique (14°26.5 0 N, 61°03.083 0 W). Collector: T. P erez. 13 June 2011. Other material examined 250312-MT4b-TP2. Grotte du Fer a Cheval at 20 m depth, le Diamant, La Martinique (14°28.067 0 N, 61°00.983 0 W). Collector: T. P erez. 25 March 2012. 131203-MT3-AE4. Grotte Couleur, Pointe Burgos at 12 m depth, Anse d 0 Arlet, La Martinique (14°29.752 0 N, 61°05.407 0 W). Collector: A. Ereskovsky. 3 December 2013. 150529-GU3-CR6. Grotte Cath edrale at 15 m depth, Anse Bertrand, La Guadeloupe (16°27.740 0 N, 061°31.837 0 W). Collector: C. Ruiz. 29 May 2015. GR34HOM30. Grotte Cath edrale at 15 m depth, Anse Bertrand, La Guadeloupe (16°27.740 0 N, 061°31.837 0 W). Collector: A. Ereskovsky. 18 May 2012. 150527-SN5-TP1. Basses Espagnoles at 12 m depth, Saint Martin (18°07.821 0 N, 63°00.270 0 W). Collector: T. P erez. 27 May 2015. 141211-SM5-CR3. Punta Gaira at 17 m depth, El Rodadero, Santa Marta, Colombia (11°13.133 0 N, 74°14.45 0 W). Collector: C. Ruiz. 12 December 2014. 141212-SM7-CR2. Punta Venado at 20 m depth, Taganga, Santa Marta, Colombia (11°16.217 0 N, 74°12.367 0 W). Collector: C. Ruiz. 12 December 2014. Diagnosis (modified from Ereskovsky et al., 2013):

  ; HMA, high microbial abundance; LMA, low microbial abundance; NA, data not available (modified from[START_REF] Muricy | Anatomy, cytology and symbiotic bacteria of four Mediterranean species of Plakina Schulze, 1880 (Demospongiae, Homosclerophorida)[END_REF]. Paratype 1: MNHN DJV181, Tintamare, Les Arches at 10 m depth, Saint Martin (18°07.588 0 N, 62°58.248 0 W). Collector: C. Ruiz, 26 May 2015. Paratype 2: MNHN DJV182, Grotte Cath edrale at 16 m depth, Anse Bertrand, La Guadeloupe (16°27.740 0 N, 061°31.837 0 W). Collector: C. Ruiz, 29 May 2015. Other specimens examined 150514-MT6-TP1, Grotte Chauve-Souris at 7 m depth, Anse Noire, La Martinique (14°32.024 0 N, 61°05.278 0 W). Collector: T. P erez, 14 May 2015. 150517-SV1-CR14, Bat Cave at 8 m depth, Bucament Bay, Saint Vincent (13°11.275 0 N, 61°16.174 0 W). Collector C. Ruiz, 17 May 2015. 150523-GU1-CR7, Cave north-west of Les Saintes at 8 m depth, Les Saintes, La Guadeloupe (15°52.984 0 N, 61°34.25 0 W). Collector: C. Ruiz & T. P erez, 23 May 2015. 131206-MT3-AE2, Grotte Couleur, Pointe Burgos at 12 m depth, Anse d'Arlet, La Martinique (14°29.752 0 N, 61°05.407 0 W). Collector: A. Ereskovsky, 6 December 2013. 150527-SN5-TP5, Basses Espagnoles at 10 m depth, Saint Martin (18°07.821 0 N, 63°00.270 0 W). Collector: T. P erez, 27 May 2015. 150528-AG3-CR1, Little Scrub at 15 m depth, Anguilla (18°17.903 0 N, 62°57.294 0 W). Collector C. Ruiz, 28 May 2015.

Figure 4 .BFigure 5 .

 45 Figure 4. Plakina nathaliae comb. nov. A, in situ picture of a specimen from Rocher du Diamant, La Martinique. B, in situ picture of a specimen from Grotte Cath edrale, La Guadeloupe. C, in situ picture of a specimen from Punta Gaira, Colombia. D, light micrograph of a transversal section through the ectosome and the choanosome of P. nathaliae. cc, choanocyte chamber; ect, ectosome; M, mesohyl.

Figure 6 .

 6 Figure 6. A, light micrograph of the ectosome and choanosome of Aspiculophora madinina gen. nov., sp. nov. B, transmission electron micrograph showing the high prokaryotic density in a part of the mesohyl located between two choanocyte chambers. C, transmission electron micrographs of vacuolar cells and pinacocytes. D, prokaryote morphotypes associated with the mesohyl of Aspiculophora madinina. Ch, choanocyte; cc, choanocyte chamber; EC, exhalant canal; exo, exopinacoderm; M, mesohyl; Pk, prokaryotes.

Figure 7 .

 7 Figure 7. Phylogenetic reconstruction of the universal Folmer portion of the CO1 gene using neighbour-joining analysis and indicating maximum-likelihood (ML) values. Bootstrap values are given for both analyses; ML values are in parenthesis. The accession numbers of each download sequence are next to the species name.

Figure 8 .

 8 Figure 8. Phylogenetic reconstruction of the I3-M11 portion of the CO1 gene using neighbour-joining (NJ) analysis and indicating maximum-likelihood (ML) values. Bootstrap values are given for both analyses; ML values are in parenthesis. The accession numbers of each download sequence are next to the species name.

Figure 9 .

 9 Figure9. Phylogenetic reconstruction concatenating the Folmer and I3-M11 fragments of the CO1 using maximumlikelihood analysis. All sequences from this study were aligned with both fragments (F+E), Folmer (F) and I3-M11 (E). The accession number of each downloaded sequence is next to the species name.

Table 1 .

 1 Comparison of spicule size and abundance between Plakina species from the Caribbean, the Mediterranean Sea and the north-east of Brazil

	Spicule characters (size in lm)	P. trilopha	P. jani	P. jamaicensis	P. coerulea	P. arletensis
	Diods (length)	A, 40-88	A, 43-100	A, 61-91	63-90	-
	Triods (actine length)	A, 12-33	A, 16-37	A, 22-36	60-75	-
	Calthrops (actine length)	C, 10-35	C, 16-38	A, 6-33	41-55	A, 20-30
	Monolophose calthrops (actine length)	R, 20-30	R, 22-38	R, 20-32	25-36	A, 13-28
	Dilophose calthrops (actine length)	R, 20-35	R, 30-35	R, 25-36	28-35	-
	Trilophose calthrops (actine length)	A, 16-27	C, 19-32	R, 22-29	15-21	C, 10-25
	Tetralophose calthrops (actine length)	C, 10-25	C, 19-25	C, 17-26	12-21	R, 5-7
	Ramification pattern of lophose calthrops	1 md, ts	1 m, 2d, ts	1 m, 2d, ts	1m, 2d, ts	1d, 2d, ts

: Plakina arletensis sp. nov. is found only in shallow water caves, where it forms a white crust with a patchy distribution on vertical walls. No indications of epibiosis or predation were observed. Taxonomic remarks Plakina arletensis sp. nov. has a growth-form typical of the 'true' Plakina species. It has mono-, tri-and tetra-lophose calthrops like the Mediterranean species P. trilopha Schulze, 1880, P. jani Muricy, Boury-Esnault, B ezac & Vacelet, 1998, P. endoumensis A, abundant; C, common; R, rare (see Muricy et al., 1998 for a description of the ramification patterns).

  Muricy, Boury-Esnault, B ezac &[START_REF] Muricy | Taxonomic revision of the Mediterranean Plakina Schulze (Porifera, Demospongiae, Homoscleromorpha)[END_REF] P. weinbergi Muricy, Boury-Esnault, B ezac &[START_REF] Muricy | Taxonomic revision of the Mediterranean Plakina Schulze (Porifera, Demospongiae, Homoscleromorpha)[END_REF] or like P. jamaicensis from the Caribbean Sea or P. coerulea Cedro, Hajdu & Correia, 2013 from northeastern Brazil. In contrast to these two sponges, the new species does not possess diods or triods (Table1). The tetralophose calthrops are heterolophose, but not candelabra-like as in Corticium. The basal actin is best described as bifurcated, with different ramification patterns in comparison with the apical actines. This type of heterolophose calthrops can be observed in other Plakina species such as P. jani and P. endoumensis[START_REF] Muricy | Taxonomic revision of the Mediterranean Plakina Schulze (Porifera, Demospongiae, Homoscleromorpha)[END_REF].Plakina arletensis sp. nov. has a well-developed mesohyl, with subectosomal cavities similar to P. trilopha, P. jani and P. kanakyRuiz & P erez, 2015 (Table

Table 2 .

 2 Comparison of principal cytological characters among Plakina species

	Plakina Plakina Plakina Aspiculophora	jamaicensis arletensis nathaliae madinina	NA + À +	À + À À	Moderate Moderate Low High	Leuconoid Leuconoid Leuconoid Leuconoid	1.8:1 1.5:1 0.7:1 1.8:1	Eurypylous Eurypylous Eurypylous Eurypylous
	Plakina Plakina Plakina	jani crypta endoumensis Plakina kanaky	+ + À À	+ + À À	Moderate Moderate Low Low	Leuconoid Leuconoid Leuconoid Leuconoid	1.8:1 1.6:1 0.7:1 0.9:1	Eurypylous Eurypylous Eurypylous Eurypylous	Pyramidal Pyramidal Cylindrical
		Plakina trilopha	+	+	Moderate	Leuconoid	1.5:1	Eurypylous	Pyramidal
		Character	Well-developed mesohyl	Sub-ectosomal cavities	Collagen density	Aquiferous system	Mesohyl/chambers ratio	Choanocyte chambers	Choanocytes

  of the CO1 gene Plakina appears as a poorly supported monophyletic group (Fig. 7), with two clades (bootstrap values of 312/47), one with P. crypta Muricy, Boury-Esnault, B ezac & Vacelet, 1998, P. monolopha and P. muricyae Cruz-Barraza, Vega & Carballo, 2014 closely related, and the other comprising three specimens of P. nathaliae, P. trilopha, P. jani and P. kanaky.

		Sample code	CO1 fragment	Accession no.
	Plakina arletensis	110613-MT6-TP1	I3-M11	KU674369.1
		140306-MT6-TP1	I3-M11	KU674370.1
		150516-MT8-CR3	I3-M11	KU674371.1
	Plakina nathaliae	110613-MT4-TP2	Folmer	KU674377.1
		141211 SM5-CR03	Folmer	KU674378.1
		141212 SM7-CR02	Folmer	KU674379.1
	Aspiculophora madinina	110613-MT4-TP11	I3-M11	KU674367.1
		131206 MT3-AE2	I3-M11	KU674368.1
	Corticium diamantense	110613MT4-TP5	Folmer	KU674376.1
	Plakinastrella onkodes	141209SM1CR01	I3-M11	KU674372.1
	Plakinastrella sp.	GR26H7	I3-M11	KU674373.1
		150514-MT6-CR5	I3-M11	KU674374.1
		150515-MT4-CR15	I3-M11	KU674375.1
		130205NC10-01	Folmer	KU674380.1
	representation lacking a sequence of the type spe-
	cies,

ACKNOWLEDGEMENTS

This work was performed in the framework of the French-Brazilian Associated International Laboratory 'LIA MARRIO' funded by the CNRS. T.P. and C.R. acknowledge the Total Foundation, and G.M., A.L. and C.D. acknowledge the Fundacão Carlos Chagas de Apoio a Pesquisa do Rio de Janeiro (FAPERJ), Coordenacão de Aperfeicoamento de Pessoal de N ıvel Superior (CAPES) and Conselho Nacional de Desenvolvimento Cient ıfico e Tecnol ogico (CNPq) for grants and fellowships. We are grateful to Jo€ el Courageot and Alexandre Alti e of the Electronic Microscope service in Aix-Marseille University, and to all the crew of the N/O Antea for their involvement in the PACOTILLES campaign in the Lesser Antilles, to Sven Zea in Colombia, to Julien Chalifour in Saint Martin and to our great friend 'Filipo' in La Martinique who took us to various diving sites and helped in the collection and preservation of most of the samples. We are also grateful to Nicole Boury-Esnault and Jean Vacelet for the great source of inspiration they represent, and for their always positive criticisms on our work. The English text was kindly thoroughly revised by Christine Morrow.


Arne Saatkamp

Frédéric Henry

Thierry Dutoit

Vegetation and soil seed bank in a 23-year grazing exclusion chronosequence in a Mediterranean dry grassland

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