Joana Ruela

Heimbürger Boavida

Ronan Becheler

Marvin Choquet

Norbert Frank

Marco Taviani

Jean-François Bourillet

Anne-Leila Leila Meistertzheim

Anthony Grehan

Alessandra Savini

Sophie Arnaud-Haond

Anton Dohrn

Jean Francois

Iii Joana Ruela

Joana Boavida
email: joanarboavida@gmail.com

Out of the Mediterranean? Post-glacial colonisation pathways varied among cold-water coral species

Keywords: Cold-water corals, Lophelia pertusa, Madrepora oculata, Marine phylogeography, Deep-sea, Last Glacial Maximum, Glacial marine refugia, Madrepora oculata. 54

come

Cold-water corals (CWC) are amongst the most charismatic marine ecosystems in the deep ocean 79 (>200 m), supporting abundant and diverse biomass (Milligan et al., 2016). Deep-reef habitats rely 80 upon the frame-building capability of several coral species, notably Lophelia pertusa and 81

Madrepora oculata (Linnaeus, 1758). The two species intermingle and anastomose in reefs in the 82

North-eastern Atlantic Ocean (NE Atlantic;Arnaud-Haond et al., 2017). Yet, ever rising 83 anthropogenic pressures in the deep-sea, like fisheries (Pusceddu et al., 2014), oil and gas exploitation 84 (Cordes et al., 2016) and deep-sea mining (Wedding et al., 2015), threaten vulnerable CWC.

85

Protection of key deep-sea ecosystems is, therefore, an urgent priority, particularly in the face of 86 global change, and strategic Marine Protected Areas, thoroughly assessed for their genetic 87 connectivity, need to be established. Nonetheless, obtaining sufficient specimens and applying 88 statistically robust sampling designs for genetic studies in the deep-sea remain challenging (Becheler 89 et al., 2017). While CWC connectivity has been assessed along the North Atlantic Ocean at the local 90 and regional scale (LeGoff-Vitry et al., 2004;Morrison et al., 2011;Dahl et al., 2012;Becheler et al., 91 2017), effective conservation warrants information at the NE Atlantic basin scale (Fenberg et al., 92 2012). Spatial distribution analyses of genetic diversity can be used to detect connectivity pathways 93 across reefs, and to define key areas for the conservation of biodiversity.

94

Similarly, the present-day geographic distribution of many terrestrial and marine species and their 95 genetic diversity is influenced by environmental gradients, contemporary dispersal and recent climatic 96 events, notably the Last Glacial Maximum, LGM, (26 -19 ka;Hewitt, 1996Hewitt, , 1999Hewitt, , 2004;;Petit et al., 97 2003;Maggs et al., 2008;Clark, 2009). Repeated Pleistocene glaciations are known to impact deep-98 sea ecosystems (Bouchet & Taviani, 1992;Sabelli & Taviani, 2014;Vertino et al., 2014). However, 99 their impact on CWC in particular remains understudied. Deep-sea species may not respond similarly 100 to coastal species, because biological assemblages, environmental gradients and dispersal patterns are 101 fundamentally different among ecosystems and geographical areas (Kousteni et al., 2015;Shum et al., 102 2015).

103

The biogeographic connectivity between Mediterranean and NE Atlantic CWC during and after the 104 Pleistocene (ca. 2.7 Ma to 12 ka) glacial events can partially be inferred from paleo-records 105 (Supplementary Paleo-history). While CWC have maintained a continuous presence in the 106 Mediterranean Sea at least since the early Pleistocene (Vertino et al., 2014), CWC in the NE Atlantic 107 may have been more affected, with consequent local demise during the LGM (Frank et al., 2011). 108

Notably, the last cold oscillation, during the Younger Dryas (12.9-11.7 ka), represented a favourable 109 period for CWC reef growth in the western Mediterranean Sea as well as in adjacent Atlantic regions 110 at the Gulf of Cadiz and African margins (Schröder-Ritzrau et al., 2005;McCulloch et al., 2010;111 Taviani et al., 2011b). In contrast, growth episodes of CWC in the NE Atlantic have been restricted to 112 warm climate stages and coral fossils are absent from strata corresponding to glacial episodes (Frank 113 et al., 2009). Radiometric dating shows that NE Atlantic CWC are younger than the ones from the 114 Mediterranean Sea, with ages estimated to be post-LGM (under 12,000 years;Freiwald & Roberts, 115 2005;Schröder-Ritzrau et al., 2005), producing a NE Atlantic CWC age-gradient from south to north 116 (summarized in Fig. 1). Given such persistent occurrence of CWC, it has been argued that the 117

Mediterranean basin may have acted as a CWC glacial refugium during range contractions in the 118

North Atlantic Ocean during Pleistocene glaciations (De Mol et al., 2002;Henry et al., 2014). Such a 119 refugium may have constituted the source for the Atlantic northward recolonisation of CWC at the end 120 of the LGM by larvae, transported with intense flows of Mediterranean Outflow Water (MOW) since 121 50 ka (Fig. 1; Voelker et al., 2006;Stumpf et al., 2010). expansion for Lophelia pertusa and Madrepora oculata. Such a common response to past-124 environmental change may not hold true in light of recent studies (Lartaud et al., 2014(Lartaud et al., , 2017) ) pointing 125 to physiological differences between both species, particularly in terms of optimal growth 126 temperatures. Dissimilar ecological strategies between the two CWC are possible. Lophelia pertusa 127 presents potential for widespread larval dispersal (Strömberg and Larsson, 2017), high fecundity 128 (Waller & Tyler, 2005) and a variable microbiome composition (Meistertzheim et al., 2016). 129

Madrepora oculata presents a different reproductive strategy, having much lower fecundity (Waller & 130 Tyler, 2005) and a resilient microbiome (Meistertzheim et al., 2016; and unknown larval 131 characteristics). Although reduced genetic connectivity has been demonstrated for L. pertusa at an 132 inter-basin scale along with moderate to high gene flow within regions (Morrison et al., 2011;Dahl et 133 al., 2012;Flot et al., 2013), the extent of CWC connectivity along the European margins and the role 134 that extant populations had in the past remain to be tested. Here we investigate the population genetic 135 diversity and structure of L. pertusa and M. oculata along the deep margins of the NE Atlantic, using 136 nuclear data (microsatellites and sequences). We discuss how past environmental variations related to 137 the LGM may have affected genetic diversity and structure of the two CWC species across this wide 138 geographic region. We hypothesise that distinct ecological requirements in the deep-sea influence 139 population structure and the distribution of genetic diversity within species. The NE Atlantic genetic group along PC2 spans samples from the Bay of Biscay to the High latitudes, 288

including Rockall bank (Fig. 3b). The most important M. oculata genetic affinities show a cline on the 289 first axis, spanning samples from the lower (Mediterranean) to the higher latitudes. The gradient is 290 visible in the group membership proportions shown on the bottom of figure 3a, particularly along the 291 Bay of Biscay canyons and along Rockall-High latitudes populations.

292

Gene diversity and the average number of alleles per locus for L. pertusa and M. oculata samples 293 were high (L. pertusa H E 0.90 +/-0.08, and 30.4 alleles per locus; M. oculata H E 0.53 +/-0.23 and 12.3 294 alleles per locus; Tables 1 and2). Regionally, average allelic richness was higher at intermediate 295 latitudes for both species (Mediterranean 7 for L. pertusa and 3 for M. oculata, Bay of Biscay 16 and 296 5, Rockall Bank 15 and 6, and High Latitudes 12 and 4, for L. pertusa and M. oculata respectively, and 297

Mid Atlantic, where only M. oculata was sampled, 3). Private alleles were present in all sampled 298 L. pertusa sites, while they were rarely present in M. oculata populations. Madrepora oculata 299 presented higher private allelic richness in the Mediterranean basin (2-4). An excess of homozygotes 300 was observed in all L. pertusa populations and in half of the M. oculata sites, particularly along the 301 NE Atlantic populations.

302

The patterns of genetic structure inferred with ITS were generally concordant with those of 303 microsatellites. Based on statistic parsimony, a haplotype network was constructed for L. pertusa, with 304 one main ITS lineage inferred as the most likely ancestral haplotype (from a total of 39 haplotypes), 305 which possibly radiated from lower to higher latitudes (>60% of samples), and many rare haplotypes 306 (over 20 unique). S2-S3). Madrepora oculata showed a regional pattern of population genetic 337 structure concordant with microsatellite analyses (high F ST between populations from distinct regions). 338

Overall, 5% of L. pertusa and nearly 40% of M. oculata genetic variation occurred among regions 339

(AMOVA; Appendix Table S5). .

340

Haplotypic and molecular diversities varied among locations (Tables 3 and4). For L. pertusa, 341 haplotypic diversity was high (up to 0.6 in the NE Atlantic and up to 0.7 in the Mediterranean Sea) 342 due to haplotype radiations. Haplotypic diversity was also high in M. oculata populations (0.3-0.7 in 343

High Latitudes, 0.9 in the Mid Atlantic site, where there were four haplotypes for five individuals). A 344 comparable pattern of variation of molecular diversity was observed for both species. 3 and4).

356

Model-based inference pointed to an ancient (up to 30 ka; Appendix Table S2 geography and the environment on population structure is difficult. Finer-scale analyses are needed to 520 estimate the contribution of such variables and putative allopatric refugia.

521

The formation of the admixed Mid Atlantic population was dated to c. 2 ka (0.7-11 ka); even 522 accounting for wide confidence interval of the estimated time, it is well within the Holocene. During 523 this period, the modern AMOC was established and can have contributed to colonisation of deep coral 524 habitat along the Mid Atlantic Ridge and to admixture with older populations either from the 525

European margins (Rockall bank, Bay of Biscay canyons) or from the NW Atlantic (which we did not 526 sample here). The southward branch of the vigorous NAC may have favoured the link between 527

European margins and Mid Atlantic. The absence of Mediterranean ancestry together with the 528 substantial presence of High latitudes ancestry in Mid Atlantic and Rockall M. oculata substantiates a 529 less important connection with Mediterranean populations via the weak flow of Mediterranean Water. 530

The fact that the European margins genetic components and haplotypes are consistently shared among 531

Mid Atlantic M. oculata makes it tempting to equate this genetic component with the colonisation or 532 at least the spread of M. oculata in the Mid Atlantic. However, such a model may be overly simplistic.

533

As shown in our analyses, the admixture patterns found in M. oculata are complex and more than a 534 single admixture event is possible, with sources on both sides of the N Atlantic. The lower coral 535 abundance and available habitat as compared to the NE Atlantic margins, may have further contributed 536 to post-colonisation drift. More detailed analyses of Mid Atlantic CWC are needed to clarify the N 537

Atlantic phylogeography of the deep-sea.

538

The colonisation of higher latitudes much more recently (c. 0.3 ka, 0.07-3 ka) from Rockall 539 populations left genetic footprints; main M. oculata haplotype shared between High latitudes and 540

Rockall but absent from Bay of Biscay. The timing difference of High latitudes colonisation among L. 541 pertusa and M. oculata suggests that differences in environmental tolerance exist: L. pertusa reached 542

the High latitudes at the end of the LGM (c. 21 ka; 9-29 ka). In fact, L. pertusa has thermal 543 acclimation in respiration and calcification mechanisms that mechanisms are absent in M. oculata 544 (Nauman et al., 2014). The Holocene colonisation of the higher latitudes of Europe (e.g., Norway; Fig. 545 1) is well documented (Schröder-Ritzrau et al., 2005). Although reassuring for interpreting our 546 findings, ABC model-based inferences rely on assumptions that cannot easily be controlled in natural 547 populations. Our findings should be interpreted as probable hypotheses rather than clear proof of past 548 demographic events. Further theoretical and experimental work are needed that can contribute to a 549 better understanding of CWC phylogeography and connectivity (e.g., early life history traits). 550

Analyses making use of high-density data will allow reconstructing the phylogeographic history of 551 CWC with more precision. Moreover, our study highlights the need to explore the genetic nature of 552 CWC found in the Western Mediterranean (Alboran Sea) and the Gulf of Cadiz to determine their 553 contribution to larval connectivity and past re-colonisation of the NE Atlantic.

Conclusion

The novel genetic data presented here from deep-sea corals from European margins opens 555 new insights into Cold-Water coral population history. Our results show a northward post-LGM 556 expansion of Lophelia pertusa and M. oculata from the west Mediterranean Sea into the NE Atlantic 557 margin. While L. pertusa moderate to high gene flow homogenized nearly the whole Atlantic genetic 558 pool, M. oculata progressed slower, particularly in High latitudes. Lophelia pertusa expanded swiftly 559 along the NE Atlantic at a rate of 0.7 to 2 km per year (considering 4,000 km in the past 10 to 30 ka). 560

The period of Atlantic-Mediterranean separation led to a strong genetic differentiation between extant 561 coral populations in the respective regions. This differentiation can be explained either by a founder 562 effect at the NE Atlantic, or an unsampled genetic source in the Mediterranean Sea or around NW 563

Africa. The remarkable mosaic of distinct genotypes of M. oculata supports the existence of a more 564 complex history, shaped by putative cryptic refugia, admixture and possible different dispersal ability.

565

With current data limitations, it is not yet possible to determine the most important factor underlying 566 the apparent lack of gene flow along the European margin for

  coral occurrence suggests a common history in terms of range contraction and 123

Figure 2 -

 2 Genetic subdivision among regional samples. (a) Microsatellites population membership 619 diagrams from TESS displaying the probability of each individual into K = 4 clusters for 620 Madrepora oculata (left) and Lophelia pertusa (right) (denoted by different colours). Each individual a horizontal line partitioned into K coloured sections, with the length of each section 622 proportional to the estimated ancestry proportion to each cluster. Sample names refer to sites indicated 623 in table and Fig. 1. Samples from the Bay of Biscay canyons (left diagram) are geographically ordered 624 from north to south according to what is shown on Fig. 1. Black horizontal lines separate adjacent sites 625 (MAt -Mid Atlantic, Med. -Mediterranean). (b) Parsimony haplotype networks of Internal 626 Transcribed Spacer ribosomal sequences for M. oculata (left) and L. pertusa (right). The size of the 627 circle is proportional to the observed number of sequences for the corresponding haplotype. Pie charts 628 illustrate the proportion of a haplotype found in each region. The length of links is proportional to the 629 number of mutations separating two haplotypes; the shortest represent a single mutation. 630 Mediterranean includes eastern and western basin samples. Genetic subdivision among regional samples. Top -Discriminant Analyses of Principal 642 Components (DAPC). A minimum-spanning tree based on the squared distances between groups 643 connects group centres (crosses). Insets show the variance explained by the principal components 644 included in the analyses (black). Because the identified optimal number of genetic clusters (K) for 645 Lophelia pertusa is K=2 (see additional inset with sample density along the first discriminant 646 function), the DAPC plot for L. pertusa shows samples color-coded by sampling site. Bottom -647 Population membership diagrams displaying the probability of each individual colour-coded by 648 sampling site. , S., Van den Beld, I.M.J., Becheler, R., Orejas, C., Menot, L., Frank, N., Grehan, A. & Bourillet, 652 J.F. (2017) Two "pillars" of cold-water coral reefs along Atlantic European margins: Prevalent association

  regression of the posterior probabilities of the tested ABC demographic scenarios.

Figure 1 -Figure 2 -

 12 Figure 1 -. Contemporary distribution of M. oculata (black diamonds) and L. pertusa (grey circles) in the NE Atlantic, (2017 UNEP database; http://data.unep-wcmc.org) and the path of the main North East Atlantic Ocean currents (adapted from Montero-Serrano et al., 2011, Somoza et al., 2014 and Cossa et al., 2018): MOW -Mediterranean Outflow Water; MW -Mediterranean Sea Water; NAC -North Atlantic Water; MNAW -Modified North Atlantic Water; EAAIW -Eastern Antarctic Intermediate Water; LSW -Labrador Sea Water; ISOW -Iceland-Scotland Overflow Water. Site abbreviations as in Table1. Dates correspond to estimated ages for coral mound growth (adapted fromSchröder-Ritzrau et al., 2005). Insets are the selected demographic history models for each cold-water coral species. ka -thousand years ago. Map created on QGIS with Mollweide's equal area projection.296x209mm (300 x 300 DPI)

Fig. 1 .

 1 Figure 2 -Genetic subdivision among global regional samples. (a) Population membership diagrams displaying the probability of each individual into K K = = 4 clusters for Madrepora oculataMadrepora oculata (left) and K = 2 for Lophelia pertusaLophelia pertusa (right) (denoted by different colours). Each individual is depicted by a horizontal line partitioned into K coloured sections, with the length of each section proportional to the estimated membership ancestry coefficient proportion (Qind) to each cluster. Sample names refer to sites indicated in table andFig. 1. Samples from the Bay of Biscay canyons (left diagram) are geographically ordered from north to south according to what is shown on Fig. 1. Black horizontal lines separate adjacent sites (MAt -Mid Atlantic, Med. -Mediterranean). The map shows the distribution of M. oculata (black diamonds) and L. pertusa (grey circles) known occurrences, according to the 2017 UNEP database (http://data.unep-wcmc.org). Map created on QGIS with GDAL bathymetry and the equal area Mollweide projection. (b) Parsimony haplotype networks based onof Internal Transcribed Spacer ribosomal sequences for M. oculataM. oculata (left) and L. pertusaL. pertusa (right). The size of the circle is proportional to the observed number of sequences for the corresponding haplotype. Pie charts illustrate the proportion of a haplotype found in each region. The length of links is proportional to the number of mutations separating two haplotypes; the shortest represent a single mutation. Mediterranean includes Ionian Seaeastern and western basin samples.

Figure 3 -

 3 Figure 3 -Genetic subdivision among regional samples. Top -Discriminant Analyses of Principal Components (DAPC). A minimum-spanning tree based on the squared distances between groups connects group centres (crosses). Insets show the variance explained by the principal components included in the analyses (black).Because the identified optimal number of genetic clusters (K) for Lophelia pertusa is K=2 (see additional inset with sample density along the first discriminant function), the DAPC plot for L. pertusa shows samples color-coded by sampling site. Bottom -Population membership diagrams displaying the probability of each individual colour-coded by sampling site.218x181mm (300 x 300 DPI)

Results 246 3.1 Core collection and dating 247 Fossils of Lophelia pertusa and Madrepora oculata were found in the gravity core. The dating of 248

  bp) were proof-read and alignedusing GENEIOUS v6.1 (Kearse et al., 2012). Nine 168 L. pertusa(Morrison et al., 2008; Becheler et al., 2017) and six M. oculata microsatellite markers 169 were amplified following Becheler et al. (2017). Products were scored using GENEIOUS v5.6.4. One L. 170 pertusa locus was discarded due to high frequency of null alleles (>30%). Clones were removed from 171 each dataset (ITS and microsatellites). Statistical power was assessed for the two coral species 172 (Appendix Supplementary methods S2). Only sites with five or more samples were kept for analyses.

	167 216 217 218 219 220 221 M. oculata 1124 173 M. oculata we used the geography-based population grouping. We compared competing scenarios in order to identify the patterns of post-LGM CWC range expansion along the NE Atlantic margins. Scenarios 1, 2 and 4 determine the pattern of colonisation of the northern edge of the NE Atlantic range, comparing models of stepping-stone colonization from the Mediterranean Sea to the Bay of Biscay and then further north (Scenario 1 -Stepwise range expansion), and models of admixture: between the Bay of Biscay, High latitudes and the west Mediterranean to produce the Rockall bank population (Scenario 2), between the High latitudes and Rockall bank to produce the Mid Atlantic 222 2.4 Phylogeographic patterns and genetic diversity 223 population (Scenario 2 M. oculata), between the Bay of Biscay and Rockall bank to produce the Mid
	174 224 175 225 176 226 177 227 178 228 179 229 180 230 181 231 182 232 233 234 235 236	F o r Atlantic population (Scenario 4 M. oculata), and between the Mediterranean Sea and the Bay of Population genetic differentiation for ITS-sequences was assessed with pairwise haplotype F ST -statistic Biscay to produce the Mid Atlantic population (Scenario 5 M. oculata). Scenario 3 examined the (Weir & Cockerham, 1984) in ARLEQUIN v3 (Excoffier et al., 2005). The distribution of genetic demographic history of the NE Atlantic and Mediterranean populations with changes in population variability within and among groups was estimated with an Analysis of Molecular Variance size since colonisation. We compared a null model of no change in population size (Scenarios 1 and 2) (AMOVA) with ARLEQUIN on four groups for L. pertusa, and five for M. oculata (1. Mediterranean, to a model of a short bottleneck (10 generations) during colonisation followed by population 2. Bay of Biscay, 3. Rockall Bank and 4. High Latitudes, and 5. Mid Atlantic). Population genetic expansion (Scenario 3). In this scenario, at each split, there is an initial reduction of the population size diversity was estimated with: i. the number of distinct and private haplotypes, ii. number of of the newly formed population, because the expansion is assumed to start with few immigrants. For polymorphic sites, iii. haplotypic diversity (Nei, 1987), and iv. the mean number of pairwise L. pertusa we used the known time of Rockall bank colonisation (approx. 9 ka; Frank et al., 2009) to differences (Tajima, 1983). Haplotype relationships were visualized with statistical parsimony calibrate divergence times (mode and 95% confidence interval) from the estimated ABC number of networks,on TCS v1.21 (Clement et al., 2000) generations; we then assumed the same generation time (est. approx. 3 years) for M. oculata. F We generated 3 to 5x10 6 simulations for each scenario tested using the combined microsatellite and o ITS datasets for M. oculata and the microsatellite dataset for L. pertusa (Appendix Supplementary r methods S2) to produce a set of pseudo-observed datasets (PODs). Effective population sizes and
	190 191 237 238 239 240 241 242	P e e r Bayesian clustering was done with TESS v2.3 (François et al., 2006; Chen et al., 2007) via the tess3r R with package (The R Foundation for Statistical Computing, 2018). parameters for the Generalized Stepwise Mutation and Kimura two parameter models were set to default values, while time of population divergence (in generations) was defined as uniform within P post-LGM periods (for model parameters see Appendix S2). The posterior probability of each scenario e was inferred with a logistic regression performed on the 1% of PODs closest to the empirical data. We empirically evaluated the power of the model to discriminate among scenarios using a Monte Carlo e r estimation of false allocation rates (type1 and 2 errors) resulting from ABC posterior probabilities-
	192 193 194 195 196 197 198 199 200 201 243 244 245	R e v i e w We used Discriminant Analysis of Principle Components (DAPC; Jombart et al., 2010) implemented in the R package adegenet (Jombart, 2008), to determine whether the genotypes of L. pertusa and M. oculata were distinct between different sampling sites. We used k-means clustering (k=2 to maximum number of sampling sites) with the Bayesian Information Criterion (BIC) to identify an optimal number of genetic clusters that describe the data, as well as the α-score optimisation procedure to identify the optimal number of principal components (PCs) to retain (retaining too many PCs can lead to overfitting the discriminant functions). The α-score optimisation showed that approximately 25 PCs needed to be retained for M. oculata (>69.3% of the total variance), and 60 PCs for L. pertusa (28.9%). Next we used DAPC to derive membership probabilities for each individual in each of the groups using location priors (see Appendix based model selection. R Supplementary methods can be found in the Appendix (S2). e w e i v 3.
	202	Supplementary methods S2).
	203	2.5 Demographic inferences
	157 204	2.2 Core collection and dating Historical fluctuations in population size for each sampled site were detected using Fu's Fs (Fu, 1996)
	205	and Tajima's D (Tajima, 1989) on ITS data from L. pertusa and M. oculata. Departures from mutation-
	158 206	Within the Guilvinec Canyon (Bay of Biscay), a 1.2 m-length sediment core (BBCO-CS01) was drift equilibrium were tested under the null hypothesis of no significant change of effective population
	159 207	collected (N46°56,045'; W005°21,642') at 815 m-depth. The core is representative of the superficial size, with ARLEQUIN. A negative value significantly different from constant population size is
	160 208	layers of the Armorican margin. Remains of coral skeleton were dated using the U/Th method at the interpreted as a signature of population expansion; the significance of Fs and D values was determined
	161 209	Institute for Environmental Physics at Heidelberg University (Schröder-Ritzrau et al., 2003). by randomization.
	162 163 210 211	Foraminifera from nearby sediments were 14 C-dated. Corals and foraminifera were subsequently The evolutionary and demographic histories of NE Atlantic and Mediterranean CWC (L. pertusa and calibrated to absolute ages using Intcal13 (Reimer et al., 2013). M. oculata) were further reconstructed using Approximate Bayesian Computations (ABC) as
	212	implemented in DIYABC v2.1.0 (Cornuet et al., 2014). We used microsatellite and ITS sequence data
	213	for M. oculata and microsatellite only for L. pertusa. Lophelia pertusa populations were grouped
	214	according to the microsatellite Bayesian clustering (cluster 1 -Eastern Mediterranean, cluster 2 -
	215	Western Mediterranean, cluster 3 -Bay of Biscay and High latitudes, cluster 4 -Rockall bank) and for
		5 6

In particular, we expected 140 that L. pertusa would present low levels of population differentiation across the NE Atlantic margins, 141 consistent with a post-LGM northward expansion from southern refugia, and maintained by present-142 day connectivity. We expected M. oculata to present stronger population differentiation, consistent 143 with lower fecundity. To these aims, we examined the genetic structure and diversity of L. pertusa and 144 M. oculata within and among the principal biogeographic provinces of the NE Atlantic and the 145 Mediterranean Sea where CWC are known to occur. 146 2. Materials and methods 147 2.1 Field collections 148 Two-hundred and seventy samples of Lophelia pertusa and 260 of Madrepora oculata were collected 149 from six regions between 250 and 1,170 m-depth: the eastern Mediterranean basin (Ionian Sea, south-150 eastern Adriatic Sea); western Mediterranean basin (Gulf of Lions); Mid-Atlantic region (Azores); 151 Bay of Biscay; Rockall Bank (off western Ireland); and the High latitudes (Iceland) (Fig. 1; Appendix 152 Table S1). In one canyon of the Bay of Biscay, Petite Sole, two sample collections were made at 153 different sections of the canyon. Samples were stored in 96% ethanol and stored frozen at -20 or -80°C 154 prior to DNA extraction. Lophelia pertusa DNA was extracted aboard using the Fast DNA®SPIN for 155 soil kit, according to the manufacturer protocol (MP Biomedicals, France); for M. oculata we used 156 cetyl trimethyl ammonium bromide (Doyle & Doyle, 1988). 164 2.3 DNA amplification and sequencing 165 The Internal Transcribed Spacer (ITS) ribosomal sequence was amplified using primers developed by 166 Diekmann et al. (2001). ITS-sequences (262 samples of L. pertusa 1130 base pair, bp, 200 samples of 183 Microsatellite genetic diversity and structure were estimated with GENETIX v4.05 (Belkhir K., Borsa 184 P., Chikhi L., 2004). The mean number of alleles per locus standardized to the lowest number of 185 samples collected; estimates of observed (H O ) and unbiased (H E ) multi-locus heterozygosity (Nei, 186 1978); the F IS -statistic and its significance (tested with 1000 permutations); and linkage 187 disequilibrium, assessed through the index ř d with MULTILOCUS v1.3 188 (http://www.bio.ic.ac.uk/evolve/software/multilocus/) were calculated. Genetic structure (pairwise 189 F ST ) was estimated with θ (Weir & Cockerham, 1984). Inference of spatial population structure CWC remains revealed ages ranging from present to 6,959 ± 169 years (U/Th corrected age). 249 3.

2 Phylogeographic patterns and genetic diversity 250

  Lophelia pertusa showed four genetically distinct groups: the eastern and the western Mediterranean 251 Sea, the Rockall bank and the remaining NE Atlantic populations (Bay of Biscay and High latitudes) 252 (Fig. 2a right). Within L. pertusa, the Mediterranean genetic ancestry, and in particular the west 253 Mediterranean ancestry (blue) can be seen in low proportions across all NE Atlantic sites. The green 254 genetic ancestry is maximized in Bay of Biscay and High latitudes populations, while the grey genetic 255 ancestry (found in the Bay of Biscay and High latitudes sites) is maximized in the Rockall bank 256 population. The grey ancestry component is present in high proportion in the High latitudes 257 populations, particularly in Hafadsjup (HAF), which comprises over half of L. pertusa ancestry. 258 Genetic differentiation was strong between east and west Mediterranean populations and weak across 259 the NE Atlantic samples, with nearly all values not significantly different from zero. The Rockall bank 260 population was differentiated from over half of the Bay of Biscay locations but not with the High 261 nearly absent from the Lampaul canyon, as well as from the Rockall bank and Mid Atlantic 266 populations (which together form the fourth cluster). M. oculata populations from the Bay of Biscay 267 harbour a strong Mediterranean genetic component (red) maximized in the northern Bay of Biscay 268 canyons (Morgat, Crozon and Petite Sole), which is absent from most individuals in the Mid Atlantic, 269 Lampaul canyon (in the Bay of Biscay) and Rockall bank populations. The NE Atlantic genetic 270 ancestry (green) is well represented in all NE Atlantic populations and in smaller proportion in some 271 Mediterranean individuals. The High latitudes genetic ancestry (grey) is found in high proportion in 272 Rockall and Mid Atlantic populations. Mean pairwise F ST between regions was high, up to 0.5 273 between the eastern Mediterranean Sea and the High Latitudes, but not significantly different from 274 zero between eastern and western Mediterranean Sea populations, while nearly all pairwise F ST values 275 were significantly different from zero. Lack of population differentiation was observed between two 276 Bay of Biscay canyons (CRZ-LMP), within one canyon (PS1-PS2), between High latitudes locations, 277and between Rockall bank and its closest High latitudes site, Londsjup (Appendix TableS4.2). 278Regional F ST was significantly different from zero for all pairwise comparisons for the two coral 279 species: Mediterranean Sea, Mid-Atlantic, Bay of Biscay, Rockall bank and High latitudes. The lowest 280 regional F ST for M. oculata was between the High latitudes and Rockall bank populations (0.051) and 281for L. pertusa between the High latitudes and Bay of Biscay populations (0.004).

	265	cluster) is
		F o r
		P e e r
		R
		e v i e w
		7

latitudes populations (Appendix Table

S4

.1). Clustering analyses showed that M. oculata was also 262 structured in four genetic clusters, but with a different organisation (Fig.

2a

). The Mediterranean Sea 263 and the High Latitudes formed two distinct clusters (displayed in red and grey on Fig.

2a

left). The 264 next two clusters are not so geographically coherent: The Bay of Biscay genetic ancestry (blue; third 282 The genetic affinities of the sampled individual corals were projected onto principal components 283 (DAPC). As expected, the first axis of L. pertusa separates Mediterranean and NE Atlantic 284 populations. Within each group, genetic variability is spread across the second axis: The Eastern 285 Mediterranean samples at one end and the Western Mediterranean samples at the other end, a 286 separation that is also clearly visible in the group membership probabilities on the bottom of figure 3b. 287

  The ancestral haplotype is nearly absent from Rockall bank L. pertusa, supporting 307 the Bayesian clustering, where High latitudes and Bay of Biscay share a genetic ancestry (green) exclusive to its CWC reefs. L. pertusa haplotypes are not particularly clustered 318 and, except for the inferred ancestral haplotype, most haplotypes are found in moderate to low 319 frequency at each site (Appendix Table S7). There are no L. pertusa region-specific haplotypes in the 320 Bay of Biscay or the west Mediterranean Sea. 321 There was some phylogeographic clustering for the M. oculata haplotype network, with one lineage 322 dominant in the Higher latitudes and Rockall bank, modestly present in distant, southern, geographic 323 locations (e.g., one samples from the Mediterranean Sea), and absent from the Bay of Biscay. A 324 quarter of all haplotypes are shared across distant geographic regions (i.e. High Latitudes and 325 Mediterranean Sea). Haplotypes found in Mid-Atlantic M. oculata are also found in all other regions 326 (one haplotype) or are restricted to the Bay of Biscay and Rockall bank (two haplotypes), with one 327 haplotype unique to the Mid Atlantic site. Private (unique) haplotypes were observed in all regions 328where M. oculata was sampled but not at all sites. Seven haplotypes were well represented, and there 329 was no obvious sign of haplotype radiation (Fig.2b). Similarly to L. pertusa, the majority of ITS 330 sequence variation for M. oculata occurred in Atlantic sites. The Bay of Biscay and Rockall bank 331 presented the highest number of haplotypes (15 and 11, respectively) and the only region-specific 332 haplotype (Appendix tableS7). Nonetheless, the highest haplotype diversity (H=0.9) was observed in 333 M. oculata from the Mid Atlantic.

	317	with two haplotypes
	308 309 310 311 312	almost absent from Rockall bank samples. Two other well-represented haplotypes were restricted to: i) the Bay of Biscay, High Latitudes and Mediterranean Sea, and ii) mostly the Rockall Bank with few Bay of Biscay samples (Fig. 2b), supporting the Rockall bank genetic ancestry (grey) as distinct from relatively close populations (Bay of Biscay and High latitudes). The Atlantic sites harbour the majority o r P e e r R of sequence variation. F e v i e w
		8

The Bay of Biscay has the highest number of haplotypes (24), with 12 313 haplotypes unique to this region, but the highest haplotype diversity (H=0.7) was found in the west 314

Mediterranean Sea (Table

3

). Ten haplotypes were observed in the Mediterranean Sea with two unique 315 to the western Mediterranean. Iceland, regardless of the high latitude and recent CWC fossil ages (8.3 316 Ka, Fig.

1

), harbours the second highest number of L. pertusa haplotypes (14; Appendix Table

S7

),

334

Along the NE Atlantic L. pertusa, populations were little differentiated while within the Bay of Biscay 335 genetic structure was weak for M. oculata (nearly all pairwise F ST not significantly different from zero; 336 Appendix Tables

  ) divergence of L. 357 pertusa east and west Mediterranean Sea populations, in agreement with DAPC. The west 358 Mediterranean population was identified as the source from which all other NE Atlantic populations 359 emerged, either directly (Bay of Biscay and High latitudes; approx. 21 ka) or through admixture 360 (Rockall bank; much later at 9 ka), agreeing with the Bayesian clustering, where the west 361 Mediterranean ancestry (blue) is found throughout the Atlantic sites, and with ITS network, where 362 around 0.2 support for scenarios including stepping-stone colonisation and 368 changes in population sizes (Appendix Figs. S3-S4). Confidence in scenario choice was high, with low 369 error rates (posterior predictive error = 0.29). Post-LGM range colonisation followed a long-range 370 model of northern range colonisation, whereby Bay of Biscay and High latitudes were colonised 371 directly from the west Mediterranean Sea populations, while at a later stage the Rockall bank was 372 colonised from admixture between Bay of Biscay, High latitudes and west Mediterranean populations 373 (Fig. 1 bottom inset). 374 Analyses with ABC pointed to the Mediterranean Sea M. oculata populations being the main source 375 population (N=8,600) from which all other NE Atlantic populations emerged. The Mediterranean 376 source population scenario, with colonisation of the Atlantic margins via Bay of Biscay agrees with 377 Bayesian clustering, where the Mediterranean genetic ancestry (red) is found in moderate proportions 378 in Bay of Biscay populations and is absent from the Mid Atlantic and Rockall populations. The 379 scenario of a Mediterranean source population with colonisation of the NE Atlantic and admixture was 380 supported with high probability (0.9) relative to low support for scenarios including changes in 381 population sizes or competing admixture scenarios (Appendix Figs. S3-S4). Confidence in scenario 382 choice was high, with low error rates (0.3). Post-LGM range colonisation along the European margins 383 followed a stepping-stone model of northern range colonisation, whereby Bay of Biscay was colonised 384 directly from Mediterranean Sea populations (22 ka), while the Bay of Biscay M. oculata later 385 colonised the Rockall bank (7 ka). The Rockall bank populations then originated other NE Atlantic M 386 oculata populations. That happened either through admixture with the Bay of Biscay (Mid Atlantic) 387 only about 2 ka, or directly (High latitudes) much more recently (0.3 ka; Fig. 1 top inset). The genetic 388 footprint of those events are seen on the main haplotype shared between High latitudes and Rockall 389 but absent from Bay of Biscay corals, by a haplotype restricted to the Bay of Biscay, Rockall bank and 390 Mid Atlantic (haplotype V in Appendix Figure S6), and by the position of Mid Atlantic samples in the 391 DAPC between Rockall and Biscay samples. We hypothesize that both 404 species were affected by glacial events differently, and/or that the geographic location of the source 405 and date of post-glacial expansion of NE Atlantic reefs differed. Verifying such hypotheses is 406 important if we are to forecast the responses of CWC facing on-going climate changes. Ritzrau et al., 2005; Eisele et al., 2011; Ramos et al., 2017; Weinberg et al., 2018). The 432 rapid increase in post-glacial Atlantic Meridional Overturning Circulation resulted in increased 433 transport of suspended particles and is speculated to have boosted the expansion of L. pertusa from 434 these southern locations (Mediterranean Sea and/or adjacent NW coast of Africa) into the NE Atlantic 435 In the temperate Eastern Atlantic, including the Mediterranean Sea, CWC growth 468 sustained glacial-interglacial cycles (Frank et al., 2011). Such a steady allopatry may have been 469 maintained by vicariance driven by ocean currents acting upon the earliest life stage of L. pertusa or 470 by local adaptation. Finer-scale analyses are needed to identify and quantify the importance of such 471 variables. All together, the data support a post-glacial recolonisation of the NE Atlantic by L. pertusa. 472 Mediterranean L. pertusa are divergent from most NE Atlantic populations (ITS, microsatellites). 473 Given that sampling is at best partial, it is difficult to speculate about the precise origin of NE Atlantic 474 populations. The Mediterranean lineages and genotypes found in low proportion across the NE 475 Atlantic reefs, the high haplotypic diversity mainly in the western basin of the Mediterranean Sea, 476 concur with ABC analyses that the glacial refugia for NE Atlantic L. pertusa colonisation may have 477 been located in the west Mediterranean Sea or in adjacent regions (e.g. the coral mounds in the Gulf of 478 Cadiz and NW Africa; Eisele et al., 2011). Lophelia pertusa populations in this region thrived during 479 glacial periods and might have provided larvae dispersing in Mediterranean Water northwards towards 480the Bay of Biscay (Fig.1). The result is the co-existence of divergent genetic lineages across the NE Atlantic, one 503 dominating the higher latitudes and another in the Bay of Biscay, both with lower frequency in other 504 regions. Such pattern is consistent with distinct areas of persistence for M. oculata, locally or 505 regionally, followed by a redistribution of divergent lineages after periods of allopatry(Petit et al., 506 2003). The low occurrence of unique haplotypes and alleles found in M. oculata is a result of the more

	F o r P e e r R e v i e w Despite the deep-sea being considered as relatively environmentally stable over time, there is evidence that CWC undergo demographic changes similarly to those experienced by their coastal water counterparts (Wilson & Eigenmann Veraguth, 2010; Sabelli & Taviani, 2014; Vertino et al., 2014; Quattrini et al., 2015). Here we focused on the LGM, and explored two scenarios that could explain the observed patterns of genetic diversity and structure for L. pertusa and M. oculata, respectively: i) Before the LGM (and to about 50 ka, which is the oldest period accounted for by existing via admixture of the Bay of Biscay group and west Mediterranean corals, around the time of the onset of the modern Atlantic Meridional Overturning Circulation (AMOC; Repschläger et al., 2017). The Mediterranean Water traveling northwards along the Iberian Peninsula and Bay of Biscay does not reach the Rockall bank (Frank et al., 2009). Instead, Rockall hydrodynamics are more influenced by northward moving Atlantic waters, including the rich North Atlantic Current (NAC). The NAC is part of the Gulf Stream, which may promote occasional genetic material from the West North Atlantic L. recent admixture and colonisation events (Mid Atlantic, High latitudes), estimated by ABC to have taken place during the late Holocene. The large-scale spatial genetic structure was shaped by the refugium-driven vicariance. Long-distance dispersal and gene flow that may disturb this pattern of population structure may be unlikely in M. oculata, if this CWC presents different reproduction and dispersal modes to those of L. pertusa, for instance, lower pelagic larval duration, brooding. A hypothetical poorer ability to disperse associated with the complex geomorphology (canyons, data)F o r P e e r R e v i e w (Schröder-452 367 408 409 410 411 412 413 457 458 459 460 461 462 507 508 509 510 511 512 513 431 pertusa. 453 The genetic distinctiveness of Rockall bank L. pertusa (seen on microsatellites and ITS loci) indicates 454 a more complicated history. The rarity of the dominant haplotype in Rockall bank suggests either 455 another source for colonisation, strong post-colonisation drift or local adaptation. Approximate 456 Bayesian Computation favours an evolutionary scenario whereby the Rockall population is colonised F o r P e e r R e v i e w 482 demographic expansion. 483 Scenario ii) F o r P e e r R e v i e w probability relative to 407 417 ii) Before the LGM, NE Atlantic populations were absent from the current northern range of their 418 modern distribution and present-day patterns of genetic variation were caused by "stepping-419 stone" northwards colonisation with modest demographic expansions and by a much later 420 admixture between NE Atlantic populations with possible contribution from other unsampled 421 populations. 422 Scenario i) 423 Geological studies report the absence of deep CWC reef growth in the NE Atlantic during glaciations 424 of the Late Pleistocene (approx. 126 -12 ka; with the Last Glacial Maximum approx. 26.5-19 ka; 425 Clark et al., 2009; Frank et al., 2009). Contrastingly, between 50-12 ka, L. pertusa reefs flourished in 426 the Mediterranean Sea (Appendix Supplementary Paleo-History). The Mediterranean Outflow Water 427 (MOW 800-1300 m depth; Price et al., 1993) volumes were smaller than at present (Rogerson et al., 428 2004) but concentrated in channels with high local flow (Zahn et al., 1997). In the Gulf of Cadiz and 429 on the Moroccan shelf, reef growth was estimated to have started around 40-50 ka (Fig. 1) when 430 glacial conditions there were particularly favourable for coral growth, unlike in the NE Atlantic 467 Contrastingly, M. oculata populations are more strongly structured and there is no evidence of a large 518 differences may have created (past) barriers to gene flow. However, separating the exact effects of reasonable. 481 519
	9 10 11 12

most Mediterranean haplotypes are shared with Bay of Biscay corals. The shared Rockall genetic 363 ancestry (grey) between Biscay and the High latitudes populations identified with Bayesian clustering 364 agrees with the admixed origin for Rockall L. pertusa found with ABC. Population size estimates were 365 much smaller for Mediterranean populations (N=2,500 to 7,000) than NE Atlantic ones (N=70,000-366 85,000). The Bay of Biscay demographic expansion scenario with admixture was supported with high 392 Discussion 393 In terms of molecular analyses, Cold-Water corals (CWC) in NE Europe have been understudied. In 394 this study we extend the available information from this area considerably, and present the first 395 inferences and empirical analyses of deep-sea coral phylogeography in the area. While the Bay of 396

Biscay genetic component presented here has been previously described in extant populations from the 397 region

(Morrison et al., 2011; Becheler et al., 2017)

, we gain further insights into its temporal depth.

398

Given their recurrent association and similarities

(Arnaud-Haond et al., 2017)

, the two main CWC reef 399 building species in the North Atlantic, Lophelia pertusa and Madrepora oculata, have been 400

hypothesised to share similar phylogeographic histories

(De Mol et al., 2002; Henry et al., 2014)

. Yet, 401 our results depart from that expectation. Far-reaching gene flow along the European margin results in 402 a poor genetic differentiation in distant NE Atlantic L. pertusa populations (Bay of Biscay and High 403 latitudes), while high differentiation characterizes populations of M. oculata. , NE Atlantic populations were absent from the current northern range of their modern 414 distribution and present-day patterns of genetic variation were caused by expansion 415 northwards, originated most likely from the west Mediterranean Sea and/or adjacent NW coast 416 of Africa, and a later Mediterranean-NE Atlantic admixture; (Eisele et al., 2011; Fink et al., 2013; Henry et al., 2014). 436 Continuous and vertical growth of CWC reefs occurred in the Bay of Biscay area during at least the 437 past 7,000 years, shown by geological analyses of the core. Genetic evidence of recent L. pertusa 438 demographic expansions in the Bay of Biscay, at the end of the LGM (approx. 21 ka), is substantiated 439 by Internal Transcribed Spacer sequences with haplotype network and conformity tests, attributed to a 440 founder event, i.e. a dominant haplotype found in all regions (from the Mediterranean Sea to the High 441 Latitudes). Such demographic expansion would have produced satellite haplotypes with low genetic 442 distance, as observed in L. pertusa. The vast nearly-panmictic ensemble occurring along the NE 443 Atlantic indicates a recent common history for Atlantic L. pertusa reefs. This is supported by studies 444 of reproduction and larval development that indicate that L. pertusa larvae have high dispersal 445 potential (Waller & Tyler, 2005; Larsson et al., 2014; Strömberg & Larsson, 2017). Despite these 446 experiments in aquaria with L. pertusa that have shown larvae swimming towards the surface, it 447 remains unknown whether larvae are neutrally buoyant and if in their natural environment remain at 448 spawning depth, concurrent with the depth of North Atlantic currents in figure 1. Larval dispersal 449 trajectories may have evolved to track specific water masses (Dullo et al., 2008), occurring at 450 intermediate depths with potential settling sites. Such high larval dispersal ability favours large-scale 451 expansion and shared genetic ancestry, such as that between Bay of Biscay and High latitudes L. 463 pertusa reefs (Morrison et al., 2011). Analyses encompassing the full distributional range of L. pertusa 464 are essential to clarify missing links in population relatedness at trans-Atlantic scales. 465 The ancient divergence of Mediterranean L. pertusa populations from the eastern and western basins 466 estimated by the ABC model to have taken place during the Last Glacial Maximum (around 24 ka) is 484 Concurrent to L. pertusa, Mediterranean Sea M. oculata populations were the main source from which 485 all other NE Atlantic populations emerged around the end of the LGM, as estimated by model-based 486 inference (Fig. 1 top inset). Mediterranean M. oculata thus represent the putative LGM refugium, 487 which may have included other unsampled adjacent populations present during glacial periods (Gulf 488 of Cadiz, NW Africa). As for L. pertusa, absence of samples from the NW coast of Africa meant that 489 inference regarding the role of this potential refugium could not be made. The estimated time for NE 490 Atlantic colonisation, approx. 21 ka, suggests that Mediterranean M. oculata represent a "stable rear-491 edge" population (Hampe & Petit 2005). Mediterranean harbours high levels of DNA diversity and the 492 greatest number of unique haplotypes and private alleles, a testament to the long-term stability of 493 Mediterranean populations (Hewitt 2000). But unlike L. pertusa, there is no substantial differentiation 494 of east and west Mediterranean M. oculata, possibly linked to differences in reproductive or early life 495 traits. Information on reproduction and early life history is much needed to reduce model uncertainty 496 and allow a better understanding of CWC past and present connectivity. 497 Analyses identified that M. oculata's northern range edge was colonised in a stepping-stone manner 498 (Bay of Biscay, followed by Rockall and later the High latitudes) instead of long-range colonisation 499 directly from glacial refugia as in L. pertusa northern populations. Unique haplotypes were almost 500 evenly spread across the distribution range, indicating that M. oculata maintained other glacial 501 (cryptic) refugia throughout its current range and underwent only localized, modest, postglacial 502 expansions. seamounts) along with extreme depth gradient present in habitats along the North East Atlantic 514 (hundreds to thousands of meters), has probably caused fragmentation, reduced gene flow and further 515 reinforced the large-scale pattern of NE Atlantic population structure in M. oculata. For instance, the 516 Lampaul canyon has the highest composition of soft substrate and near absence coral framework 517 compared to the other Bay of Biscay canyons studied here (van den Beld et al., 2017). Such

Table 1 -

 1 M. oculata populations. Further CWC 567 from the North Atlantic are needed in order to conclude, to what extent these signals of migration and 568 admixture are representative of N Atlantic CWC as a whole. Nonetheless, the contrasting patterns of 569 genetic diversity observed here strongly support the existence of a different influence of past differing present-day dispersal of L. oculata and M. oculata. Our study 571 provides an important warning for managers that even taxa with seemingly similar ecological roles 572 and tolerances may well differ in their response to global change. Therefore, multi-species models are 573 required to ensure conservation measures such as truly representative and connected networks of 574 Marine Protected Areas. Genetic diversity at microsatellite loci for Lophelia pertusa. n -sample size; Âr -allelic richness; Âr(5) -standardized allelic richness to the lowest 586 sample size (N=5); Âp -private allelic richness; He and Ho -expected and observed heterozygosity respectively; Fis -departure from HWE. P-values below 587 0.05, 0.01 and 0.001 are respectively represented by *, ** and ***.

	570 environmental changes and 575 Author contributions:
	576	Designed research RB, SAH,
	577	Performed research RB, SAH, JB, A-L C, OM, MC
	578	Contributed new reagents or analytic tools SAH, JFB, NF, MT, AS, AG, JB
	579	Analysed data RB, JB, SAH
	580 581 582	Wrote the paper JB, RB, SAH F Data Accessibility Statement o r Microsatellite data are accessible online at GenBank under accession numbers: KF944311, KF944317,
	583	r e e P KF944323, KF944330, KF944331 and KF944344.
		R
		e v i e w
		13

Table 3 -

 3 Genetic diversity and neutrality tests for ITS at each location of Lophelia pertusa. Asterisk (*) -P-values under 0.05. NA -Not applicable (Lampaul 594 canyon had no polymorphic sites). Contemporary distribution of M. oculata (black diamonds) and L. pertusa (grey circles) in 601 the NE Atlantic, (2017 UNEP database; http://data.unep-wcmc.org) and the path of the main North 602 East Atlantic Ocean currents (adapted from Montero-Serrano et al., 2011, Somoza et al., 2014 and 603 Cossa et al., 2018): MOW -Mediterranean Outflow Water; MW -Mediterranean Sea Water; NAC -604 North Atlantic Water; MNAW -Modified North Atlantic Water; EAAIW -Eastern Antarctic 605 Intermediate Water; LSW -Labrador Sea Water; ISOW -Iceland-Scotland Overflow Water. Site 606 abbreviations as in Table 1. Dates correspond to estimated ages for coral mound growth (adapted from 607 Schröder-Ritzrauet al., 2005). Insets are the selected demographic history models for each cold-water 608 coral species. ka -thousand years ago. Map created on QGIS with Mollweide's equal area projection.

	No.			Haplotype	Nucleotide
	Samples	No.	No. Private	diversity	diversity	Tajima's
	(n)	Haplotypes	haplotypes	(H)	(π)	D	FS

  Grorud-Colvert, K., Guidetti, P., Jenkins, S.R., Jones, P.J.S., Lester, S.E., McAllen, R., Moland, E., Ronan Becheler is a postdoctoral researcher in evolutionary ecology and population genetics. His 852 research interests concern the evolutionary consequences of reproductive systems, with a special 853 emphasis on partial clonality. He has worked on the assessment of dispersal in coastal and deep-sea 854 species. Currently, he studies the evolution of reproductive strategies and local adaptation in temperate 855 macroalgae.856Joana Boavida is a postdoctoral researcher interested in biodiversity-related disciplines, from 857 taxonomy to biogeography and macroecology, typically using corals as model systems.

	701 801 802 849 850 851 858 859 860 861	F o r F o r Mauritania. Deep-Sea Ecosystems Off Mauritania (ed. by A. Ramos), J.L. Sanz), and F. Ramil), pp. 481-525. Springer Netherlands. xi. Biosketch xii. Appendices An appendix shows supporting information E.J., F o r F o r xiii. Supporting information is supplied in a separate file F o r TABLE S1 -SUMMARY OF SAMPLING LOCATIONS
	862	S2 -ADDITIONAL METHODS	P e P e P e P e e P
			e r e r e r e r e r
			R R R R R
			e v e v e v e v e v
			i i i i i
			e e e e e
			w w w w w
			21 23 24

653

of Madrepora oculata with Lophelia pertusa, from reef to colony scale.

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Zibrowius, H. (1980) Les Scléractiniaires de la Méditerranée et de l'Atlantique nord-oriental, Institut

BOBECO, IceCTD, and CE98. We thank Franck Lartaud and Nadine Le Bris 41 N. Le Bris) for providing Mediterranean samples. Thanks to Lars-Eric Heimbürger for english edits. 42 JB was supported by the European Union's H2020 research and innovation program under grant 43 agreement Number: 678760 (ATLAS). This work is a contribution to the European Union's FP7 and 44 Horizon 2020 projects, respectively CoralFISH (under grant agreement no. 213144) and ATLAS 45 (under grant agreement no. 678760), samples from the Adriatic Sea through the EU Coconet program. 46 This is Ismar-CNR, Bologna scientific contribution n. 1960. This contribution reflects the authors'

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(2016) Environmental impacts of the deep-water oil and gas industry: a review to guide management 675 strategies. Frontiers in Environmental Science, 4, 1-54.

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(2011) Genetic discontinuity among regional populations of Lophelia pertusa in the North Atlantic Ocean.


Cosimo Micheletti

Pierpaolo Minei

Marco Carlotti

Virgilio Mattoli

Francesco Muniz- Miranda

Anna Perfetto

Ilaria Ciofini

Carlo Adamo
email: carlo.adamo@chimieparistech.psl.eu

Giacomo Ruggeri

Andrea Pucci
email: andrea.pucci@unipi.it

Prof Andrea Pucci

Prof Carlo Adamo

Mechanochromic LLDPE Films doped with NIR Reflective Paliogen Black

The perylene bisimide derivative Paliogen Black (P-black) has been proposed as a new chromogenic probe which shows visible (VIS) and near-infrared (NIR) responses after mechanical solicitations of host linear low-density polyethylene (LLDPE) films. P-black has been reported to display strong absorption in the VIS spectrum and unusual reflective and cooling features in the NIR region. Uniaxial deformation of the 2.5, 5 and 10 wt% Pblack/LLDPE films yields a dichroic absorption under polarized light with color variations attributed by the computational analysis to the distinct anisotropic behavior of the transition dipole moments of P-black chromophores. When LLDPE films are deformed, P-black aggregates reduce their size from ∼30-40 μm to ∼5-10 μm that, in turn, causes reflectivity losses of about 30-40% at the maximum elongation. This gives rise to warming of 5-6 °C of the locally oriented film placed in contact with a black substrate under the illumination with an IR lamp for 5 seconds. These features combined with the high sensitivity of the VIS-NIR response towards mechanical solicitations render P-black as a new solution to detect uniaxial deformations of plastic films through both optical and thermal outputs.

Introduction

Nowadays, smart materials with chromogenic features are widely employed in the detection of external stimuli such as deformation, temperature alterations, and light exposure. [START_REF] Bamfield | Chromic Phenomena: Technological Applications of Colour Chemistry, 3rd Edition[END_REF] Such optical sensors are based on the predictable relationship between the presence of the relevant stimulus and changes in the wavelength of light absorbed and/or emitted by the sensing system. The introduction of chromogenic species into polymers in the form of dispersed or covalently bonded chromophores have been reported as the most exciting and versatile procedure for conferring color features that are strongly depending on the environment variations. [START_REF] Pucci | [END_REF]3] Moreover, the possible formation of dye aggregates opens a wealth of exciting opportunities for the development of polymers with targeted chromogenic features. Notably, molecular aggregates can display entirely new features with respect to the isolated dye molecules and result extremely sensitive to external solicitations thus supporting smart materials as sensitive optical sensors. [4] Polymers that respond to mechanical stimuli through variations of their optical features are called mechanochromic. Mechanochromism in polymers was reported for the first time by Weder in 2002 [5] and it has been supported by using diverse polymeric materials and different classes of aggregachromic dyes and fluorophores. [6][7][8][9][10][11] Notably, the optical responses towards mechanical solicitations are consistent with variations in the absorption and/or emission of these dye/polymer assemblies due to modifications of their interphase interactions and chromophore orientation towards the stress direction. [START_REF] Pucci | [END_REF]3,7,12,13] Many practical applications can be predicted, e.g. mechano-and pressure sensors, [14,15] indicators of mechano-history, security plastics and papers and data storage devices. [16][17][18][19][20] and new solutions are provided everyday. [21][22][23][24][25][26][27] Among the different chromogenic dyes, perylene bis-imide derivatives (PBIs) are one of the most investigated since their optical features result strongly depending on molecular packing among the chromophoric units. [4,[28][29][30] The disaggregation phenomena of PBIs assemblies triggered by polymer deformation causes a clear modification of the their optical features with very evident changes especially in emission. [7,31] Dyes embedded into plastic films are also reported to display a well-defined dichroic pattern in polarized light owing to the oriented chromophore assemblies towards the drawing direction of the film. [8,32] Very recently, there has been a growing interest in the application of PBIs as Near-Infrared (NIR) transparent and reflective organic pigments for the development of cool organic coatings for different surfaces. [33][34][START_REF] Qu | The Optical Properties of Black Coatings and Their Estimated Cooling Effect and Cooling Energy Savings Potential[END_REF][START_REF] Kaur | [END_REF] Painting building roofs with coatings based on cool pigments, is indeed one of the most accessible solutions to reduce the warming effect caused by the sunlight exposure, which is mainly due to the absorption of the NIR contribution of the solar spectrum. This approach has been recently reported to reduce the energy demand and therefore beneficial for downgrading the urban heat island (UHI) phenomenon. [37] One of the most effective PBIs with NIR reflective features is Paliogen ® Black L0086 (P-black, Figure 1 left panel up) that is distributed by BASF as pigment for solar heat management in paints. [START_REF] Basf | Paint it cool! Pigments for solar heat management in paints[END_REF] P-black mimics the transparent and NIR reflective features of certain green plants [START_REF] Akbari | [END_REF] thanks to the high-symmetry of PBI that favors the formation of crystalline supramolecular assemblies promoted by the intermolecular π-π interactions among perylene nuclei. [34,[START_REF] Kaur | [END_REF][40][41][42][43] Its supramolecular structure preserved within the polymer coatings allowed NIR reflectances close to 50% and consistent cooling effects. [33,44] Being the NIR reflective features strongly depending on the supramolecular packing, polymer drawing may promote the exfoliation of the dispersed PBIs aggregates, thus causing the loss of NIR reflection that in turn substantially reduces the cooling effect of film surfaces.

In this work, we propose for the first time the use of P-black as a pigment for low-density polyethylene (LLDPE) aimed to endow the derived films with a responsive behavior to mechanical deformations both in the visible and in the NIR region of the spectrum of light.

The external stress applied to the LLDPE film is thus detectable as color variations in the visible and reflectance reduction in the NIR, this last causing a prompt temperature variation of the film surface under IR irradiation.

Experimental part

Materials

Paliogen ® Black L0086 (P-black) was provided by BASF and used without further purification. Linear low density polyethylene (LLDPE, Dowlex SC 2107, Melt flow index, 190 °C/2.16 kg 2.3 g/10 min, d = 0.917 g/cm 3 ) was supplied by Dow Plastics, USA. The remaining chemicals were obtained from Sigma-Aldrich.

P-black/LLDPE film preparation

A desired amount of P-black was dispersed under vigorous stirring in a 5 wt.% LLDPE solution in toluene at 80 °C. After sonication for 5 min at 400 W and 24 kHz with Hielscher's UP 400 S probe, the solvent was removed by rotary evaporation and the obtained solid blend eventually compression molded at 180 °C for 5 min into thin film with a thickness between 120 and 150 μm.

Methods

The thickness of the LLDPE films was measured with a CM1S dial indicator (Borletti, Milan, Italy) with ruby movement bearing. Differential scanning calorimetry (DSC) was performed on LLDPE films under nitrogen atmosphere by using a Mettler Toledo StarE System, equipped with a DSC822c module and following the procedure reported in the literature. [45] Solid-state drawings of the binary films were performed on a thermostatically controlled hot stage at 90 °C. The draw ratio (Dr), defined as the ratio between the final and the initial length of the samples, was determined by measuring the displacement of ink-marks printed onto the films before stretching. UV-Vis spectra of P-black/LLDPE films were recorded at room temperature over the wavelength range of 350-800 nm in polarized light with the help of a Perkin-Elmer Lambda 650 equipped with motor-driven Glan-Taylor linear polarizers.

UV-Vis-NIR reflectance measurements of P-black/LLDPE films were recorded at room temperature over the wavelength range of 350-2500 nm by using an Agilent Cary 5000 spectrophotometer equipped with a 150 mm integration sphere. A commercially available 100W IR lamp (Kerbl, Buchbach, Germany) was used for the heating tests. The Pblack/LLDPE films were placed over the black surface of a Leneta ® checkerboard chart and irradiated at a distance of 20 cm for 5 seconds and the temperature measured by means of a FLIR™ E6 infrared thermo-camera (FLIR, Wilsonville, OR, USA). The thermal images were opened by the FLIR Tools software (version 2.1) by using the iron palette image setup. The optical microscope analysis was accomplished on a Nikon Eclipse Ni-E equipped with a Thorlab LPVISE200-A polarizer and a Nikon DS Ri2 camera. Particle analysis was performed using the public domain ImageJ 1.52k software version image analyzer program developed by the National Institutes of Health (USA) and available on Internet at https://imagej.nih.gov/ij/index.html.

Computational details

P-black molecules have been investigated computationally to analyze and interpret their optical and structural features. We adopted various models to elucidate the available experimental data in both solvent/vacuum medium and in condensed phases.

Solvent/vacuum calculations

The molecular structure of P-black molecules has been relaxed in the ground electronic state (GS) using the Gaussian 16 package, [START_REF] Frisch | Gaussian 16[END_REF] adopting the B3LYP exchange and correlation functional [START_REF] Lee | [END_REF]48] in conjunction with 6-31+G(d,p) basis set and Grimme's D3-BJ dispersion correction: [49] this level of theory proved to be suitable in a previous investigation of some of us on a similar system (dye Paliogen Red) [41] . All relaxed structures are potential energy minima, as all normal modes have a positive frequency. Calculations of electronic excited states have been performed with the TD-DFT approach using the range-separated exchange and correlation functional CAM-B3LYP, [50] to better describe charge-transfer transitions and the spectral shape. Solvent effect was simulated using an implicit solvation model (polarizable continuum model). [51]

Crystal-embedded calculations

Periodic calculations have been carried out with the Crystal 17 [52] code at the B3LYP-D3 level with a modified all-electron 6-31+G(d,p) basis set to match that of non-periodic calculations. Numerical DFT integration has been performed considering 75 radial points and 974 angular points, ensuring an error on the integrated electron density to an accuracy of 10 -5 e per unit cell. The Coulomb and exchange series were truncated with threshold values of 10 - 7 , 10 -7 , 10 -7 , 10 -7 , and 10 -21 . A tolerance of 10 -7 a.u. has been used for the convergence of the self-consistent field procedure. Cell parameters and atomic positions have been let free to relax during structural optimization. Convergence was determined from the root mean square and absolute value of the largest component of the forces and displacements, considering default values (0.00045 and 0.00067 au for the forces, and 0.0018 and 0.0027 a.u. for the displacements, respectively). The crystalline structure of P-black belongs to the triclinic P1 space group, with flat perylenetetracarboxylic diimide portions packed in stacks through π-π interactions, with experimental lattice parameters of a = 4.09 Å, b = 9.71 Å, and c = 17.11 Å, α = 98.82°, β = 91.07°, γ = 101.58°. [53] The optimized lattice parameters obtained are a = 3.99 Å, b = 9.78 Å, c = 16.96 Å, α = 96.72°, β = 90.04°, and γ = 101.67°. In addition, the flat portions of neighboring molecules are found at about 3.49 Å apart, with respect to an experimental value of 3.59 Å. To create a more cubic-like box, we adopted a 7 × 3 × 2 monoclinic supercell, resulting in a simulation box of volume ∼28 × 29 × 34 Å 3 .

Starting from the optimized crystal structure obtained, a large supercell was built and partitioned into three zones, according to the SC-Ewald approach. [54] At the inner first zone treated at the QM level, two different clusters with either one, two, or three P-black molecules have been considered. The intermediate second zone consisted of point charges fixed at the value of the Mulliken atomic charges of the periodic calculation, while the point charge values of the outer third zone have been adjusted to impose the exact Madelung potential of the infinite periodic system on the first two zone sites. Further details can be found in ref 51. [55] Vertical TD-DFT calculations with the aforementioned models (embedded monomer and dimer of P-black) have been carried out with Gaussian 16, thus including the effects of the crystal environment. The embedded dimer model was also used to test the dependence of computed spectra on the intermolecular distance and relative translation. All calculations (both embedded and not) have been carried out using the Pople's 6-31+G(d,p) basis set, and dispersion forces were modeled employing Grimme's D3-BJ correction.

Results and discussion

P-black is an insoluble PBI pigment that was dispersed by ultrasonication in toluene containing the 5 wt% of linear low-density polyethylene (LLDPE). LLDPE films containing different concentrations (2.5, 5 and 10 wt%) of P-black were prepared by compression molding of the respective LLDPE mixtures. The presence of P-black did not substantially altered the thermal properties of LLDPE films, whose crystallinity and melting point were only slightly enhanced at the highest pigment content of 10 wt% (Table S1). No thermal transitions attributed to P-black were detected up to 300 °C in agreement with the thermal behavior of PBI pigments. [43,56] The UV-Vis spectra P-black/LLDPE films show absorption maxima centered at 430 and 680 nm (Figure S1a), which correspond to that displayed in toluene dispersion. Those bands progressively widen with concentration until reaching almost a continuous contribution all over the visible spectrum at the highest pigment content of 10 wt%. Absorbance flattening at the highest P-black content was attributed to saturation phenomena. Moreover, P-black/LLDPE films did not display any emission behavior, thus suggesting the de-excitation pathways was mainly governed by the typical aggregationcaused quenching phenomenon of PBI insoluble pigments. [57] Calculations were employed to investigate the nature of the electronic transitions that originate the P-black color as a function of the local environment (solution, embedded in matrix). To this end, we have simulated P-black molecules in vacuo, in trichlorobenzene (TCB) solvent, and in an electrostatic field that mimic a crystal environment, refer to Computational detail for the technical part. In Figure 1 (left panel) we report the computed shapes of the optical spectra obtained from a two-molecules model. This latter model has been chosen as it is able to describe not only absorption from a single molecule but also to include in the simplest way excitonic effects, if they are present. This two-molecules model has been proven to be relevant in the case of a similar compound (Paliogen Red). [41] Of note the calculations including the electrostatic field of the crystal environment (hereafter referred to as embedded model) are here considered as the simplest model to simulate absorption within the LLDPE matrix since micro-crystalline aggregates are formed in this matrix and the effect of the low polarity polymer matrix on the absorption properties of this micro-crystal is expected to be negligible.

The calculations carried out in gas and solution phases yield very similar spectral shapes, with just a hypsochromic shift observed when going from vacuum to solution. The corresponding transitions have a local character, without any appreciable inter nor intra molecular charge-transfer (Figure 1, right panel), as measured also according refined quantum descriptors. [58] Two main peaks can be located, around ~610 nm and ~410 nm for the solution phase: these values are within 0.2 eV from the observed peaks in solid phase, in agreement with the accurate level that may be expected from TD-DFT calculations and in agreement with the known tendency of the CAM-B3LYP functional to overestimate excitation energies. [59] A more detailed analysis of the computational data in vacuum and in solution can be found in the Supporting Information (see Figures S8-S12). The model expected to reproduce the absorption behavior in an aggregate phase, referred to as crystal-embedded and described in the computational details in the crystal embedded calculation section, yields instead a less structured spectrum, with significant absorption in the 300 -700 nm range of wavelengths, thus spanning a large portion of the visible range (as expected from a black material). The nature of the first excitation (I* in Figure 1) is also different, with a more pronounced intermolecular charge-transfer character, a behavior found also in the TD-DFT study of so-called Paliogen Red molecule (or Pigment Red 179) [41] when simulated embedded into a crystal. The D CT [58] quantum descriptor for charge transfer suggests an hole-electron distance of ~3.15Å, to be compared with an intermolecular distance between P-black molecules in the crystal of ~3.4 Å. Models which include just a single P-black molecule or do not take into account the effect of the environment yields a vanishing value for the D CT descriptor. A more detailed description of the embedded model can be found in the Supporting Information (see

Figures S13-S14 and Table S1-S2

).

UV-Vis response to mechanical drawing

The P-black/LLDPE films were then oriented by uniaxial drawing at different draw ratios up to a maximum of 6 (elongation = 500%). The anisotropic behavior of the stretched films was evaluated by UV-Vis spectroscopy in polarized light (Figure 2), i.e. at different polarization angle with respect to the drawing direction of the film. The mechanical drawing of the P-black/LLDPE films conferred to the plastic materials a well-defined dichroic behavior under polarized light. It was worth noting that by varying the direction of the polarization angle with respect to the drawing direction of the films the absorption bands experienced an opposite behavior (Figure 2a-c). Notably, the band at 430 nm increased its intensity whereas that at longer wavelengths (i.e., 680 nm) decreased its contribution, flanked by the existence of a clear isosbestic point at about 570 nm, thus supporting the existence of two different populations of chromophoric assemblies. The behavior was confirmed for all the films investigated, therefore suggesting that dichroic assemblies of pigment molecules were generated also at the highest pigment content of 10 wt%, notwithstanding the reduced absorbance variations caused by possible signal flattening due to saturation phenomena (Figure 2c). We did not find such behavior in unstretched samples (Figure S1b), only negligible absorbance variations were detected in polarized light and possibly attributed due to uneven orientation of P-black aggregates during film formation by compression molding.

Notably, the mechanochromic response was clearly visible simply by observing the oriented portions of the LLDPE films under a linear polarizer (Figure 2d). The color changed accordingly to the absorption variations from yellow-green (0°) to dark-cyan (90°).

The effect provided by the P-black content and the draw ratios on the chromogenic behavior was also studied in terms of the absorbance variation ΔA (Table 1 and Figure S2). with ΔA values mostly similar for all the draw ratio investigated. This phenomenon could be possibly attributed for the former to a maximum chromophore orientation already gathered during the early stages of uniaxial deformation and for the latter to the difficulties of orienting the large content of P-black assemblies (i.e., 10 wt%) along the drawing direction and confined in the amorphous phase between polymer crystallites even at the highest Dr = 6.

Nevertheless, absorbance flattening caused by saturation phenomena in LLDPE films containing the 10 wt.% of P-black cannot be excluded, thus affecting ΔA linear variations.

It was worth noting that the absorption band at about 430 nm appeared more sensitive to uniaxial deformation than that at about 680 nm for all the P-black/LLDPE films investigated.

This behavior was firstly attributed to the absorbance flattening issue, since the absorption band at higher wavelength was also the more intense. Nevertheless, such phenomenon can be traced back to the transition dipole moments of the optical excitations. Considering as the simplest model to simulate absorption of P-black within the LLDPE the embedded crystal model, since micro-crystalline aggregates are formed in this matrix and the effect of the low polarity polymer matrix on the absorption properties of this micro-crystal is expected to be negligible, we analyze the transition dipole moments of all the most intense S n ←S 0 transitions. Here it is worth to recall that even if the computed transitions energies are expected be overestimated (i.e., predicted at shorter wavelengths with respect to what experimentally observed) due to known well documented limitations of the employed level of theory (i.e., the CAM-B3LYP exchange-correlation functional used which overestimates transition energies due to its high content of Hartree-Fock exchange) the nature of the electronic transition is expected to be correctly reproduced. Therefore dipole moments, depending on ground and excited state densities are expected to be qualitatively reliable.

The computed dipole moments show a certain extent of variability as reported in Table S3 of the Supporting Information. In particular, transitions #11 and #27 expected to contribute to the higher energy band show significantly different absorptions if oriented differently in a single crystal. In fact, this anisotropy increases when a translation is made along the x-axis (see Tables S3 andS4 of the Supporting Information), suggesting that the second band of the spectrum is more sensible to the orientation with respect to incoming light when subject to a lateral stress, as experimentally observed.

Optical microscopy taken on pristine and oriented P-black/LLDPE films (e.g., those containing the 5 wt% of P-black, Figure S3) revealed that the mechanical drawing was particularly effective in reducing the size of P-black aggregates from about 30-40 μm to 5-10 μm and in orienting them towards the drawing direction. Notably, microscopy images taken on oriented films at Dr = 6 and in polarized light (Figure S4) confirmed the results gathered from UV-Vis spectroscopy (Figure 1). It was worth noting that all the oriented samples displayed color variations in absorption as a function of the polarization angle (i.e., 0° and 90°) with P-black 5-10 μm-sized aggregates mostly oriented towards the orientation direction of the film. The dichroic behavior appeared more evident for the oriented P-black/LLDPE film containing the 2.5 and 5 wt% of pigment and in agreement with the data reported in Table 1. The highest P-black content of 10 wt% strongly affected the light transmission of the film and rendered difficult the determination of the aggregates anisotropy throughout the film sample.

Nevertheless, notwithstanding the high absorption of the films, optical microscopy was effective in revealing the dichroic behavior of P-black/LLDPE films at the early stages of mechanical deformation and containing the highest pigment content (10 wt% of P-black, Figure S5). Notably, the uniaxial elongation corresponding to Dr = 1.2 conferred LLDPE film a chromogenic response in polarized light, thus suggesting that even at very small LLDPE deformations the supramolecular structure of P-black assemblies resulted effectively affected. This result was particularly relevant since it demonstrated the sensitivity of the designed P-black/LLDPE system in providing a prompt mechanochromic response towards uniaxial deformation.

NIR response to mechanical drawing

We then investigated the influence of the P-black content and the polymer uniaxial drawing on the film reflectivity in the visible and NIR region. In agreement with literature data, the reflectance spectra were dependent on the optical features of the P-black chromophoric unit, i.e. a strong absorption in the visible region and maximum reflectivity around 800 nm flanked by a long tail throughout the NIR region (Figure 3). [34,42] Figure 3. UV-VIS-NIR reflectance spectra of P-black/LLDPE films at different P-black content. The black dashed lines delimit the visible region (400-700 nm).

Notably, reflectance values progressively raised from 10% to about 51% on passing from the neat LLDPE film (gray line) to that doped with the 10 wt% of pigment (red line) thanks to the NIR reflective features of P-black aggregates. [33,44] NIR reflectances close to 50% are particularly beneficial for the development of dark organic coatings with cooling characteristics since the affected wavelength region represents about the 25% of the total solar energy. [START_REF] Kaur | [END_REF] Visible reflectances of the pigment doped films (i.e., from 400 to 750 nm)

were lower than that of the neat LLDPE due to the strong absorption of P-black aggregates. The mechanical drawing of the P-black/LLDPE films (Dr = 6) effectively affected the reflectance of the films with decreasing of about 30-40% at around 800 nm. This phenomenon might be possibly addressed to a combination of effect: the first resides on the fact that uniaxial drawing caused P-black dilution throughout the oriented film portion; the second consist on the partial exfoliation of the pigment aggregates during deformation as revealed by optical microscopy (Figure S3). The reduction of the average aggregate size of Pblack assemblies has been recently reported to adversely affect pigment reflectivity and, in turn, the pigment cooling characteristics. [44] The films were then placed over the black surface of a Leneta ® checkerboard chart and irradiated by means of an IR lamp for 5 seconds to investigate the different cooling features of the oriented and pristine portions of the P-black doped LLDPE films. Thermal images taken during lamp irradiation revealed consistent temperature raising of the oriented LLDPE film of about 5-6 °C and in agreement with reflectance variations. In summary, the addition of P-black endowed LLDPE film with NIR-reflective and cooling characteristics as demonstrated by the progressive temperature reduction from 38.7 °C to 32.2 °C on passing from the neat LLDPE film (Figure S6) to that doped with the 10 wt% of pigment (Figure 4).

As soon as the mechanical drawing was applied to the films, reflectance reduction of about 20-30% caused temperature raising close to 6 °C. Thickness variations occurred during film drawing supposedly contribute to the thermal response specifically for the highly oriented LLDPE samples. Anyway, temperature variations were also detected for the initially oriented 10 wt% P-black/LLDPE film (Dr = 1.2) with the deformed film portion 4.5 °C warmer than the pristine counterpart (Figure S7).

Conclusions

This work enabled the use of P-black as an unique pigment that once embedded into LLDPE films provided combined optical variations in the visible and reflectivity response in the NIR after mechanical uniaxial deformation. Notably, LLDPE films containing the 2.5-10 wt.% of P-black showed a dichroic absorption profile in linearly polarized light with clear isosbestic points and color variations from yellow-green to dark-cyan. This feature was addressed by density functional theory calculations to the different anisotropic behavior of the transition dipole moments of the P-black chromophoric assemblies within the oriented LLDPE matrix.

Mechanical drawing was also responsible in reducing the average size of P-black aggregates from ∼30-40 μm in the pristine film to ∼5-10 μm after maximum film deformation at Dr = 6, thus also causing substantial reflectivity decrease of ∼30-40% due also to the concomitant Pblack dilution all over the oriented portion of the LLDPE film. Such reflectivity loss was ingeniously exploited since the oriented film in contact with a black substrate displayed a noteworthy warming of 5-6 °C maximum when illuminated with an IR lamp for 5 seconds.

Both optical and thermal outputs revealed very sensitive to mechanical drawing thus opening a new avenue for the direct determination of mechanical deformations of plastic films.

Figure 1 .

 1 Figure 1. Left panel up: chemical structure of Paliogen ® Black L0086 (P-black). Left panel down: computed optical spectra at the TD-CAM-B3LYP level of theory in TCB (black), vacuo (red), and embedded into the crystal field (blue); the curved are smoothed with Lorentzian functions of half width at half height of 0.33 eV, while vertical lines are actual vertical S n ←S 0 transitions. Right panel: main orbitals involved into the first (I) and second (II) calculated band, for the solution/gas calculations (black) and the crystal-embedded one (blue).

Figure 2 .

 2 Figure 2. UV-vis absorption spectra as a function of the polarization angle for Pblack/LLDPE oriented films containing a) 2.5 wt%, b) 5 wt%, c) 10 wt% of pigment; d) photos of the same 5 wt% P-black/LLDPE oriented film taken by superimposing a linear polarized oriented parallel (0°) and perpendicular (90°) with respect to the drawing direction. Dr = 6 in all cases.

Figure 4 .

 4 Figure 4. UV-VIS-NIR reflectance spectra of P-black/LLDPE films at different P-black content before (pristine) and after uniaxial deformation at Dr = 6. Thermal images of the same films with temperatures recorded for the oriented and unoriented portion of the films after 5 second exposure to an IR lamp (nominal power 100W, distance from the sample 20 cm)

Table 1 .

 1 Absorbance variations at 430 and 680 nm for P-black/LLDPE films at different pigment content and draw ratio (Dr). ΔA = A 90°-A 0°

	Entry	Dr = 3	Dr = 4	Dr = 6
		ΔA 430	ΔA 680	ΔA 430	ΔA 680	ΔA 430	ΔA 680
	2.5 wt% P-black/LLDPE	0.43	-0.30	0.51	-0.40	0.43	-0.30
	5 wt% P-black/LLDPE	0.20	-0.18	0.63	-0.41	0.78	-0.45
	10 wt% P-black/LLDPE	0.16	-0.10	0.18	-0.12	0.22	-0.16
	The data reported in Table 1 revealed that the dichroic phenomenon was already obtained at
	the smallest deformation extent (Dr = 3) and particularly evidenced in the LLDPE films
	containing the lowest 2.5 wt% content, i.e. reaching absorbance variations of 0.43. P-

black/LLDPE films containing the 5 wt% of the pigment revealed as the most sensitive towards mechanical drawing, with ΔA values progressively raising with draw ratio. Notably, a maximum of ΔA ∼ 0.8 was reached at the highest Dr = 6. Conversely, LLDPE films containing the 2.5 and 10 wt% of P-black showed lower sensitivity towards uniaxial drawing

Acknowledgements

This work was supported by the MIUR-PRIN 20179BJNA2. This project has also received funding from the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Program (grant agreement no. 648558 STRIGES CoG).