We found that the Q1/Q2 length ratio quantitatively encodes vocalization frequency ranges, from the infrasonic to the ultrasonic, displaying striking convergent evolution patterns. Thus, species emitting ultrasonic vocalizations converge with bats in having a low ratio, whereas species vocalizing in the low-frequency/infrasonic range converge with elephants and whales, which have higher ratios. Similar, taxon-specific patterns were observed for the FOXP2-related protein FOXP1. At the molecular level, we observed that the FOXP2 polyQ tracts form coiled coils, assembling into condensates and fibrils, and drive liquid-liquid phase separation (LLPS). By integrating evolutionary and molecular analyses, we found that polyQ length variation related to vocalization frequency impacts FOXP2 structure, LLPS, and transcriptional activity, thus defining a novel form of polyQ length-based molecular encoding of vocalization frequency.The transcription factor FOXP2, a regulator of vocalization- and speech/language-related phenotypes, contains two long polyQ repeats (Q  •1 and Q2) in FOXP2 vary broadly across mammalsThe lengths of two polyQ repeats (Q•1/Q2 length ratio correlates quantitatively with vocalization frequency rangesThe Q•The FOXP2 polyQ repeats form α-helical coiled coil structures and promote LLPS•Evolutionary polyQ length variation regulates FOXP2 structure, LLPS, and function Zoology; Molecular biology; Evolutionary biology In particular, it is unclear how specific features of vocalization, such as its frequency range in each species, are encoded at the genetic and molecular levels. Some genes related to vocalization, speech, language, and their evolution, have been identified,,,FOXP2 and its paralog gene FOXP1, both encoding for transcription factors, are involved in neural development and function,,Mutations in both genes can give rise to various forms of speech and language impairments in humans.,,,,,,,FOXP2 mutations can cause the ‘FOXP2-related speech and language disorder’ (FOXP2-SLD) with childhood apraxia of speech (CAS) as a core phenotype.FOXP1 mutations can also lead to distinct speech and language phenotypes in the context of a broader neurodevelopmental ‘FOXP1-related disorder’, with prominent dysarthria and motor planning deficits, linguistic-based phonological errors, and generally more severe language and social impairments than those observed in the FOXP2-SLD.,,,The molecular evolution of FOXP2 has also been associated with the emergence of specialized vocalization phenotypes. Indeed, two amino acid substitutions in human FOXP2, which are not present in the great apes, and intronic variants, have been related to the acquisition of speech and language.Interestingly, both the FOXP2 and FOXP1 proteins contain two major polyglutamine (polyQ) repeats. One of these in FOXP2 represents the longest polyQ repeat in the human proteome.,,PolyQ tracts can form α-helical coiled coils (CCs) that mediate protein oligo-/poly-merization and interactions,,,,,,,,,The evolutionary variation of polyQ and other amino acid repeats, in terms of occurrence and length, can have an evolutionary impact by adjusting, as ‘tuning knobs’, the transcriptional activity of developmental proteins.,PolyQ repeats are enriched in human proteins with speech- and language-related roles like FOXP2,,,,Vocalization and hearing frequency ranges varied extensively throughout phylogenesis, and similar specialized forms of vocalization/hearing developed in different branches of the mammalian evolutionary tree by convergent evolution. Thus, high- and low-frequency specialist species, vocalizing/hearing in the ultra- or infra-sonic ranges, respectively, are found in multiple orders including Primates, Rodentia, Chiroptera, Cetartiodactyla, and Afrosoricida.prestin and other hearing-related proteins display similar amino acid substitutions which regulate protein function.,,,,Convergent evolution of organismal phenotypes between distant species/taxa can arise from convergent changes at the molecular level. For instance, in chiropteran and cetacean species using ultrasonic echolocation, Limited knowledge exists on convergent molecular evolution in vocalization-related proteins. Lee et al.Thus, in search of possible genetic and molecular encodings of vocalization frequency, we focused here on the evolution of FOXP2 and its polyQ repeats in Mammalia, a taxon characterized at the same time by highly diversified forms of vocalization and by a considerable degree of polyQ length variation in FOXP2.To identify a possible link between FOXP2 polyQ repeat lengths and vocalization frequency, we initially focused on Chiroptera 1’ and ‘Q2’ , display relatively limited variation in Primates. Conversely, Q1 and Q2 vary extensively across chiropteran species, in terms of both total (Q1+Q2 sum) and relative (Q1/Q2 ratio) lengths.Indeed, the two major N-terminal polyQ repeats, i.e., ‘Q1, which shorten this repeat compared to other related species.Notably, several species in both taxa exhibit a recurrence of interspersed proline residues and/or polyproline stretches in QThe order Chiroptera comprises the Yinpterochiroptera and Yangochiroptera suborders B. ‘Micro1’ . Yangochiroptera also include ‘megabats’ that do not echolocate (‘NE’) using laryngeal USVs but navigate relying on sight and rudimentary echolocation, using sonic wing- or tongue-generated clicks.For simplicity, we group FM USVs into ‘FM1 in Vespertilionoidea, and FM2 in Noctilionoidea, are prevalent in each chiropteran superfamily, several species demonstrate phenotypic convergence by employing USV types distinct from those of their closest relatives (as highlighted by arrowheads in Megaderma spasma (Rhinolophoidea) emits FM calls, Pteronotus parnellii (Noctilionoidea) CF calls, and Mormoops blainvillei (Noctilionoidea) uses FM1 calls. USV-emitting bats exhibit morpho-functional specializations, including an increased relative cochlear sizeNotably, the use of these different USV types is not strictly determined by phylogeny in Chiroptera.1 and Q2 lengths in chiropteran FOXP2 orthologs, calculated their total (Q1+ Q2 sum) and relative (Q1/Q2 ratio) lengths, and studied their variation in relation to phylogeny and USV types (right columns). This analysis corroborated the initial evidence of generalized polyQ variation in Chiroptera. A comparable analysis was conducted for two analogous polyQ repeats (‘Q1’ and ‘Q2’) in FOXP1, with length variation being restricted to only a few species  rather than their phylogenetic relationships . The threshold values for these groups are highlighted in Remarkably, this graph demonstrates a clear clustering of the 53 species that were analyzed into four distinct groups based on their USV types , cluster together with Yangochiroptera species that also emit FM calls. Similar cases of convergence can be observed across the FM1 and FM2 clusters. Mormoops blainvillei (Noctilionoidea) clusters with FM1 Vespertilionoidea species, while Molossus molossus (Vespertilionoidea) clusters with FM2 Noctilionoidea. Taphozous melanopogon  groups with other FM2 species belonging to Noctilionoidea and Rhinolophoidea.Indeed, it is worth noting how species belonging to the same Yinpterochiroptera suborder are differentially distributed among all four clusters based on their USV types. Specifically, Pteropodidae (NE) and Rhinolophoidea species utilizing CF USVs form two distinct clusters, well separated from those of FM species. Strikingly, the three Rhinolophoidea species emitting FM rather than CF USVs  that do not converge with CF Rhinolophoidea. However, the lengths of FOXP1 polyQ repeats in both species vary considerably compared to most chiropteran species. Notably, this is also observed in other species that demonstrate USV type convergence with those from different orders (1/Q2 ratio is either below 0.5 (as in P. parnellii) or is ≥2 (as in N. leporinus) due to prevalent Q2 or Q1 elongation, respectively ,1/Q2 is < 3.5 (and Q1+ Q2 < 60), as typical of USV-emitting bats (Homo sapiens (Q1/Q2 = 4) and other species devoid of ultrasonic vocalization/hearing. We observed convergence with NE bats only in a single species, H. glaber, a subterranean rodent which lost sensitivity to ultrasound.FOXP2 ortholog sequence alignments revealed a striking recurrence of the polyQ variation patterns and proline insertions, as found in USV-emitting bats, also in Euarchontoglires species with known ultrasonic vocalization/hearing  of similar length (Q1/Q2 = 0.85) can be found in most Euarchontoglires species .Sum-terplots C highligCarlito syrichta, Microcebus murinus, and Callithrix jacchus, displaye polyQ convergence with USV-emitting bats. Notably, C. syrichta, the only Primate known to emit purely ultrasonic calls,Galeopterus variegatus, whose ultrasonic vocalizations have been recently demonstrated,1/Q2 ratio below 3.5.In Primates, three USV-emitting species, i.e., Microtus ochrogaster,Octodon degus,Cricetulus griseus.Typhlomys cinereus, a blind rodent recently discovered to rely on echolocation for spatial navigationprestin protein.In Rodentia, several species have FOXP2 polyQ lengths in the range of USV-emitting bats, such as P. parnellii and M. lyra in having a low FOXP1 Q1/Q2 (<0.5). This ratio goes below 0.2 upon extreme Q2 expansion, as evident in some species with the highest vocalization/hearing frequencies, such as the mouse, the rat and the echolocating species T. cinereus. Species like Jaculus jaculus and Marmota marmota, adapted to desert and/or fossorial life, which are sensitive to both low-frequency and ultrasonic acoustic signals with a bimodal frequency sensitivity,,1/Q2 ratio  also converge with some USV-emitting bats in having a FOXP1 Q1/Q2 ≥ 2  is an extreme case of low polyQ sum and ratio, clustering with USV-emitting bats. Strikingly, O. princeps displays accelerated evolution in the hearing-related protein prestin similar to echolocating bats,The pika O. princeps in the wild and studied its inner ear. We found evidence of spontaneous calls extending into the ultrasonic range, well above 20 kHz  and height (height/width)M. musculus, tend to have auditory sensitivity to higher frequencies ,Strikingly, these two patterns of evolutionary phenotypic convergence toward either ultrasonic or infrasonic vocalization/hearing are paralleled, at the molecular level, by FOXP2 polyQ variations of opposite sign B and 3C.,,,Felis catus, Neovison vison, and Enhydra lutris), Eulipotyphla  and Afrotheria  have Q1/Q2 ≤ 3.5. , Elephas maximus (Afrotheria), and Ceratotherium simum (Perissodactyla) all have Q1/Q2 > 4.5 (Homo (Q1/Q2 = 4) and other species with sonic vocalization/hearing ranges (Hippopotamus amphibius (Cetartiodactyla) is a borderline case (Q1/Q2 = 4.38). Other large animals in Artiodactyla/Perissodactyla, also had Q1/Q2 > 4, except for some camelids, consistent with their ability to hear ultrasonic frequenciesConversely, large species that evolved infrasonic vocalization/hearingQ2 > 4.5 B, occupyg ranges C. HippopPhyseter catodon and Tursiops truncatus, which use (ultra)sonic clicks for underwater echolocation, do not cluster with USV-emitting bats, but have FOXP2 polyQ repeats like infrasonic Mysticeti ,1/Q2 ratio <0.5.In all these taxa, some species known to produce USVs also converge with some USV-emitting bats and most rodents in having a low FOXP1 Qio <0.5; C. These M. lyra (Chiroptera) and T. cinereus (Rodentia) show convergence for both FOXP2 and FOXP1.Overall, these findings show that FOXP2 polyQ lengths vary and converge in opposite ways in mammalian species with high versus low frequency laryngeal vocalization/hearing, in the entire range from the infra- to the ultra-sonic. Interestingly, some USV-emitting species such as The previous findings suggested that polyQ parameters may overall encode vocalization frequency ranges in mammals, and we tested this hypothesis for a sample of >100 species with available data.1/Q2 ratio (in 0.5 unit bins) and calculated for each group the mean minimum and maximum vocalization frequency as found in the literature (1/Q2 and mean minimum (r = 0.93) or maximum (r = 0.91) vocalization frequency across groups . Thus, a lower FOXP2 Q1/Q2 ratio is associated with a higher range of vocalization frequency and vice versa. Because acoustic allometry principles indicate that the vocalization frequency range is inversely related to body mass,1/Q2 correlates with body mass in mammalian species . Similar findings were obtained when Chiroptera were not included in the analysis  = 65.65; max. freq.: F = 26.25 and F = 30.05 without Cetacea; body mass: F = 85.22).When we compared quantitatively the mean vocalization frequencies (minimum and maximum) and body mass across species groups with low (≤3.5), intermediate (3.5–4.5), and high (>4.5) Q1/Q2 ratio (≤3.5) have a significantly higher vocalization frequency (min. and max.) than species with intermediate, more human-like (3.5–4.5), or high (>4.5) ratios, as well as a lower body mass (post hoc test). Conversely, species with a high ratio display significantly lower minimum frequency and higher body mass  in comparison with the other two groups. They also display lower maximum frequency than the low-ratio group  and the intermediate group , contraction (Miniopterus) and contraction by proline/polyP insertion (Rhinolophus).To determine the possible CC structure of the FOXP2 polyQ repeats and the impact of their variation, we first obtained structural predictions of the CC propensity of human FOXP2 and chiropteran orthologs with representative polyQ variation patterns A, i.e., cc1-4 (with (1 – P-score) ≥ 0.8)Cc2 and cc3, overlap with Q1 and Q2, respectively, whereas cc4 is a known leucine zipper.cc1 and cc4 are conserved across orthologs, the extension of cc2 and cc3 varies with polyQ length . The Cynopterus ortholog displayed the longest CC with Q-Q hydrogen bondsRhinolophus forms a random coil loop between Q1 and Q2.Paircoil2 identified in human FOXP2 four high-probability CC regions, i.e., Q length A–S5D, Pr1Q2 peptides (Rhi fer) and medium (Min sch) polyQ length. While the first displayed good solubility, Min-sch-Q1Q2 was already much less soluble, producing an opalescent solution indicating the prevalence of oligo-/poly-mers.Min-sch-Q1Q2and Rhi-fer-Q1Q2.To experimentally test these predictions, we chemically synthesized Qpeptides A. Given Min-sch-Q1Q2 in saline buffer displayed α-helical structure with a random coil component .The more soluble ructures A. Notablructures A, consis,,,Together with the CD results, these findings indicate that CC α-helical peptides containing the FOXP2 polyQ repeats can assemble into LLPS-like mesoscale condensates and elongated fibrils, with transitional forms, as illustrated in ,The previous results, and existing evidence that CCs and amino acid repeats can drive LLPS,Toward this aim, we first tested whether FOXP2 can form discrete foci in cells, and then whether these foci have recognized, distinctive properties of LLPS-driven condensates .Figure 6, upper panels). These results were confirmed upon expression of GFP-tagged FOXP2, which formed similar foci that became larger and more defined in a time-dependent manner , as typical of proteins undergoing intracellular LLPS.Using immunocytochemistry and super-resolution confocal microscopy (Airyscan) to reveal the subcellular distribution of FOXP2, we found that the endogenous protein forms numerous discrete foci of variable size in the nuclei of both HEK293 and Neuro-2a cells .Based on this morphological evidence, we further tested whether the observed FOXP2 nuclear foci exhibit typical features of LLPS-driven condensates.These findings indicate that FOXP2 can form LLPS-driven condensates in the cellular context.in vitro analyses strongly suggest that the polyQ repeats may drive FOXP2 LLPS. To test this hypothesis in the cellular context, we determined whether the isolated polyQ-bearing CC N-terminal domain (NTDCC) can recapitulate the LLPS behavior of FOXP2. When we expressed GFP-tagged NTDCC in cells, we found that it forms rounded foci as full-length FOXP2. The foci were also present in the cytoplasm, consistent with the fact that the FOXP2 nuclear localization signal is in the C-terminal domain,. 1 ± 0.11, n = 12, p < 0.001, t-test). Larger condensates were apparently less sensitive to 1,6-HEX, consistent with possible transitions to more solid-like configurations.Our CD and AFM CC was expressed in cells as a fusion protein with mCherry-Cry2  = 34.84, F = 22.91, F = 11.75, respectively, p < 0.001 in all instances). NTDCC-mCh-Cry2 condensation started within seconds after photoactivation and peaked after 2–4 min  post hoc test), whereas control cells expressing mCh-Cry2 did not display significant LLPS . Moreover, LLPS was reversible, reinducible, and condensates displayed coalescence , or of the NTDCC devoid of it (ΔQ1Q2-mCh-Cry2). We compared the relative capacity of these two fragments to induce LLPS with that of the whole NTDCC by measuring condensate formation in cultures 2 min after photoactivation relative to untreated control cultures  = 45.50, F = 180.66, F = 45.50, respectively, p < 0.001 in all instances) in the induction of condensates upon photoactivation in comparison with basal levels.To determine the contribution of the polyQ repeats to the NTDnstructs A for thecultures G. Overal1Q2-mCh-Cry2 in comparison with the NTDCC, showing that the polyQ repeats have an important role in driving FOXP2 LLPS . Indeed, the polyQ region alone (Q1Q2-mCh-Cry2) was still able to drive significant LLPS induction , although at lower levels in comparison with the whole NTDCC . Thus, also the Q-rich regions flanking the Q1Q2 repeats  in which the polyQ repeats were either replaced with those of other species representative of the observed polyQ variation patterns, i.e., Cynopterus sphinx (Cs), Miniopterus schreibersii (Ms), or Rhinolophus ferrumequinum (Rf), or deleted (ΔQ1Q2 variant). These variants were expressed as full-length proteins or as NTDCC fragments, in fusion with either GFP or mCh-Cry2  with that of evolutionary variants with low (Rf) or high (Cs) polyQ length  = 23.10, p < 0.001, one-way ANOVA; mean relative cell area occupied by condensates: 1 ± 0.04, n = 33 fields, for Hs; 1.41 ± 0.08 for Cs, n = 12; 0.78 ± 0.04 for Rf, n = 46; p < 0.02, for both the Cs vs. Hs and Rf vs. Hs comparisons;  = 222.34, p < 0.001). Similar results where obtained when only the NTDCC domains of the three constructs were expressed  = 25.19, p < 0.001, one-way ANOVA; mean relative cell area occupied by condensates: 1 ± 0.06, n = 32, for Hs, 1.55 ± 0.10 for Cs, n = 26, 0.75 ± 0.07 for Rf, n = 31, p < 0.03, for both the Cs vs. Hs and Rf vs. Hs comparisons, NK post hoc test; F = 32.408, p <0 .0001, one-way ANOVA, for the mean condensate size). Consistent with these findings, the three NTDCC variants displayed differential ability to undergo triggered LLPS  = 75.62), photoactivation  = 478.19), and their interaction  = 75.62). Moreover, significant differences  were found between all constructs and the control (mCh-Cry2), showing that all variants displayed significant LLPS induction upon photoactivation, and between Hs and either Cs or Rf.We first compared the ability to form condensates of the GFP-tagged human FOXP2  = 23.932, p < 0.001, repeated measures ANOVA; Rf FRAP curve displayed, in comparison with the Hs one, a significantly lower time constant  and a higher mobile fraction .Moreover, FRAP experiments further revealed that the mobility of molecules within condensates was higher for the Taken together, these findings indicate that the evolutionary polyQ variation in FOXP2 can modulate the mobility of the protein and its capacity to undergo LLPS.Finally, we determined the possible regulatory effect of the evolutionary polyQ variation on the transcriptional activity of FOXP2. FOXP2 regulates several target genes, including SRPX2, another speech-/language-associated gene,Hs, Rf, Ms, Cs, and ΔQ1Q2) as in Roll et al.luc+) under the control of the SRPX2 promoter (SRPX2-pGL3-luc), and a third vector for the CMV promoter-driven expression of Renilla luciferase  as a transfection control. The empty pcDNA4/HisMax vector was transfected to measure basal luciferase levels in control cultures  = 29.10). Post hoc comparisons confirmed, as expected based on previous observations,Hs-FOXP2 induces a significant reduction of Luc+ expression in comparison with control cultures, consistent with the repressive role of FOXP2 on SPRX2 , SRPX2 repression also correlated with the Q1/Q2 ratio . Therefore, this analytical approach offers powerful quantitative and qualitative tools in molecular evolution studies.In molecular evolution studies, convergence is generally defined in terms of single amino acid substitutions which can only be associated qualitatively, as a binary feature, to the presence of a certain phenotype, such as ultrasonic vocalization.,,,A growing body of evidence indicates that polyQ repeats are enriched in neural proteins, displaying complex evolutionary dynamics throughout phylogenesis.Pteronotus parnellii and Noctilio leporinus we observed polyQ convergence with USV-emitting rodents in FOXP1. P. parnellii (Noctilionoidea), which emits CF USVs, also has a unique Q-to-P substitution extending the predicted loop between Q1 and Q2, as in CF-emitting Rhinolophoidea. Strikingly, similar polyQ convergence patterns in FOXP2 and/or FOXP1 were found between species in most mammalian orders. Thus, these findings identify a first case of extensive molecular convergence associated with vocalization-related organismal phenotypes.The patterns of polyQ convergence between species in relation to vocalization/hearing frequency can be detected within and between mammalian orders. This is already evident in Chiroptera, in which we analyzed >50 species, eight of which display phenotypic convergence in USV type with species not belonging to their same superfamily. In six of them, this is paralleled by polyQ convergence in FOXP2, while in prestin) in taxa with high-frequency vocalization/hearing, i.e., echolocating Chiroptera and Cetacea.,,,,,,In mammals, multiple cases of molecular convergence of single residue substitutions are known in hearing-related proteins  highlighted the predictive power of FOXP2 polyQ lengths for vocalization/hearing frequency ranges. Indeed, its polyQ repeats converge with those of USV-emitting bats, in striking parallelism with the known pika-bat similarity in the evolution of the hearing-related protein prestin.O. princeps emit calls extending into the ultrasonic range and has morphological cochlear parameters indicative of high-frequency hearing,prestin.The case of the pika  recapitulated the phase separation behavior of the entire protein. Moreover, the NTD alone was also able to recruit FOXP1 into condensates. These findings concurrently identified the NTD, and its polyQ repeats as the drivers of FOXP2 LLPS. Remarkably, human FOXP2 mutants in which the polyQ repeat lenghts were changed to mimic their evolutionary variation displayed differential ability to undergo LLPS as a function of total and relative polyQ lengths.The AFM findings of condensate-like structures were confirmed in the cellular environment. Indeed, both endogenous and exogenously expressed FOXP2 form discrete, 1,6-HEX-sensitive intranuclear foci which undergo rapid FRAP, as typical of LLPS-driven condensates.,,These findings are consistent with the emerging ability of polyQ and other amino acid repeats in transcription factors to drive LLPS, as shown for the polyQ/polyA repeats of RUNX2.1+Q2 and Q1/Q2 were associated with higher repressive activity on the target SRPX2 promoter and vice versa. Only two studies have previously analyzed the possible regulatory roles on transcription of the FOXP2 polyQ repeats with conflicting results.,This interpretation is supported by our observation that, besides LLPS, the FOXP2 transcriptional activity is also related to the total and relative polyQ lengths. Indeed, lower QRemarkably, our results are instead similar to those obtained in experiments studying a FOXP1 variant devoid of its polyQ repeats, which displayed enhanced repressive activity.,,,,,,,,,A growing body of evidence indicates that polyQ and other amino acid repeats can modulate in a length-dependent manner, the structure, interactions, and function of proteins.Taken together, our findings uncover a quantitative molecular encoding of vocalization frequency in the relative lengths of polyQ repeats in FOXP2, highlight widespread patterns of vocalization-related polyQ convergent evolution, and rationalize mechanistically how polyQ length variation can have phenotypic impact by regulating the molecular function of FOXP2.These results and analytical approaches contribute to a quantitative, mechanistic understanding of vocalization and its evolution.Our analyses of the molecular evolution of FOXP2 and FOXP1 polyQ repeats in relation to vocalization frequency-related parameters were deliberately focused on Mammalia, given their considerable degree of polyQ length variability and their highly diversified vocalization modalities and frequency ranges. Thus, the study did not include some non-mammalian taxa, such as Aves, in which a considerable number of species are known to emit highly sophisticated and diversified forms of vocalization. We are currently exploring whether the results of our analyses can also apply to these non-mammalian taxa.ferdinando.fiumara@unito.it).Further requests for information, resources, and reagents should be directed to and will be fulfilled by the lead contact, Ferdinando Fiumara  supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and, for Neuro-2a cells, MEM non-Essential amino acids solution .HEK293 cells  and Neuro-2a cells  were maintained following standard procedures at 37°C with 5% CO,www.uniprot.org/) or NCBI (www.ncbi.nlm.nih.gov/protein/). For certain species, polyQ lengths were manually determined by translating genomic sequences identified through BLAST searches on the NCBI genome platform (https://www.ncbi.nlm.nih.gov/genome/) using human FOXP2 or FOXP1 sequences as the query. The list of species for which FOXP1/2 polyQ repeats were analyzed, their name abbreviations, and the corresponding protein or genomic sequence IDs are listed in 1’ and ‘Q2’) corresponding in sequence alignments to the two major repeats flanking the hinge peptide ‘ALQVARQLLL’ in human FOXP1 or ‘HPGKQAKE’ in human FOXP2. In a few predicted FOXP2 sequences of Cetacea species, the hinge peptide is extended by a likely spurious 22-residue peptide , which is not present in other cetacean or mammalian orthologs. It is encoded by a genomic sequence immediately downstream of a conserved splice site located at the end of the sequence encoding the canonical hinge octapeptide. This splice site may have been missed in the computational identification of FOXP2 exon-intron junctions in certain Cetacea genomes. Sequencing of FOXP2 cDNAs from Cetacea will be required to address this uncertainty in a conclusive manner. Anyhow, the presence or absence of this peptide does not change Q1 and Q2 lengths in these predicted ortholog sequences, which were therefore included in our analyses.PolyQ lengths were quantified using a polyQ analyzer Perl script1+Q2 sum and the Q1/Q2 ratio. In the case of multiple available sequences/isoforms for each species, we analyzed the sequence with the longest polyQ tracts. The Q1 repeats of FOXP2 are interrupted by one or more proline residues in several species. In these species, given the structural effect of proline residues,,1 length as the length of the uninterrupted polyQ repeat upstream of the first proline residue, while fragmented repeats between this proline residue and the hinge peptide were not taken into account. In a few cases, a single histidine residue was occasionally found within Q1. Given the polar nature of the residue, it was considered as being part of the polar polyQ stretch rather than an interruption of it.For each ortholog, we calculated the Qhttp://phylopic.org). M. unguiculatus and O. degus silhouettes were derived from Wikimedia .Sequence alignments were generated using MultAlin.https://www.utoledo.edu/al/psychology/research/psychobio/comphearinglab.html). For 4 species, i.e., Dipodomys merriami, Marmota monax, Onychomys leucogaster, and Spalax ehrenbergi, we used audiograms of closely related species in the same genus, i.e., Dipodomys ordii, Marmota marmota, Onychomys torridus and Spalax galili, respectively. The vocalization frequencies for non-chiropteran species of interest were derived from Charlton and Reby,https://opendata.eol.org/dataset/all-body-size-data; Vocalization modalities, vocalization frequency/bandwidth, and body mass for Chiroptera were derived from Collen.1/Q2 ratio . In 1/Q2 ratio .To calculate the correlation between the mean values of polyQ-related parameters and vocalization-/hearing-related parameters D, and 3EμCT) scans encompassing the cranial region of species of interest were obtained from the MorphoSource database , we analyzed archived recordings originally taken as part of a monitoring project  of a resident population at talus slopes in the Columbia River Gorge . Pika vocalizations were recorded using an acoustic microphone with some sensitivity to ultrasonic signals (up to 25 kHz) connected to a SM3BAT recorder (Wildlife Acoustics). The recordings that were analyzed, showing evidence of pika vocalizations extending in the ultrasonic range with harmonics >20 kHz , using a microphone located near Herman Creek . The vocalization spectrograms were analyzed using the Kaleidoscope software (Wildlife Acoustics).To analyze spontaneous vocalizations of wild American pikas . Predictions of coiled coil structures were obtained using Paircoil2.R. ferrumequinum (1–240), M. schreibersii (1–232), and C. sphinx (1–264) FOXP2 orthologs, were obtained using RaptorXPredictions of FOXP2 secondary structure were obtained using DeepCNFR. ferrumequinum and M. schreibersii orthologs were chemically synthesized (Thermo Fisher Scientific) with N-terminal acetylation, C-terminal amidation and purity >95%. Peptides were dissolved in a saline buffer 2, and stored at −80°C. The Miniopterus-derived peptide formed an opalescent solution whose concentration was difficult to measure spectrophotometrically. Thus, peptide concentrations in circular dichroism experiments were estimated based on peptide weight. This may overestimate the concentration of soluble, optically active chiral material and thus underestimate the ellipticity signal.θ] was calculated as [θ] = θ × mw/(10 × (n–1) × c × pl), where θ is the measured ellipticity, mw is the molecular weight of the peptide, n is the number of amino acids in the peptide, c is the concentration of the peptide (mg/mL), and pl is the cuvette pathlength (cm).Synthetic peptides encompassing the FOXP2 polyQ region , near the resonance frequency (190 kHz), mounted on an Easyscan2 AFM (Nanosurf) equipped with a high resolution 10-μm scan head, within a shielded and insulated enclosure placed on an antivibration platform, as in Pelassa et al.The pcDNA4/HisMax-FOXP2 plasmid encoding human FOXP2 and the pGL3-SRPX2 containing the SPRX2 promoter were kindly provided by Dr. P. Szepetowski .1, Q2, and the hinge peptide were replaced by the corresponding sequence of chiropteran FOXP2 orthologs of interest, were obtained using the In-Fusion cloning system (Clontech-Takara). Briefly, two PCR products with overlapping ends (15 bp overlaps) were obtained using the PrimeSTAR-GXL-SP DNA Polymerase (Clontech). The first product consisted of the pcDNA4/HisMax-FOXP2 plasmid sequence devoid of the region encoding the two major polyQ repeats (residues 152–209 in the human protein). The second product contained a fragment of FOXP2 orthologs of species of interest , M. schreibersii (Ms), or R. ferrumequinum (Rf)) overlapping with the human FOXP2 polyQ region fragment (a.a. 152–209) in FOXP2 ortholog sequence alignments.Plasmids encoding chimeric polyQ variants of human FOXP2, in which the sequence encoding QCs and Ms orthologs, these products were PCR-amplified using long, partially overlapping forward and reverse primers, encoding overall for the entire region of interest (as derived from NCBI). For the Rf ortholog, the second product was PCR-amplified from a plasmid encoding the region of interest previously obtained by gene synthesis . All the three primer pairs used for the Cs, Ms, and Rs polyQ region contained 15 bp extensions, corresponding to the ends of the pcDNA4/HisMax-FOXP2-derived PCR product, suitable for cloning using the In-fusion system. Finally, the latter PCR product was alternatively combined with one of the Cs, Ms or Rf polyQ fragments in cloning procedures using the In-Fusion system to generate the pcDNA4/HisMax-FOXP2-Cs, -Ms, and -Rf plasmids: These plasmids were used, together with the original plasmid encoding the Homo sapiens (Hs) ortholog, that we called pcDNA4/HisMax-FOXP2-Hs. In a fifth construct (ΔQ1Q2), the polyQ region  was deleted by re-circularizing a pcDNA4/HisMax-FOXP2-derived PCR product upon ligation using a T4 ligase (NEB). These five plasmids  were then used for luciferase assay experiments. Moreover, they served as templates for the PCR amplification of sequences encoding full-length human FOXP2 polyQ variants  with suitable 15 bp extensions for their In-Fusion cloning into the pAcGFP1-N In-Fusion Ready vector  for the expression of the GFP-tagged polyQ variant proteins. These constructs were used for studying the subcellular distribution of FOXP2 polyQ variants  and their transcriptional activity (luciferase assays). A set of plasmid constructs was similarly generated, using pAcGFP-N1-In-Fusion-Ready as the recipient plasmid, for the expression of GFP-tagged N-terminal domains (NTDs) of human FOXP2 and its polyQ length variants , corresponding to the first 244 residues of human FOXP2 in ortholog protein alignments, which encompass the first three CC regions in the Paircoil2 prediction .optoDroplet system for a spatiotemporally controlled induction of protein LLPS in living cells, we generated a vector for the cellular expression of proteins of interest fused to mCherry  and to an A. thaliana Cry2 fragment .1Q2 (a.a. 146–221) and ΔQ1Q2), and its polyQ variants  as described above.To use the All PCR products in the cloning procedures were separated by agarose gel electrophoresis and bands of interest were purified using QIAquick (Qiagen) or the NucleoSpin (Macherey-Nagel) kits. Plasmids were amplified by transformation of Stellar (Clontech) chemically competent cells and purified using the QIAprep Spin Mini-/Maxi-prep kits (Qiagen), or the NucleoSpin plasmid Mini kit (Macherey-Nagel), or NucleoBond Xtra MIDI EF kit (Macherey-Nagel). For all plasmids that were generated, the coding sequences of FOXP2 and tags were verified by Sanger sequencing.optoDroplet and FRAP experiments, cells were plated into poly-L-lysine-coated dishes obtained starting from Falcon 50 mm plastic cell culture dishes (Corning) that were perforated to produce a central hole (10 mm) then closed by a glass coverslip glued using Sylgard 184 (Dow Corning).For immunocytochemistry and imaging of fluorescently tagged proteins, cells were plated on poly-L-lysine-coated (0.1 mg/mL) glass coverslips within 24-well culture plates. For Cell (co-)transfections with plasmid DNA were performed in 24 h after cell plating using either Fugene-6 (Promega) or Lipofectamine 2000 (ThermoFisher) with a 3:1 (μl:μg) ratio between the transfection reagent and plasmid DNA (∽0-5-1 μg per well in 24-well plates). Alternatively, transfection was performed in non-adherent cells in suspension immediately before platingHEK293 and Neuro-2a cells, fixed 24–48 h after plating, were permeabilized , rinsed in PBS (3X), and incubated in blocking solution  in PBS (1 h at RT). After rinsing in PBS (3X), the cells were incubated with a primary anti-FOXP2 antibody  for 1 h at RT, rinsed in PBS (3X), and then incubated with fluorescently labeled (Alexa 488) anti-rabbit secondary antibodies for 1 h at RT. After rinsing in PBS (3X), coverslips were mounted on slides. In some experiments, coverslips were also incubated with DAPI (Sigma) for 15 min at room temperature and then rinsed with PBS (3X) before mounting.Confocal fluorescence imaging was performed using Olympus FV300 or Leica TCS SP5 confocal microscopes. Super-resolution imaging analyses were performed using a ZEISS LSM 800 Airyscan microscope system using default Airyscan settings. In general, each experiment was replicated at least three times and images were collected from multiple coverslips per experiment.z axis caused major distortions of the typical fluorescence recovery profiles and/or sudden disappearance of the foci from the ROI. These measurements, for both the Hs and Rf constructs, were not included in the analysis. Data were analyzed using Easy-FRAPt-half and immobile fraction calculations.FRAP experimentsad hoc CellProfilerThe spontaneous formation of condensates by fluorescently tagged human FOXP2, its fragments, and polyQ variants, was quantified by confocal fluorescence imaging in HEK293 cells 48–72 h after transfection. Maximum intensity projections of z stack fluorescence confocal images of microscopy fields  were converted into 8-bit images using ImageJ (NIH). Image brightness and contrast were adjusted to highlight either the foci or the entire nuclei/cell profiles of the fluorescent cells. These images were used to automatically quantify, for each field, the surface area occupied by condensates and that occupied by nuclei/cells using To define the sensitivity to 1,6-hexanediol  of the FOXP2-GFP and FOXP2-NTD-GFP foci, which is typical of LLPS-driven condensates,The triggered induction of LLPS of protein fragments of interest was obtained using the optoDroplet system.CC fragments), occupied by condensates in photoactivated cultures comparison with non-photoactivated control cultures.To quantify the overall LLPS induction in cell populations, the cell cultures were photoactivated 48–72 h after transfection using blue light (488 nm), illuminating the entire coverslip, emitted by the 100 W fluorescent lamp of an Eclipse TE200 microscope (Nikon) with suitable dichroic filters for 5–7 min. Two minutes after photoactivation, the cells were rinsed in PBS and fixed for subsequent fluorescence confocal imaging to detect the mCherry red fluorescence. Control cells were treated in the same way, except that photoactivation was omitted. LLPS-driven condensate formation was quantified, as described above, by calculating for each construct the relative area of nuclei (for full-length FOXP2 or its polyQ variants), or cells (for FOXP2 NTDz level of the cell layer) was monitored using 561 nm laser light, every minute before (5 scans) and after (20 scans) a 60 s photoactivating pulse delivered by fast-scanning the sample with blue light . In some experiments, to test LLPS reinduction, a similar pulse was delivered 20 min after the first one and red fluorescence was monitored again for 20 min. LLPS-driven condensate formation was quantified, as described above, by calculating the relative area of nuclei (for full-length FOXP2 or its polyQ variants), or cells , occupied by condensates in cultures before and after photoactivation. For each nucleus/cell, all values were normalized to the mean value in the scans before the first photoactivating pulse.To quantify the kinetics of LLPS induction, reversibility, and re-induction, we performed live-cell imaging experiments using an Olympus FV300 confocal microscope. In these experiments, the culture medium was replaced with HBSS, pH 7.4 (Sigma) at RT and the red mCh fluorescence of 2–10 cells within a magnified confocal microscopy field (at an intermediate i) the pcDNA4/HisMax plasmid, either empty (control condition) or encoding human FOXP2 (Hs), or one of its polyQ variants ; (ii) the pGL3-SPRX2 plasmidiii) the pRL-CMV vector (Promega) as a transfection control. For transfecting 8 culture wells, we mixed 1125 μg of the empty pcDNA4/HisMax plasmid (or equimolar amounts of the same vector encoding human FOXP2 or its polyQ variants), 255 μg of pGL3-SPRX2, and 120 μg of pRL-CMV. The DNA mix was transfected in a 3:1 μg:μl ratio with Fugene-6 or Lipofectamine 2000, following the manufacturers’ protocols. Forty-eight hours after transfection, firefly and Renilla luciferase activity were sequentially measured using the Dual-Glo luciferase assay system  and a GloMax microplate reader (Promega). For each well, the firefly luciferase luminescence was first normalized against the Renilla luciferase luminescence to correct for transfection efficiency. Then, all luminescence values were further normalized to the average value measured in the experimental control group expressing the empty pcDNA4/HisMax plasmid. In some luciferase assays, GFP-tagged FOXP2 variants were expressed and the pEGFP-C1 vector, instead of pcDNA4/HisMax, was transfected in control cultures. At least 18 wells from three independent experiments were analyzed for each FOXP2 variant construct. Each experiment included control wells for normalization (pcDNA4/HisMax control), for a total of 116 control wells across all the luciferase assay experiments.For luciferase transcription reporter assays, HEK293 cells were plated into 96-well white culture plates (VWR). Twenty-four hours after plating, the cells were co-transfected with three plasmid vectors, i.e., (www.morphosource.org). Ochotona princeps vocalization recordings were analyzed using Kaleidoscope Lite 5.4.7 software (Wildlife Acoustics). Digital endocasts of the left cochlea and lateral semicircular canals, as shown in Basicranium μ-CT stacks shown in n), are reported above, where appropriate, in the t test, one- or two-way ANOVA, ANOVA for repeated measures, Newman-Keuls post hoc test. and other tests, were performed where appropriate, as detailed in the pandas, matplotlib, and numpy packages).Data are expressed as mean ± standard error of mean (SEM). The details concerning the specific parameters that were quantified in each analysis , the number of experimental groups, their composition  and numerosity (μCT) scans were obtained from the MorphoSource database (https://www.morphosource.org) The scan Media IDs .The accession numbers of protein or nucleotide (genomic) sequences of the FOXP2 and FOXP1 orthologs that were analyzed are reported in


Furthermore, PDDbF displayed an elongation at break as high as 732%, around five times higher than that of the 2,5-furandicarboxylic acid counterpart, stressing the unique features of the bisfuranic class of polymers compared to monofuranic ones. The enhanced ductility combined with the relevant thermal properties makes PDDbF a highly promising material for flexible packaging.The continuous search for novel biobased polymers with high-performance properties has highlighted the role of monofuranic-based polyesters as some of the most promising for future plastic industry but has neglected the huge potential for the polymers’ innovation, relatively low cost, and synthesis easiness of 5,5′-isopropylidene bis-(ethyl 2-furoate) (DEbF), obtained from the platform chemical, worldwide-produced furfural. In this vein, poly) (PDDbF) was introduced, for the first time, as a biobased bisfuranic long-chain aliphatic polyester with an extreme flexibility function, competing with fossil-based polyethylene. This new polyester in-depth characterization confirmed its expected structure (FTIR,  In the past decades, an increasing social, environmental, legislative, and ultimately economic demand for a more sustainable model of development  has witnIn this vein, ubiquitous biomass fractions such as cellulose, lignin, polysaccharides, proteins, and vegetable oils have emerged as an inexhaustible source of raw materials to prepare a wide portfolio of valuable, and, in many cases, innovative building-block chemicals for polymer synthesis. Among them, 2,5-furandicarboxylic acid (FDCA) and various alkyl 2-furoates (including bisfuranic derivatives) are some of the most important ,9,10,11.FDCA has been exploited in both fully aromatic polymers (mainly polyesters) ,17,18,19Of particular interest are those with very long alkyl-chains (≥C8) due to the combination of rigidity imparted by the furan ring and the extreme softness associated with the aliphatic moieties. Despite this feature, there are only a few reports on this type of polyesters, focusing mostly on the optimization of their synthesis or in the study of their thermal properties ,29,30,31m) of 111 °C and a sub-ambient glass transition temperature (Tg~−22 °C). Despite the relatively moderate melting and glass transition temperatures, FDCA-based long-chain aliphatic polyesters display a combination of (bio)degradation and mechanical properties, which makes them suitable for a broad range of applications [Other works focused instead on the assessment of properties of such polyesters ,26,28; fications ,26. Moreications . Fibers ications .g and amorphous character [g, showing the same typical trend already mentioned for FDCA-based polymers decreasing with the increasing number of methylene groups [Less extensive research has been dedicated to bisfurans, such as 5,5′-isopropylidene bis-(ethyl 2-furoate) (DEbF), obtained with high purity, through a simple and cost-effective process from the platform chemical furfural ,35, albeharacter , oppositharacter ,41. Highe groups . To the 1H and 13C NMR), thermal , and mechanical (tensile testing and DMTA) properties were assessed.Additionally, to the best of our knowledge, bisfuranic long-chain aliphatic polyesters have never been reported before, albeit their anticipated enhanced flexibility, imparted by the combination of a long-aliphatic chain spacer and the less-rigid DEbF bisfuranic structure, with a symmetrical carbon between the two furan rings. This kind of polyester will be of interest for flexible packaging, an ever-growing sector . Therefo−1) was found to be in accordance with a high molecular weight polymer ). PDDbF comprises both stiff units ) and soft aliphatic chain , promptly synthesized by applying a two-step melt polycondensation procedure, as described in the experimental section and depicted in 1H and 13C NMR spectroscopies  related to 2,5-disubstituted furans. In addition, the FTIR spectrum also exhibits a band at 2852 cm−1 associated to the C-H stretching of the methylene groups of the aliphatic diol moiety and two very intense bands near 1716 and 1295 cm−1 assigned to the C=O and C-O-C stretching vibrations of ester groups, respectively.The detailed chemical structure of PDDbF was studied using FTIR, oscopies  and composcopies . The FTI1H NMR  of 341 °C (c.a. 83% weight loss), followed by a smaller peak near 430 °C. Comparing with linear low density poly(ethylene) (LLDPE), the thermal stability of PDDbF is much higher  [The thermal stability of the as prepared PDDbF was evaluated by TGA. The thermogram of  250 °C) .g) at −6 °C . This moderately low Tg is due to the high number of methylene groups in the aliphatic chain, which is associated to the high flexibility of the macromolecular chain. Nevertheless, this Tg is still higher than that of LLDPE [gs (−1 vs. 50 J g−1), which is indicative of a lower crystallinity degree and that the crystallization of PDDbF is hampered by the bisfuranic moiety [The thermal behavior of the PDDbF below the degradation temperature was studied by DSC analysis. The heating thermogram  agrees wof LLDPE . This efof LLDPE  or poly.Dynamic mechanical behavior of PDDbF was investigated by DMTA and its storage modulus (E′) a, loss m6 and 1.86 × 107 Pa, respectively), allowed to study the elastic behavior of PDDbF. Typically, this polyester had a lower E’ than other aromatic polymers incorporating shorter aliphatic moieties , hence being less stiff [Importantly, the E’ value, measured at room temperature (20 °C) and in the region of the glass transition  with similar molecular weight , which i6, 99.5% D), and deuterated chloroform  were purchased from Merck . All solvents used were analytical grade. All chemicals were used as received, without further purification.1,12-Dodecandiol , ethyl 2-furoate (99%), titanium (IV) tert-butoxide , deuterated dimethyl sulfoxide (DMSO-d1H and 13C NMR spectroscopic techniques (DEbF was synthesized according to a procedure described elsewhere . Brieflychniques .−3 mmol) were charged into the glass reactor. The reaction started by heating progressively from 150 to 170 °C, during 3.5 h, and kept at that maximum temperature, under a nitrogen atmosphere, for 1 h. Before starting the second step, the reaction temperature was decreased to 150 °C, and an additional quantity of DEbF  was added and the temperature was slowly increased again until 170 °C under vacuum for 3 h. Afterwards, the temperature was raised to 210 °C, and then, during 2 h, progressively increased up to 230 °C and kept at that maximum temperature for at least half hour. Finally, the obtained product was dissolved in chloroform, precipitated in an excess of cold methanol, filtered, and dried. PDDbF film specimens were prepared by melting procedure at c.a. 165 °C, placing the samples in a Teflon rectangular mold (50 × 10 × 1 mm3).PDDbF was synthesized by melt polycondensation, according to a previously reported procedure . In the −1 after 128 scans in the range of 500–4000 cm−1.Attenuated total reflection Fourier transform infrared (ATR FTIR) spectra were obtained using a PARAGON 1000 PerkinElmer FTIR spectrophotometer equipped with a single horizontal Golden Gate ATR cell. The resolution was 8 cm1H and 13C Nuclear Magnetic Resonance spectroscopy  analyses of samples dissolved in CDCl3 were recorded using a Bruker AMX 300 spectrometer operating at 300 or 75 MHz. All chemical shifts were expressed in parts per million (ppm) and reported downfield from 0.00 ppm using tetramethylsilane (TMS) as an internal reference.Intrinsic viscosity−4Mv0.68 ,53,54.−1 at a constant heating rate of 10 °C min−1 from 25 to 800 °C.Thermogravimetric analyses (TGA) were carried out with a Shimadzu TGA50 analyzer equipped with a platinum cell using platinum pans to encapsulate the samples (ca. 7 mg). Thermograms were recorded under a nitrogen flow of 20 mL min−1 in the temperature range of −50 to 250 °C. Two heating/cooling cycles were performed. The first heating scan was used to erase the thermal history of the polymer, and the second heating scan was performed after rapid cooling from the melted polymer.DSC thermograms were obtained with a Setaram DSC92 calorimeter. Scans were carried out under dry nitrogen, with a heating rate of 10 °C minX-ray diffraction (XRD) patterns were acquired using a Philips X’pert MPD diffractometer operating with CuKα radiation (λ = 1.5405980 Å) at 45 kV and 40 mA. Samples were scanned in the 2θ range of 3 to 60 °C, with a step size of 0.026° and a time per step of 69 s.3) were recorded with a Tritec 2000 DMTA Triton equipment operating in the single cantilever bending geometry. Tests were performed in multifrequency mode (1 and 10 Hz), from −100 to 200 °C, at 2 °C.min−1.Dynamical mechanical thermal analysis (DMTA) of thick samples , and a maximum decomposition temperature of 341.4 °C, much higher than the same parameter for LLDPE  . All in


When combinedwith comparisons with existing terrestrial reference materials (SuwanneeRiver fulvic acid and Pony Lake fulvic acid), these results suggestthat TRM-0522 is a useful and otherwise unavailable reference materialfor use in marine DOM biogeochemistry.Recent methodological advances have greatly increasedour abilityto characterize aquatic dissolved organic matter (DOM) using high-resolutioninstrumentation, including nuclear magnetic resonance (NMR) and massspectrometry (HRMS). Reliable DOM reference materials are requiredfor further method development and data set alignment but do not currentlyexist for the marine environment. This presents a major limitationfor marine biogeochemistry and related fields, including natural productdiscovery. To fill this resource gap, we have prepared a coastal marineDOM reference material (TRM-0522) from 45 m deep seawater obtained∼1 km offshore of Sweden’s west coast. Over 3000 molecularformulas were assigned by direct infusion HRMS, confirming samplediversity, and the distribution of formulas in van Krevelen spacewas typical for a marine sample, with the majority of formulas inthe region H/C 1–1.5 and O/C 0.3–0.7. The extractedDOM pool was more nitrogen (N)- and sulfur (S)-rich than a typicalterrestrial reference material (SRFA). MZmine3 processing of ultrahigh-performanceliquid chromatography (UPLC)-HRMS/MS data revealed 494 resolvablefeatures  over a widerange of retention times and masses. NMR data indicated low contributionsfrom aromatic protons and, generally speaking, low lignin, humic,and fulvic substances associated with terrestrial samples. Instead,carboxylic-rich aliphatic molecules were the most abundant components,followed by carbohydrates and aliphatic functionalities. This is consistentwith a very low specific UV absorbance SUVA As a result, DOM chemical compositionremains poorly characterized.7Dissolved organic matter(DOM) is a complex mixture of thousandsif not millions of compounds.12 Complex but reliable reference materials are necessary in orderto compare such methods and, critically, to evaluate the scope andimplications of the resulting data sets that are generated. Typically,materials from the International Humic Substances Society (IHSS) areused, which are long-established, internationally known and trusted,and can be purchased on the scale of hundreds of milligrams. The maindownside of these materials is that they are all sourced from terrestrial,often very brown water environments, with the exception of Pony Lakefulvic acid (PLFA) which came from an Antarctic lake with no higher-orderplants but which has now been depleted with no plans to restock (thatwe are aware of). The introduction of a marine DOM reference materialhas been discussed,13 and a marine referencefor dissolved organic carbon (DOC) concentration analysis has beenused successfully for some time14 but isnot available at sufficient concentrations or volumes as to be usefulas a DOM characterization standard. Thus, a suitable reference materialfor marine DOM studies does not currently exist. The lack of a marineDOM reference material is problematic and hinders method developmentand data set alignment in marine biogeochemistry within the DOM characterizationfield and in related subjects such as natural product discovery andtrace metal speciation analysis.In recent years, severaladvanced methods have been developed whichhave allowed progress to be made, such as those using nuclear magneticresonance (NMR) and high-resolution mass spectrometry (HRMS), includingtandem mass spectrometry (HRMS/MS) and coupling to ultrahigh-performanceliquid chromatography (UPLC).In order to fill this resourcegap, we have prepared and characterizeda marine DOM reference material from 45 m deep seawater obtained fromthe Tjärnö Marine Laboratory (Gothenburg University)on the west coast of Sweden, which is representative of a coastalmarine environment. The material was collected in this first effortin May 2022 from 1500 L of seawater using a commercially availableaqueous-compatible C18 column (C18-AQ hereafter), and after sampleclean-up, totaled 1.06 g in mass. We have given the sample a uniquedesignation “Tjarno Reference Material—May 2022”(TRM-0522), allowing for future efforts to collect and distributemore material. In this article, we characterize the material usingabsorbance, fluorescence, HRMS, UPLC-HRMS/MS, and NMR.22.13) was obtained from Sigma-Aldrich . Hippuric acid, fuscidicacid, chloroform (CHCl3) containing 100–200 ppmamylenes as the stabilizer, deuterium oxide (D2O), and25% ammonium hydroxide (NH4OH) solution were obtained fromSigma-Aldrich . Suwannee River fulvicacid  and PLFA (batch number 1R109F) wereobtained from the IHSS .LC–MS-grade methanol(Supelco LiChroSolv hypergrade for LC–MS) and fuming hydrochloricacid (37% HCl) were obtained from Merck (UK). Acetonitrile (ACN) wasobtained from Sigma-Aldrich (Supelco LiChroSolv hypergrade for LC–MS),formic acid (FA) was obtained from VWR ,and 25% ammonia (NH2.2Text S1), which draws water from a 45 m deepintake located ∼1.0 km off-shore of the nearest land mass onthe west coast of Sweden at 58°52.843 N 11°06.378 E .Seawater was obtainedfrom the Tjärnö Marine Laboratory seawater system (Text S2) PuriFlash C18-AQ F1600 flash column (Interchim) for ∼22h each day. Accumulated salts were then removed by flushing the columnwith ∼2 column volumes (4 L) acidified MilliQ. The column wasthen flushed with a further 4 L acidified MilliQ water. During theelution process, a fresh batch of seawater was filtered and acidified,after which the process was repeated.The DOM extraction processis summarized in Eluted MeOH was collected into a series of ashed(4 h at 550 °C)glass Duran bottles and chilled overnight, along with a portion ofacidified MilliQ which came through the column on either side of MeOH. The reason we collected the MilliQfraction was to ensure that no MeOH  was missed. A total of 1500 L of coastal seawater was pumpedthrough the column over 5 days.2.3Text S3 and resulted in a dried DOM mass of 1058 mg. The dried DOMwas redissolved in 100 mL of 10% MeOH in MilliQ water with 0.1% NH3, aliquoted for distribution, and dried down using a Speedvac or freeze drier . A 1 mgsubsample was rediluted in 41 mL of MilliQ water, giving a DOM concentrationof 24.7 mg L–1. DOC concentration was 10.1 mg L–1, equivalent to 41% DOM. Aliquots were stored in a- 20 °C freezer in 1.5 mL Eppendorf tubes ,8 mL amber glass vials , and 40 mL clear glassvials .Each eluted samplewas dried down into a round-bottom flask using a rotary evaporator,and the dried material was transferred into an 8 mL amber glass vialusing small volumes of ACN and ultrapure water (MilliQ). A residuewhich could not be dissolved in ACN or ultrapure water was removedusing 50% MeOH, which was then removed using a rotary evaporator.We suspected that this residue was the leached column material andso additional processing steps (mostly liquid–liquid extractionbetween basic water and chloroform) were added to ensure its completeremoval. Details of these steps are given in 2.42.4.1–1 using KPH, and subsequent KPH standardswere all within 5% of their calibrated value.DOC was measured using a Sievers M9 TOC analyzer and quantified asnonpurgeable organic carbon against MilliQ blanks and a potassiumhydrogen phthalate (KPH) standard. Replicate measurements had a coefficientof variation of ≤2%. The instrument was calibrated between1 and 50 mg L–1 scan speed and a 2 nm slit width using a Lambda40 UV–vis spectrophotometer  anda 1 cm pathlength quartz cuvette. Samples were blank corrected againstMilliQ water. DOC specific absorbance at 254 nm (SUVA254), a commonly used metric for aromaticity where a higher number isassociated with a greater aromatic content, was calculated by dividingabsorbance at 254 nm (in cm–1) by DOC concentration(in mg L–1) and multiplying by 100.15 Spectral slopes between 275 and 295 nm (S275–295) and 350 and 400 (S350–400) and the ratio betweenthe two (SR) were also calculated.16 S275–295 and SR are both inversely related to averageDOM molecular weight.Absorbance wasmeasured between 200 and 600 nm at 1 nm intervals and with a 240 nmmin17 and normalized to Raman units.18 Common fluorescence indices [humification (HI),19 fluorescence (FI),20 and diagenetic freshness (β/α)21 were determined alongside Coble peaks,22 which give a broad characterization of compound class . All corrections and calculations were performedusing the FDOMcorr toolbox23 for MATLAB.24Fluorescence was measured at excitation(ex) wavelengths between250 and 445 nm at 5 nm intervals and emission (em) wavelengths between300 and 600 nm at 4 nm increments using a Horiba Fluoromax-4 spectrophotometer in ratio mode and a 1 cm pathlength quartzcuvette. Slit widths were set to 5 nm, with a 0.1 s integration time.The resultant excitation-emission matrix (EEM) was blank correctedusing the EEM of MilliQ water, corrected for inner-filter effects,2.4.2–1 with 50% methanol  in ultrapure water . These solutions wereinfused at 10 μL min–1 into a high-resolutionmass spectrometer  equipped witha heated electrospray ionization source operating in negative ionmode at 100 °C with a setting of 25 units of sheath gas and 3kV. The S-lens was set to 60, and capillary temperature was 250°°C. The MS resolution was set to 140,000 at 200 Da, and transientswere measured between m/z 150 and1000, with 369.11911 and 525.19775 sets as lock masses. 500 transientswere collected and averaged, and a wash solution of 50% methanol:water (LCMS grade) was infused between samples until the blank signalstabilized. Formulas were assigned allowing 4–50 C, 4–100H, 2–40 O, 0–2 N, 0–1 S, and 0–1 13C. Combinations of N, S, and 13C were not allowednor were double-bond equivalence minus O greater than 10. A 5th orderpolynomial calibration was performed using a series of expected ions,as given in a previous paper,25 and formulaswere assigned if the mass offset was less than 1.5 ppm.Dried samples were accurately weighed and dissolved/diluted to 50mg DOM L2.4.3–1. 10 μl of the sample containing 50 μg of DOM was injectedonto a UPLC C18 column  at a flow rate of 0.4 mL min–1 using a Vanquish UPLC (Thermo Fisher). Three mobile phases wereprepared: LCMS-grade water with 0.1% FA (A), LCMS-grade acetonitrilewith 0.1% FA (B), and 90:10 LCMS-grade MeOH in LCMS-grade water with0.05% FA and 0.25% NH3. The gradient was run as follows:0–1 min = 1% B, 0% C; 1–9 min = 1–99% B, 0% C;9–10 min = 99% B, 0% C; 10–10.1 min = 99–1% B,0–99% C; 10.1–11.4 min = 1% B, 99% C; 11.4–11.5min = 1% B, 99–0% C; and 11.5–15 min = 1% B, 0% C. AnOrbitrap Q Exactive mass spectrometer was used, operating in negativemode for one analysis (triplicates plus blank) and positive mode foranother (triplicates plus blank). Data were collected in a data-dependentmode, with the top five peaks sent for HCD fragmentation, and areavailable at the MassIVE data repository . MZmine3 was used to align MS1-level LC–MS features26 and find chromatographically resolved peaks10 (MZmine3 project available in the Supporting Information), and Global Natural ProductSocial Molecular Networking (GNPS) jobs were run on positive and negativemode fragmentation data27 to cluster andlibrary match fragmentation patterns at the MS2 level.28A 1 mg dried aliquot was dissolved in 200 μLof 10% LCMS-grade methanol in LCMS-grade water, sonicated for 5 min,and vortexed to give a concentration of 5000 mg L2.4.413C/15N/1H 5 mm-ZCryoProbe. The samples were dissolved in D2O and referencedto the residual solvent peak at 4.79 ppm in the 1H NMRspectrum or to MeOH at 49.50 ppm which was added as an external referencefor the 13C NMR spectrum. 1H NMR spectra weregathered over 330 scans, with a 3.28 s acquisition time and a 15.3s relaxation delay. 13C NMR spectra were gathered over9600 scans, with a 0.92 s acquisition time and an 8 s relaxation delay.NMRspectra were acquired at 298 K on a Bruker 500 MHz spectrometer usinga TXO CRPHe TR-33.1–1 seawater extracted. DOC accounted for 41% ofthe extracted DOM, equivalent to 0.29 g of DOC L–1 seawater extracted (24.2 μM SPE-DOC). DOC typically comprises∼50% DOM in humics29 which are likelyto be highly phenolic and aromatic in character and ∼45% infreshwaters, where humics are mixed with the rest of the aquatic DOMpool.30 The aged, recalcitrant DOM typicallyfound in seawater is thought to be dominated by alicyclic and linearcompounds which are rich in carboxylic acids,31 andthe increased abundance of oxygen from carboxyl groups likely decreasesthe percentage of carbon in marine DOM relative to the terrestrialsystems.The final dry DOM mass was 1.06 g, equivalent to 0.71 g of DOML–1, giving a SUVA254 of 1.52 L mg C–1 m–1. SUVA254 typically ranges from 1.0 to4.0 L mg C–1 m–1 in surface waters,32 although values >6.0 L mg C–1 m–1 have been reported for samples with a strongterrestrial signal33 or with a high ironcontent,34 and values <1 L mg C–1 m–1 have been reported for samplesdominated by fresh DOM production . Coastal ocean samples typically exhibit valuesof around 1.5–3.5 L mg C–1 m–1, while open ocean samples tend to be closer to 1.0 L mg C–1 m–1.36 Thus, the SUVA254 value of TRM-0522 (1.52 L mg C–1 m–1) is within the expected range and indicates a considerablylower degree of aromaticity relative to other available referencematerials, e.g., Suwannee River natural organic matter , SRFA (4.20 mg C–1 m–138), and PLFA (2.0–3.2 L mg C–1 m–139). S275–295 and S350–400 were0.18 and 0.17, respectively, giving an SR of 1.05, which is lowerthan SR typically reported for marine samples, for example, Helmset al. (2008) reported SR values of 9.4 for the Sargasso Sea, droppingto 3.9 on the continental slope and 1.5 at the shelf break, relativeto SRNOM which has an SR of around 0.70.16 The SR of TRM-0522 describes the extracted rather than the bulksample, and so an SR of 1.05 suggests that the extracted DOM fractionis biased toward higher-molecular-weight compounds. An absorbancespectrum and EEM for TRM-0522 diluted to a concentration of 10.1 mgL–1 are shown in Figure S3, with the associated Coble peaks listed in Table S1. TRM-0522 fluoresces highly in the peak T and M regions,indicative of protein-like and marine humic-like DOM, with a smallerbut notable contribution of peak A, terrestrial humic-like fluorescence.FI, HIX, and β/α were 1.49, 1.59, and 1.67, respectively.FI and HIX are indicative of the degree of autochthonous and humifiedmaterial in a sample and together indicate that TRM-022 contains DOMwith a low degree of humification which is typical of marine samples.40 β/α provides a measure of diageneticstatus, with higher values indicating less diagenetically altered(i.e. “fresher”) DOM, and has previously been reportedas < 0.7 in freshwaters41 and between0.65 and 1.3 in coastal waters,42 whichindicates that TRM-022 contains DOM which is less diagenetically alteredthan is typical.Absorbance at 254 nm was 0.152 cm3.225 and so the true diversity of the TRM-0522 samplecould not be completely assessed.43HRMS analysis yielded >3000 peaks 1. In ter44 with the majority offormulas in the region H/C 1–1.5 and O/C 0.3–0.7 ,numbering 233 (negative) and 261 (positive). Unlike the typical broadlyeluting features found in the geochemically degraded DOM background,these features are well resolved and can be used as reference peaksfor method development, validation, and drift correction if TRM-0522is included in metabolomic data sets. The number of library hits foundusing GNPS molecular networking was surprisingly low (only five perionization mode and most of these known laboratory contaminants).This low number of hits may be due to the fact that so much of DOMis not characterized and annotated47 andmay also be due to the extraction method used here being poor at retainingparticularly hydrophilic compounds. Possibly, the pipeline and gravelfiltration method used to collect coastal water for the aquarium activitiesat the research site also filters out many known metabolites. However,hundreds of resolvable features were found at the MS1 and MS2 level, with relatively minor variations in intensity. We cannotrule out that this peak is derived from lignin in TRM-0522, but thelack of aromatic C–H protons attributable to lignin makes itdifficult to conclusively state that this methoxy ether peak is ligninderived. The relative amounts of each accurately definable area (regionsA–E) are presented in By examiningthe entified4, with r1H NMR spectra of TRM-0522 retain thekey featuresthat have previously been described for marine and freshwater DOM.50 Specifically, regions have beenattributed to carbohydrates and aliphatic compounds.31 The aliphatic region has been described as including bothpeptide-derived aliphatics but also material derived from linear terpenoids(MDLT).51 Roughly, the ranges displayedon 49 Of note is a pronounced feature at 3.52–3.76ppm, which in the majority of previously reported samples is not observedas a distinct peak.50 However, this peak is seen in spectra of samples isolated at severaldepths in the Atlantic ocean31 and is possiblyderived from methoxy-ether functionalities.6 Consistent with other marine water samples, TRM-0522 lacks strongsignals attributable to alkenes or aromatics, indicating a low contributionfrom lignin, humic, and fulvic substances. This also corresponds wellto our finding of very low SUVA254 (1.52 L–1 g cm–1).The 13C NMR spectrum, as well as some highly branched aliphatic signals that neighbor,but are not directly attached to, heteroatoms. Region C, from 65 to95 ppm, contains predominantly signals attributable to oxygen-substitutedcarbon atoms, such as ethers and ring carbons in carbohydrate molecules.Region D, from 95 to 110 ppm, contains dioxygenated carbons, suchas those from the anomeric position of carbohydrates. Next, regionE consists of aromatic carbon atom signals and covers the range of110–145 ppm, while region F consists of phenolic carbon atomsignals and spans 145–165 ppm. Finally, signals for carboxylicfunctionalities, including acids, amides, esters, and ketones, arefound in region G, covering 165–220 ppm. A peak correspondingto residual methanol can be observed at 49.00 ppm.In the spectrum4, seven 13C NMRspectrum of TRM-0522 agrees with the analogous 1H NMR spectrum.Furthermore, the general regions and rough peak shapes correspondto previously reported spectra.57 While there are observable peaksin the aromatic/olefinic region, they are relatively minor when comparedto those derived from saturated carbon functionalities. This is consistentwith the relatively low content of lignin, humic, and fulvic substancesin oceanic samples.57 A carbonyl signal is observed in the regiontypically associated with carboxylic acid and amide functionalities(∼190–170 ppm), while no detectable signal is associatedwith other carbonyl functionalities . In agreement with the 1H NMR spectrumanalysis, a strong band corresponding to the alcoholic carbons insugar compounds (95–75 ppm) is observed. While the anomericsignals are obscured by the residual water peak in the 1H NMR spectrum, a peak attributable to the anomeric carbons of carbohydratesis observed in the 13C NMR spectrum (∼105–95ppm). The remaining two regions contain strong aliphatic signals,with the signal reducing in intensity outside the 55–15 ppmrange, consistent with the presence of cyclic and linear terpenoid-derivedmaterial, with cyclic material tending toward the higher chemicalshift range, and linear material tending toward the lower chemicalshift range.51Generally,information extrapolated from the 13C NMRcomparison, data was used from the U.S.geological survey.58 The most obvious differencebetween the 13C NMR spectra of the materials is a significantaromatic peak spanning 110–145 ppm in the SRFA spectra, a regionin which only relatively minor resonances can be observed in the TRM-0522spectra. This finding reinforces the lack of the relatively photolabilehumic, fulvic, and lignin substances in TRM-0522 and suggests thatthe material is unlikely to be photoreactive. While other regionsin the 13C NMR spectra of the two samples are relativelysimilar, differences are noted with the increased peak height in regionsattributable to sugars in TRM-0522 and an increased peak height indicative of substances attributableto carbonyl functionalities in SRFA .For 1H NMR spectrumof SRFA was obtained . Aromatic hydrogensare typically associated with humic and fulvic substances, as wellas lignin. In SRFA, these hydrogens should predominantly come fromfulvic acid, but the presence of lignin cannot be ruled out as thearomatic window is broad and covers the signature peak for ligninat 6.57 ppm. Second, the ratios of the A, B, and C regions remainalmost identical between spectra of SRFA and TRM-0522 samples, suggestingthat the relative amounts of MDLT, CRAM, and complex polysaccharidesremain consistent between the two samples.3.513 The smallcartridge size typically used has thus far presented a barrier toextraction of marine DOM on the scales required to produce meaningfulquantities of reference material.13 Thelarge-scale extraction method detailed in this article and appliedin a coastal setting could be applied to open waters with little needfor further development.We note ongoing discussionwithin the IHSS on the need for more diverse DOM standards, includingfor open ocean marine DOM.14 as a DOM standard is technicallyfeasible, but we consider this to be an inefficient option: assuminga typical DOC concentration of 0.5 mg L–1, a C contentof 50%, and an extraction efficiency of 50%; a 1 mg of DOM extractionwould require 100 × 20 mL vials at a cost of $140 USD. TRM-022is currently available at a cost of approximately $25 USD per 1 mgand has been well characterized using several common high-resolutionmethods . Thus, TRM-022 fills a resourcegap for a marine DOM reference material.Use of the existing marine DOC referencematerialTRM-0522 is not anopen ocean marine standard but rather a DOMstandard obtained from a coastal marine setting. While it is compositionallyvery different from available terrigenous standards , we do acknowledge some terrigenous input. However, until suchtime as a fully marine, open water DOM standard can be made available,TRM provides the research community with a reference material whichis considerably more similar to oceanic waters than the existing terrigenousstandards are. Once a fully marine DOM standard exists, we believethat a coastal marine sample will remain of great value, particularlywith regards the growing number of land–ocean studies whichmust operate across fresh and marine waters. In this context, TRMfills an important niche between a fully terrigenous and a fully marinesample.4The results presented inthis paper demonstrate that TRM-0522 isa useful and reasonably representative reference material for usein marine DOM biogeochemistry, particularly in a coastal setting.TRM-0522 is now available to the research community, and we encourageits use in marine biogeochemical studies including DOM characterization,natural product research, and metabolomics. During our first effort,we have made 700 mg of marine DOM reference material available, withfuture, larger-scale extractions planned. 50 mg of the current batchis available free of charge for researchers with financial barriersto purchase. A fixed extraction point associated with an establisheduniversity research laboratory  was selected to ensure ongoing access, consistencyof source, and a long-term supply. Thus, we intend to make TRM widelyavailable and encourage its day-to-day analytical use. Enquiries canbe sent to the corresponding author (JAH).


The data indicated three phases for the mangrove development: (1°) mangrove expansion on tidal flats with estuarine organic matter between >4420 and ~2870 cal yrs BP, under the influence of the mid-Holocene sea-level highstand; (2°) mangrove contraction with an increased contribution of C3 terrestrial plants between ~2870 and ~84 cal yrs BP due to an RSL fall, and (3°) mangrove expansion onto the highest tidal flats since ~84 cal yr BP due to a relative sea-level rise. However, significant mangrove areas were converted to fish farming before 1984 CE. Spatial-temporal analysis also indicated a mangrove expansion since 1984 CE due to mangrove recolonization of shrimp farming areas previously deforested for pisciculture. This work mainly evidenced a trend of mangrove expansion due to RSL rise preceding the effects of anthropogenic emissions of CO2 in the atmosphere and the resilience of these forests in the face of anthropogenic interventions.Predictions of the effects of modern Relative Sea-Level (RSL) rise on mangroves should be based on decadal-millennial mangrove dynamics and the particularities of each depositional environment under past RSL changes. This work identified inland and seaward mangrove migrations along the Ceará-Mirim estuary  during the mid–late Holocene and Anthropocene based on sedimentary features, palynological, and geochemical (δ Global mangrove distribution, which occurs along tropical and subtropical latitudes, has changed in the Holocene and Anthropocene ,2,3. The13C, δ15N), and elemental (C\N) analyses along topographically referenced cores sampled from the highest and lowest tidal flats of the Ceará-Mirim estuary with historical low (Landsat) and high (QuickBird and Drone) spatial resolution images and aerophotogrammetric data acquired by drones. Drone data allowed us to identify the coastal vegetation structure associated with the topographical gradients of the coastal plain. The application and analysis of drone images (3 cm resolution) permitted the identification of small patches of mangroves and their changes in much greater detail [Besides natural forces, the anthropogenic conversion of mangrove forests to land meant for aquaculture/agriculture has caused global mangrove loss . The manr detail ,28,29,302, causing a significant impact on the wetlands vegetation (2), with Rhizophora mangle and Avicennia germinans, occur along ~5 km of the estuarine channel on tidal flats between 1 and 2 m above mean sea-level (amsl). Rhizophora trees occur mainly on lower tidal flats (~1 m amsl), showing the tallest trees , while the smallest Rhizophora  inhabit middle elevations (~1.7 m amsl)  and present the highest trees  on lower surfaces (~1.7 m amsl), causing a transition with the lowest Rhizophora. The tallest Avicennia trees (~10 m) are spread among the tallest Rhizophora on the lowest flats (~1 m amsl). Some Laguncularia  are spread in smaller numbers, mainly among Avicennia trees on the highest flats. Herbaceous vegetation (4.6 km2), represented by Poaceae, Cyperaceae, Borreria and some Arecaceae, occurs on the highest tidal flats (2–3 m amsl). Arecaceae trees inhabit seasonally and permanently inundated flats (~7 km2) by freshwater, while the coastal plateau presents wooded steppe savannah mainly characterized by Cyperaceae and Poaecea, with few shrubs primarily characterized by Anacardiaceae and Malpighiaceae  a. Avicenghiaceae .Avicennia trees led this mangrove migration onto the highest tidal flats. The transition zones show advances and retreats of the mangrove/herbaceous vegetation, where there is the coexistence of Poaceae, Cyperaceae, and Avicennia shrubs. It is noteworthy that isolated and elevated sandy tidal flats (~2 m amsl), presenting a circular morphology with herbaceous vegetation and without human interference, also show intermittent advances and retreats of Avicennia shrub limits, but with an expansion trend between 1984 and 2018. By contrast, a progressive mangrove expansion has occurred mainly onto lower limits of tidal flats (~1–~1.7 m amsl) with abandoned shrimp farms. These lower surfaces have been consistently invaded, mainly by Rhizophora.Satellite images indicated a mangrove expansion trend between 1984 and 2018, with 315,800 ha in 1984 increasing to 443,400 ha (2000), 404,800 ha (2005), 483,200 ha (2011), 435,100 ha (2016), and 590,200 ha (2018). Mangroves expanded mainly onto herbaceous flats and abandoned shrimp farms . The manThe C−14 dating for the NAT−3 and NAT−5 cores are exhibited in 13C, δ15N, TOC, TN, and C:N data allowed us to propose three facies associations to determine sedimentary environments [The radiography of these cores allowed us to identify internal sedimentary structures. As recorded in ronments : (A)—TidCyperaceae (0–33%), Poaceae (0–23%), Euphorbiaceae (0–20%), Typhacea (0–4%), and Cucurbitaceae (0–4%), predominate in this facies association. Trees and shrubs (35 to 71%) are mainly represented by Fabaceae (0–40%), Moraceae/Urticaceae (0–25%), Rubiaceae (0–38%), Myrtaceae (0–23%), Bignoniaceae (0–5%), Melastomataceae (0–5%), Meliaceae (0–4%), Myristicaceae (0–4%), Malpighiniaceae (0–3%), and palms, formed by Arecaceae (0–6%). Mangrove pollen (15 to 47%) are represented by Rhizophora (0–35%), Laguncularia (0–40%), and Avicennia (0–3).This facies association presents massive sand (Ms) . This facies association occurs at the basis of the NAT−5 in intervals of 150–130 cm depth . Herb po13C and δ15N values vary between −29.7‰ and −26.8‰ (mean 28.2‰) and from −0.84 to 1.6‰ (mean 1.22‰), respectively. TOC and TN values are between 3.74 and 17.58% (average 10.66%) and 0.12 and 0.54% (average 0.33%), respectively. The C:N values occur between 21.2 and 45 (The δ2 and 45 .Rhizophora (8–30%). Herbs (45–82%) are mainly characterized by Cyperaceae (40–68%), Poaceae (0–7%), and Typhacea (0–3%). Trees and shrubs (17–38%) are characterized by Fabaceae (0–34%), Anacardiaceae (0–22%), Euphorbiaceae (0–20%), Bignoniaceae (0–8%), Moraceae/Urticaceae (0–20%), Rubiaceae (0–15%), Myrtaceae (0–8%), Melastomataceae (0–6%), Meliaceae (0–5%), Theaceae (0–7%), and Ilex (0–4%). Arecaceae pollen occurs with 1–5%. The δ13C and δ15N values are between −29.2 and −28.7‰ (mean 28.95‰) and −0.91 and 2.91‰ (mean 1‰), respectively. TOC and TN present values between 3.44 and 17.58% (mean 10.51%) and 0.06 and 0.43% (mean 0.24%), respectively. The C:N values are between 27.6 and 34.14 (These deposits are characterized mainly by wavy (Hw) heterolytic bedding (Hl) and by massive mud (Mm)  with bioturbation, represented by plant fragments . Roots and 34.14 .Cyperaceae (26–50%), Poaceae (0–27%), Euphorbiaceae (0–20%), Typhacea (0–25%), Mimosa (0–6%), and Asteraceae (0–5%). Trees and shrubs (25–40%) are represented by Fabaceae (0–22%), Anacardiaceae (0–24%), Moraceae/Urticaceae (0–10%), Rubiaceae (0–5%), and Myrtaceae (0–10%). Mangroves, represented by Rhizophora (0–1%), and palms, characterized by Arecaceae (0–3%), were also identified. The δ13C and δ15N values are ~−28.9‰ and ~0.58‰, respectively. TOC and TN present percentages between 4.25 and 20.4% (mean 12.32%) and 0.43 to 0.83% (mean 0.63%), respectively. The C:N values are between 24.5 and 28.67 (This facies association consists of massive mud (Mm) and wavy (Hw) heterolytic bedding  and the presence of fragments of plants and roots . It occund 28.67 .The interpretations of this study are based on robust multi-proxies. Environmental proxy indicators have the potential to provide evidence for large-scale climatic, sea-level, and depositional system changes. However, the interpretation of a proxy record is complicated by the presence of “noise” in which environmental information is immersed and there is a variety of possible distortions of the underlying environmental information ,41; thenRhizophora, Avicennia, and Laguncularia, typical elements that occur along the margin of a tidal channel (facies association A). Isotopic data revealed that organic matter was sourced from terrestrial C3 plants and estuarine organic matter >4420 cal yr BP. The second phase  in the NAT−5 site (2.5 m above mean sea-level) is characterized by the establishment of the mixed tidal flat dominated by herbaceous vegetation and mangrove expansion, represented by Rhizophora. This phase is represented by estuarine organic matter (Multi-proxy data suggest four phases of mangrove development: The first stage is represented by massive sand (Sm) with a gradual transition to wavy heterolithic beddings (Hw), suggesting that flow energy decreased ,44. Thesc matter a. The thc matter . The fouc matter .13C and δ15N) and C/N analyses combined with mangrove pollen grains along stratigraphic profiles have been used to record trends of RSL changes [13C, δ15N, and C/N changes may indicate an RSL trend [RSL changes are related to land-based reference ,51, main changes ,57,58, b changes ,60,61,62SL trend ,30, accuSL trend , which aSL trend ,63.15N values oscillated between 45 and 18 and 0 and 3, respectively, suggesting a blend of aquatic and terrestrial organic matter. A paleoecological study revealed mangrove establishment in the study area at ~6920 cal yr BP [In this context, RSL rose on the northeast coast of Brazil during the early and mid Holocene ,16,64 wial yr BP  after a al yr BP , while mal yr BP . The higal yr BP ,18,19, cStudies have indicated a gradual RSL fall in southeastern, northeastern, and northern Brazil since the mid-Holocene high sea-level stand ,16,18,6715N values decreased from 3 to 0, indicating a decreased trend in the aquatic influence. The core NAT–3, sampled from a tidal flat lower (~1.5 m amsl) than core NAT–5 (~2.5 m amsl), presented an increased trend of mangrove pollen percentage from 15 to 35% during the last ~600 cal yrs BP, probably due to an RSL fall, which favored mangrove expansion on lower surfaces.The transition from mid-Holocene high sea-level stand to RSL fall triggered feedbacks associated with tidal range, vertical sediment accretion, and tidal water salinity along the estuarine channel . MangrovAfter the mid-Holocene sea-level highstand , RSL fell immediately and eventually rose again about 2100–1100 cal yr BP, resulting in a second coastal retreat in the Holocene . This cu13C, as recorded along the northern and northeastern Brazilian coast throughout the late Holocene [15N (from 3 to 0) values, suggesting a decreased trend in the aquatic influence, then a significant impact of a relative sea-level fall in the source of sedimentary organic matter in the NAT−5 site  during the last ~2170 cal yr BP. Furthermore, climate trends in northeastern Brazil during the mid–late Holocene are still open to debate. In the Maranhão littoral, ~800 km distant from the study area, paleoecological studies revealed a drier climate between ~10,000 and ~4000 cal yr BP [Mangrove retraction on the highest tidal flats of the study area may not be exclusively influenced by an RSL fall. An increase in rainfall regime on mangroves and over the Ceará-Mirim River basin may cause a decrease in the estuarine salinity indicators and mainly affect the values of δHolocene ,6. This al yr BP , and a wal yr BP ,72. Analal yr BP ,74,75,76al yr BP ,78,79. Hal yr BP ,82,83. TAvicennia trees  occur on tidal flats between 1.7 and 2 m amsl. The lowest tidal flats (~1 m amsl) are dominated by Rhizophora  that lasted roughly from 1300 until 1850 CE [In this context, an RSL rise caused an increase in the tidal inundation frequency as the upper limit of the tides reached progressively higher surfaces. This process caused salinization of the higher flats, but with a gradual decrease in porewater salinity along the topographic gradient as the time available for evaporation on tidal flats decreased ,24,30,84 warming ,87,88,89 1850 CE ,91,92. T 1850 CE ,93,94,95This trend of relative sea-level rise can be recorded even during the last decades ,24,30, bThis study integrated planialtimetric and stratigraphic data, following a methodology flow chart divided into three phases : (1) spa2 and flows in a west–east direction, with a length of 120 km crossing the coastal plateau/plain. It is influenced by semidiurnal saline and dynamic mesotides with a mean spring and neap tidal range of ~2.30 m and 0.85 m, respectively. Near the mouth, it presents a transition zone under marine and fluvial influence, dominated by sand–clay and silty sediments [Rhizophora mangle, Avicennia schaueriana, Avicennia germinans, and Laguncularia racemosa. Herbaceous vegetation is represented by typical species of the brackish environment, with halophytes that tolerate a high degree of salinity, mainly characterized by Sesuvium portulacastrum and lresine vermicularis [Sesuvium portulacastrum, Conocarpus erectus, Laguncularia racemosa, Avicennia germinans, and Spartina alterniflora [Borreria [Fabaceae, Rubiaceae, Arecaceae, Poaceae, and Cyperaceae families [The researched area, situated in the mouth of the Ceará-Mirim River in northeastern Brazil , is charediments . The coaediments ,84. Mangicularis . Some torniflora . The traBorreria . Floodplfamilies ,101. Herfamilies —Figure 12, 0.09 ha) are indistinguishable. Therefore, the mapping of mangrove/herbaceous vegetation and mangrove/abandoned shrimp farms transition will be limited by the uncertainties caused by the pixels with mixed coverages . The spectral response of these coverages depends on the average reflectance involving the different targets in the pixel [Landsat 5 and 8 satellites (resolution of 30 m), with three bands , were acquired from INPE , Global Land Cover Facility Project Website—(GLCF), and US Geological Survey (USGS). These images (1984–2018) were used to identify vegetation and geomorphological units by an unsupervised classification with the PCI software. Ground control points using a GPS  provided a reliable indicator to subsidize the classification obtained by satellite images. Kappa index, with a sampling of 300 pixels for the classification, in random mode, followed the methodology of  to validhe pixel .Planialtimetric data obtained by drone images (0.03 m) were processed by the Agisoft Metashape 1.6.2 software . The data obtained by drone images were integrated and assessed using the Global Mapper 22 Software . The digTwo sediment cores  were acquired via a Russian sampler in a transition restinga/mangrove , and in mangrove vegetation  a. RussiaThe sediment cores were radiographed to characterize sedimentary aspects. Samples of ~0.5 g, obtained using a scale with a precision of 1 mg and at 10 cm intervals (46 samples) along the studied cores, were necessary to guarantee the quality of grain size analysis by laser diffraction (SHIMADZU SALD 2101) at the Laboratory of Coastal Dynamics of the Federal University of Pará UFPA (Brazil). The sampling considered the heterogeneity of the beddings to evidence the alternations in the grain size fractions, which was not always possible due to the millimeter thickness of the mud/sand layers. Hydrogen peroxide and ultrasound were applied to eliminate organic matter and disaggregate sediment particles, respectively. Grain sizes classification followed : sand  for palynological analysis. The conventional analytical procedures of pollen in the samples followed the criteria of [Lycopodium spore capsule (grains/cm3). Pollen databases were used to help identify pollen and spores [Forty-four sediment samples of 1 cmteria of . Pollen d spores ,115,116,d spores .13C, δ 15N, total organic carbon (TOC), and total nitrogen (TN) analyses for the samples with an accuracy of 0.09% and 0.07%, respectively. Our δ13C and C/N data were analyzed according to sedimentary organic matter determined for C3 terrestrial plants (δ13C: −32–−21‰ and C/N > 12), C4 plants (δ13C: −17–−9‰ and C/N > 20) [13C: −25–−30‰, and C/N: 6–10) [13C: −24–−18‰, and C/N: 6–10) [15N values between ~0‰ and ~10‰, respectively [Rhizophora (δ13C: −31.8–−30.2‰), Avicennia (δ13C: −30.4–−29.1‰), and Laguncularia (δ13C: −30.4–−29.2‰) [A total of 90 samples 10–60 mg) were obtained at 5 cm intervals along the cores. HCl (4%) was applied to remove carbonate . The Sta mg were /N > 20) ,119, freN: 6–10) , and marectively . Mangrov–−29.2‰) .In order to obtain a chronological model, three sediment samples (~10 g) were taken for C-14 dating. Predominantly muddy layers accumulated by vertical accretion were sampled for C-14 dating. Roots, plants debris, and seeds were physically removed from the sediment samples by a stereomicroscope to avoid carbon rejuvenation ,122. The2 in the atmosphere. However, significant mangrove areas were converted to fish farming before 1984 CE. Spatial-temporal analysis revealed a mangrove expansion since 1984 CE caused by mangrove recolonization of shrimp farming areas previously deforested for pisciculture, evidencing the resilience of these forests in the face of anthropogenic interventions.Multi-proxy data revealed mangrove migrations along a topographic profile of a tidal flat of the Ceará-Mirim estuary, northeastern Brazil, during the mid–late Holocene and Anthropocene. Mangroves expanded on the highest tidal flats with estuarine organic matter between ~4500 and 2870 cal yrs BP, under the influence of the mid-Holocene sea-level highstand. However, a mangrove contraction occurred after ~2870 cal yr BP with an increased contribution of C3 terrestrial plants, probably due to an RSL fall in the late Holocene. Mangroves have recolonized the highest flats of the study area during the last ~84 cal yr BP due to a relative sea-level rise, probably related to the end of the LIA, preceding the effects of anthropogenic emissions of CO