First assessment of genetic diversity, phylogeographic relationships, and population structure of the brown seaweed Ericaria amentacea from Italian coasts using cytochrome oxidase subunit I (COI) gene

First assessment of genetic diversity, phylogeographic relationships, and population structure of the brown seaweed Ericaria amentacea from Italian coasts using cytochrome oxidase subunit I (COI) gene | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 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Data may be preliminary. 27 January 2026 V1 Latest version Share on First assessment of genetic diversity, phylogeographic relationships, and population structure of the brown seaweed Ericaria amentacea from Italian coasts using cytochrome oxidase subunit I (COI) gene Authors : Maha Moussa , Sarra Choulak 0000-0003-0088-315X [email protected] , Valentina Asnaghi 0000-0003-1659-2613 , Daniele grech , Khaled Said , Mariachiara Chiantore , and Sonia Scarfi Authors Info & Affiliations https://doi.org/10.22541/au.176949737.74841877/v1 156 views 82 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Ericaria amentacea is an endemic key seaweed species in the coastal ecosystem of the Mediterranean basin. Given its ecological role and sensitivity to environmental conditions decline, it has been designated as protected species. Scarce local population genetic studies have been performed on this species on Italian coasts. For the first time, mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and analyzed for 42 Italian E. amentacea specimens, collected from five localities along Ligurian and Sardinian coasts. In addition, 9 COI sequences of the same species were retrieved from National Center for Biotechnology Information (NCBI) investigating South Italy coasts. Polymorphism results revealed high values of haplotype diversity (Hd) and very low nucleotide diversity (π). Thus, these results suggest that our brown seaweed E. amentacea populations may have undergone a bottleneck followed by rapid demographic expansion. This inference is strongly confirmed by the results of neutrality tests and “mismatch distribution”. The important number of haplotypes between localities and the high genetic differentiation (Fst = 0,79) of the current E. amentacea populations could be maintained by the limited gene flow Nm (0.48). Moreover, results indicated that genetic variation is high, with most of it distributed among populations (79.39 %). Both haplotype Network and biogeographic analysis showed a structured distribution according to the geographic origin. E. amentacea populations are subdivided into continental versus insular populations. Indeed, these findings have important implications for conservation and restoration actions to counteracting the potential decline of E. amentacea populations that require a deep knowledge of their genetic structure. First assessment of genetic diversity, phylogeographic relationships, and population structure of the brown seaweed Ericaria amentacea from Italian coasts using cytochrome oxidase subunit I (COI) gene Maha MOUSSA 1,2,3 , Sarra CHOULAK 3* , Valentina ASNAGHI 1,2 , Daniele GRECH 2,4 , Khaled SAID 3 , Mariachiara CHIANTORE 1,2 , Sonia SCARFI 1,2 1 Department of Earth, Environment and Life Sciences (DISTAV), University of Genoa, Italy 2 National Biodiversity Future Center (NBFC), Piazza Marina 61, 90133 Palermo, Italy 3 Laboratory of Genetics, Biodiversity and Bioresources Valorisation (LR11ES41), Higher Institute of Biotechnology of Monastir, University of Monastir, Tunisia 4 International Marine Centre (IMC), Loc. Sa Mardini, Torregrande, 09170 Oristano, Italy *Sarra CHOULAK : [email protected] Abstract Ericaria amentacea is an endemic key seaweed species in the coastal ecosystem of the Mediterranean basin. Given its ecological role and sensitivity to environmental conditions decline, it has been designated as protected species. Scarce local population genetic studies have been performed on this species on Italian coasts. For the first time, mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and analyzed for 42 Italian E. amentacea specimens, collected from five localities along Ligurian and Sardinian coasts. In addition, 9 COI sequences of the same species were retrieved from National Center for Biotechnology Information (NCBI) investigating South Italy coasts. Polymorphism results revealed high values of haplotype diversity ( H d) and very low nucleotide diversity ( π ). Thus, these results suggest that our brown seaweed E. amentacea populations may have undergone a bottleneck followed by rapid demographic expansion. This inference is strongly confirmed by the results of neutrality tests and “mismatch distribution”. The important number of haplotypes between localities and the high genetic differentiation ( F st = 0,79) of the current E . amentacea populations could be maintained by the limited gene flow Nm (0.48). Moreover, results indicated that genetic variation is high, with most of it distributed among populations (79.39 %). Both haplotype Network and biogeographic analysis showed a structured distribution according to the geographic origin. E. amentacea populations are subdivided into continental versus insular populations. Indeed, these findings have important implications for conservation and restoration actions to counteracting the potential decline of E. amentacea populations that require a deep knowledge of their genetic structure. Keywords : Ericaria amentacea , COI mtDNA, phylogeography, Genetic diversity, Italian coasts. INTRODUCTION Canopy-forming brown macroalgae are foundational species in temperate coastal ecosystems, providing structural habitat, influencing hydrodynamics, and supporting diverse communities of invertebrates and fishes (Thibaut et al., 2015; Bulleri et al., 2022). The decline of these macroalgal forests has been linked to coastal degradation, pollution, and climate change, resulting in loss of biodiversity and reduced ecosystem functionality (Mineur et al., 2015 ) . Among these key species, Ericaria amentacea , class Phaeophyta order Fucales (Agardh, 1820; Molinari and Guiry 2020, Figure 1), plays an essential ecological role along the Mediterranean coasts, including Italy, where it forms dense intertidal belts and acts as a habitat engineer (Falace et al., 2018; Mangialajo et al., 2008). Despite its importance, information about its genetic structure, population connectivity, and phylogeographic history remains scarce. The taxonomy of the Cystoseira sensu lato complex, to which Ericaria amentacea belongs, has been historically challenging due to pronounced morphological plasticity and frequent phenotypic overlap between species, often influenced by local environmental conditions (Orellana et al., 2019; Bermejo et al., 2018). This has complicated efforts to delineate populations and understand their evolutionary relationships using morphology alone. At the same time, increasing anthropogenic pressures such as coastal urbanization, eutrophication, and thermal stress are fragmenting populations and threatening their persistence (Thibaut et al., 2005; Susini et al., 2007). Assessing genetic differentiation and connectivity is therefore crucial to identify vulnerable populations, reconstruct dispersal pathways, and design effective conservation measures such as marine protected areas or restoration programs (Buonomo et al., 2017; Sales et al., 2019). Previous molecular work on Ericaria amentacea has used a variety of marker types and sampling scales and consistently points to limited dispersal, pronounced population structure at relatively small spatial scales, and strong sensitivity of local populations to human impacts (Thibaut et al., 2016). Early allozyme/RAPD-based studies detected appreciable within-population diversity but also substantial differentiation among nearby sites, suggesting restricted effective dispersal of recruits in this fucoid (RAPD; Buonomo et al., 2017). Regional population genetic analyses in the northwestern Mediterranean likewise reported strong and often non-linear genetic structuring among Ericaria stands, with weak or no isolation-by-distance in some comparisons, a pattern interpreted as the combined outcome of short-distance propagule dispersal, local extinctions/recolonisations, and habitat discontinuities created by coastal development (Susini et al., 2007, Thibaut et al., 2017; Buonomo et al., 2017). More recent molecular and applied studies have extended these patterns and begun to scale up sampling and methods. Population-level surveys that integrate genetic data with demographic and transplantation experiments confirm that local demographic declines and restoration outcomes are tightly linked to the genetic composition and connectivity of donor and recipient stands. Experimental transplant and restoration case studies report limited stress responses to transplantation but emphasize the need to consider genetic structure when choosing donor sources for restoration (Thibaut et al., 2015). At broader spatial scales, recent, larger multi-site genetic surveys (including high-replication sampling across dozens of sites) reinforce the picture of predominantly short-range, multi-generation dispersal shaping genetic structure, while identifying regional hotspots and potential contact zones that may represent glacial refugia or corridors of secondary contact (Thibaut et al., 2016). Taxonomic reassessments and barcoding resources (i.e., AlgaeBase; Molinari and Guiry 2020) have also clarified nomenclature (involving the modification of the species name from Cystoseira amentacea var. stricta to Ericaria amentacea ) and improved the interpretability of molecular datasets for this complex (Chemello et al., 2022; Reynes et al., 2025). Mitochondrial cytochrome oxidase subunit I (COI) has become a standard molecular marker for studying genetic diversity and phylogeography in brown algae because of its high interspecific resolution, maternal inheritance, and extensive reference datasets (Robba et al., 2006; Saunders and McDevit, 2012). COI sequences have successfully revealed cryptic diversity, post-glacial recolonization patterns, and barriers to gene flow in several macroalgal taxa across the Mediterranean and Atlantic (Hoarau et al., 2007; Draisma et al., 2010; Neiva et al., 2012). However, to date, no comprehensive COI-based study has focused specifically on E. amentacea populations distributed along the Italian coasts, leaving gaps in understanding regional differentiation and historical connectivity between major basins such as the Tyrrhenian, Adriatic, and Ionian Seas. The present study provides the first assessment of genetic diversity, phylogeographic relationships, and population structure of Ericaria amentacea along the Italian coasts using the mitochondrial COI gene. Through extensive sampling and molecular analyses, we aim to (1) quantify patterns of genetic diversity within and among Italian populations, (2) identify phylogeographic barriers and potential historical glacial refugia or contact zones, and (3) infer levels of gene flow and connectivity relevant to conservation management. The outcomes will contribute to a baseline framework for monitoring genetic diversity and guiding conservation strategies for E. amentacea and other Mediterranean canopy-forming brown algae. Figure 1. Ericaria amentacea forest in the intertidal zone along the Ligurian coast (photo by Maha Moussa in May 2025, Pontetto, Genova - Italy). MATERIAL AND METHODS Sampling and genomic DNA extraction A total of 42 specimens of E. amentacea were collected, between April and June 2023, from 5 sampling locations along the Italian coasts (Figure 2, Table 1). These samples covered the north board of Italy (Liguria region; Pontetto, Bergeggi and Bonassola) as well as the Sardinia (Corona Niedda and Torre dei Corsari localities). From each specimen, a branch approximatively 7-8 cm long was preserved in 100 % ethanol and stored at 4°C for subsequent DNA extraction. Genomic DNA was extracted from 20–40 mg of the algal tissues using the CTAB method described by Zuccarello and Paul (2019), with some modifications. DNA quantity and quality evaluations were carried out using a NanoDrop One/One c spectrophotometer (ThermoFisher, Milan, Italy) and agarose gel electrophoresis, respectively (Sambrook et al., 1989). A set of E. amentacea COI sequences was retrieved from the National Center for Biotechnology Information (NCBI). These data covered the south of Italy (Sicily (Capo Milazzo) and Pantelleria islands and Apulia region). Figure 2. Geographical distribution of E. amentacea samples. 2.2 Mitochondrial DNA amplification and sequencing The mitochondrial fragment of the cytochrome oxidase subunit I (COI) gene was amplified using a pair of primers; COI-Frwd: 5’-CCAACCAYAAAGATATWGGTAC-3’ and COI-Rev: 5’-GGATGACCAAARAACCAAAA-3’) (Saunders and McDevit, 2013). PCR reactions were performed in a total volume of 25 μL including 2 μL (20 ng/μL) of DNA, 2.5 μL PCR Buffer (10x), 3.2 μL MgCl 2 (20 mM), 0.5 μL of each primer (10 μM),0.25 µL dNTP mix (20mM), 1.25 μL BSA (10 mg/mL), 0.25 μL (1U/μL) of Taq DNA polymerase, and sterile double-deionized H 2 O. PCR amplifications were performed in an Eppendorf AG Thermocycler, programmed to perform an initial denaturation at 94°C for 4 min; followed by 38 cycles at 94°C for 1 min, 50 °C for 30 s, and 72 °C for 1 min; and a final extension at 72 °C for 7 min. PCR amplicons were screened for specific fragment size on 1.5% agarose gel electrophoresis and subsequently purified using a Gel and PCR Clean-up (MACHEREY-NAGEL, Germany) purification kit. The agarose gel was photographed by iBright 1500 imager (Invitrogen, Thermofisher). Amplified PCR products were sequenced using the Sanger method (Sanger et al., 1977) at Eurofins Genomics Germany GmbH (Ebersberg, Germany) and aligned using MUSCLE (Edgar, 2004) implemented in MEGA version 11.0 (Tamura et al., 2021). Table 1 Information on brown seaweed E. amentacea sampling including collection region, collection site, number of specimens (N), site collection depth, and geographic coordinates. Italian continents Liguria_Bergeggi (1) 10 Tide level from 0 to 20 cm 44° 14′ 55″ N, 8° 26′ 37″ E Liguria_Pontetto (2) 10 Tide level from 0 to 20 cm 44° 22′ 33″ N, 9° 4′ 31″ E Liguria_Bonassola (3) 8 Tide level from 0 to 20 cm 44° 10′ 60″ N, 9° 34′ 60″ E Apulia (6) 3 - Data from NCBI Italian Islands Sardinia: *Torre dei Corsari (4) * Corona Niedda (5) 8 6 Tide level from 0 to 20 cm 39° 40′ 45.18″ N, 8° 27′ 05.06″ E 40° 12′ 45.47″ N, 8° 27′ 31.21″ E Pantelleria (8) 2 - Data from NCBI Sicily_Capo Milazzo (7) 4 - Data from NCBI 2.3 Data and Statistical Analysis Analysis of genetic variability The level of DNA polymorphism, the number of haplotypes (H), haplotype diversity (Hd; Nei, 1987), and nucleotide diversity (π; Tajima, 1983; Nei, 1987) were estimated across the entire dataset using DnaSP version 5.10 (Librado and Rozas, 2009). Theta (per site) from Eta, the average number of pairwise differences (K), transition/transversion bias (R), and the number of variable and parsimony-informative nucleotide sites were calculated using MEGA version 11.0 (Tamura et al., 2021). Inference of demographic history The demographic history of the Italian brown seaweed E. amentacea was investigated. Mismatch distribution analysis was performed using DnaSP version 5.10 (Librado and Rozas, 2009) for all datasets. To assess deviations from neutrality, demographic expansion, or the detection of selection signatures, additional tests were carried out based on the total number of mutations, including Tajima’s D (Tajima, 1989), Fu’s Fs (Fu and Li, 1993), the raggedness index (rg), and Ramos-Onsins and Rozas’s R² (Ramos-Onsins and Rozas, 2002). These analyses were performed using coalescent simulations implemented in DnaSP, with 1000 simulated resampling replicates. Phylogeographic structure analyses To infer the relationships of E. amentacea haplotypes, a haplotype network was constructed in PopART version 1.7 software (Leigh and Bryant, 2015) using median-joining method (Bandelt et al., 1999). Phylogenetic reconstructions were built using Neighbor-Joining (NJ) method implemented in MEGA version 11.0 (Tamura et al., 2021). A sequence from Ericaria zosteroides (GenBank accession numbers: OK480329.1) was used as an outgroup. Genetic differentiation analyses The analysis of molecular variance (AMOVA; Excoffier et al., 1992) was conducted using Arlequin version 3.5 (Excoffier and Lischer, 2010) to assess the level of genetic differentiation among Italian populations of E. amentacea . Two supplementary AMOVA tests were performed. In the first analysis, genetic variation among populations was examined according to geographic proximity: Liguria_Bergeggi/ Liguria_Bonassola/ Liguria_Pontetto/ Sardinia_Torre dei Corsari/ Sardinia_Corona Niedda/ Sicily_Capo Milazzo/ Apulia / Pantelleria. The second analysis compared continental versus insular populations, grouped as follows: Liguria_Bergeggi/ Liguria_Bonassola, Liguria_Pontetto / Apulia (continental) versus Sardinia_Torre dei Corsari / Sardinia_Corona Niedda / Sicily_Capo Milazzo / Pantelleria (insular). All AMOVA analyses were performed with 10000 permutations under null distributions. The extent of genetic differentiation among populations was further estimated using fixation indices F ST (Wright, 1931), GST (Nei, 1975), and NST (Lynch and Crease, 1990) as well as gene flow Nm (Hudson et al., 1992). These values were calculated with 1000 data permutations using DnaSP version 5.10 (Librado and Rozas, 2009). RESULTS 3.1 Genetic diversity The Ericaria amentacea mitochondrial cytochrome oxidase (COI) subunit I dataset, composed of 51 sequences, has a final alignment length of 610 bp, of which 585 sites are conserved and 25 are variable (Table 2). Among these variable positions, 14 are informative on parsimony and 11 are unique sites, corresponding to a total of 28 mutations detected. The number of polymorphic sites (S = 25) and the total of 17 haplotypes indicate relatively high genetic variability within the studied populations. The diversity of haplotypes (Hd = 0.851) and nucleotides (Pi = 0.00597) suggests moderate to high intra-species genetic variation, in line with expectations for a species with geographically structured populations but retaining multiple lineages in its range. The mean number of differences per pair (K = 3.64) supports the existence of distinct haplotype groups within Italian populations, reflecting both historical isolation and current narrow gene flow. Table 2 Summary of polymorphism of COI sequences. Number of sequences 51 Alignment length (bp) 610 Conserved sites 585 Variable sites 25 Parsimony informative characters 14 Singleton variable sites 11 Total number of mutations 28 Number of polymorphic sites (S) 25 Number of haplotypes (H) 17 Haplotype diversity (Hd) 0.851 Variance of haplotype diversity 0.00165 Nucleotide diversity (Pi) 0.00597 Theta (per site) from Eta 0.01020 Average of pairwise differences (K) 3.64471 Transition/transversion bias (R) 1.06 3.2 Evolutionary and demographic history The neutrality tests applied to the dataset produced predominantly negative values for different statistics (Table 3). Tajima’s D was negative (D = -1.36603), but not significant, indicating a little excess of low frequency polymorphisms compared to neutral expectations (Tajima, 1989). Similarly, the D* (-1.89830) and F* (-2.03313) of Fu and Li were negative but not significant, suggesting a small deviation from neutrality (Fu and Li, 1993). In contrast, Fu’s Fs was strongly negative (Fs = -4.536), this result is generally linked to an excess of rare and reliable alleles in the context of recent population expansion (Fu, 1997). Demographic indices have strengthened the neutrality tests (Table 3). The value of R2 was low (R2 = 0.0674), representative of population expansion (Ramos-Onsins and Rozas, 2002). The index of distribution disconformity was also low (r = 0.0275), indicating a pattern of disconformity consistent with expectations in case of rapid population expansion (Harpending, 1994). Overall, although the non-significant values of Tajima’s D and the statistics of Fu and Li suggest that neutrality cannot be formally rejected, the combination of a strongly negative Fu’s Fs and low R2 and discordance values indicates that the population has likely experienced a recent demographic expansion. The mismatch distribution for Ericaria amentacea populations from Italy showed a unimodal curve, with a single clear peak at low pairwise differences. This pattern closely fits the expected distribution under a model of sudden population expansion (Rogers & Harpending, 1992) (Figure 3). Table 3 Tajima’s D , Fu’s F S, Ramos-Onsins and Rozas’s ( R 2), Fu and Li’s D* and Fu and Li’s F* neutrality tests and mismatch distribution raggedness index ( r ) for the entire E. amentacea samples. All data set -1.36603 ns -4.536 0.0674 0.0275 -1.89830 ns -2.03313 ns *ns = non-significant Figure 3. Mismatch distribution of pairwise nucleotide differences using DNAsp for Ericaria amentacea populations from Italian coasts. 3.3 Genetic differentiation analyses The analysis of molecular variance (AMOVA) highlighted a marked and notable genetic structure among the populations studied (Table 4). According to a global analysis, the majority of genetic diversity (79.39%) was distributed among E. amentacea populations, whereas only 20.61% was maintained within populations (Φ ST = 0.79391, p < 0.05). This indicates a restricted gene flow and significant genetic differentiation among populations. The division of the data set into continental and island populations highlighted an even more pronounced structuring (Table 3). Most of the genetic variation (64.92%) was due to disparities between the two groups, while 21.19% were due to the variation existing between populations within each group, and only 13.89% were preserved within populations (ΦCT = 0.64917; ΦSC = 0.60393; ΦST = 0.86105; p < 0.05). These observations confirmed the important genetic differentiation of island populations, in accordance with the consequences of geographical isolation and the restricted gene transfer. Table 4 Molecular variance analysis (AMOVA) of , * P < 0.05. Source of variation Fixation index Sum of squares Variance components Pourcentage of variation Among populations ΦST= 0.79391 417.217 7.899 79.39085* Within populations 88.175 2.051 20.60915* Total 505.392 9.950 100 AMOVA groups: continent vs islands Liguria_Bergeggi / Liguria_Bonassola / Liguria_Pontetto / Apulia vs Sardinia_Torre dei Corsari / Sardinia_Corona Niedda vs Sicily_Capo Milazzo vs Pantelleria Among groups ΦSC = 0.60393 ΦST= 0.86105 ΦCT= 0.64917 318.180 9.580 64.91716* Among populations within groups 99.037 3.127 21.18764* Within populations 88.175 2.051 13.89519* Total 505.392 16.610 100 Pairwise estimates of genetic differentiation (FST) and gene flow (Nm) revealed a pronounced genetic structuring among populations (Table 5). Most comparisons showed very high levels of differentiation, with FST values ranging from 0.68 to 0.92. For instance, Sardinia_Corona Niedda and Sicily_Capo Milazzo were almost completely differentiated (FST = 0.92), and similarly high values were observed between Sardinian, Sicilian, and Pantelleria populations. These results indicate strong isolation and minimal gene flow among insular populations. In contrast, continental Ligurian populations displayed low levels of differentiation. Pairwise comparisons between Liguria_Pontetto and Liguria_Bonassola (F ST = 0.04) and between Liguria_Pontetto and Liguria_Bergeggi (F ST = 0.05) revealed low genetic structuring. The corresponding Nm values (4.87 and 4.37, respectively) indicated significant gene flow among close Ligurian populations, consistent with their geographic proximity. Overall, Nm estimates were close to zero for most inter-island and island–mainland comparisons, confirming that gene exchange is rare across major geographic barriers. Negative Nm values observed in some comparisons (e.g., Liguria_Bergeggi vs. Liguria_Bonassola) are generally interpreted as artefacts of estimation and should be considered indicative of negligible migration. The overall pattern therefore reflects high genetic isolation among islands, contrasted with higher connectivity within the Ligurian mainland region. Table 5 Pairwise comparisons of genetic differentiation, estimated from haplotype frequencies ( F ST , above the diagonal) and gene flow ( N m , below the diagonal). Pantelleria 0 1 0.81 1 0.88 1 0.86 0.81 Apulia 0 0 0.69 1 0.88 1 0.76 0.69 Liguria_Bergeggi 0.06 0.11 0 0.76 0.86 0.68 1(-0.03) 0.05 Sardinia_Corona Niedda 0 0 0.08 0 0.88 1 0.72 0.76 Sardinia_Torre dei Corsari 0.05 0.03 0.06 0.03 0 0.92 0.84 0.80 Sicily_Capo Milazzo 0 0 0.11 0 0.02 0 0.76 0.69 Liguria_Bonassola 0.04 0.08 0 (-7.62) 0.05 0.05 0.08 0 0.04 Liguria_Pontetto 0.06 0.11 4.37 0.07 0.06 0.11 4.87 0 3.4 Phylogeographic structure analyses Haplotype network analysis revealed a complex yet geographically structured genetic pattern among E. amentacea populations along the Italian coasts (Figure 4), supporting the phylogenetic divergence observed in the NJ tree (Figure 5). A total of 17 haplotypes were identified, indicating a clear separation between all Italian coast populations, as well as island-continental populations (Ligurian, Apulian vs Sicilian, Sardinian and Pantelleria). The central and most common haplotype (Hap 2), found predominantly in Ligurian samples from Pontetto, Bergeggi, and Bonassola, likely represents a probable ancestral or dominant haplotype from which peripheral variants derive. The star-like structure surrounding this central node indicates that Ligurian populations had recently expanded or exhibited significant connectedness, consistent with their geographic continuity. In contrast, southern groups displayed different and spatially restricted haplotypes, indicating significant regional isolation. Samples from Sicily (Capo Milazzo) and Apulia contained distinct haplotypes (Hap 9, Hap 7, and Hap 10) that were not detected in any other location. Similarly, Sardinian populations were separated into two groups corresponding to the northern (Corona Niedda) and western (Torre dei Corsari) coasts, with each group distinguished by isolated haplotypes (Hap 17 and Hap 13), indicating intra-island differentiation caused by habitat discontinuities. The Pantelleria population occupied a terminal position in the network, represented by an isolated haplotype (Hap 11) that was linked to the main cluster by numerous mutational steps, highlighting the South-Mediterranean island’s substantial genetic isolation and role as a peripheral refugium for E. amentacea . Figure 4 The median-joining haplotype network constructed from COI sequences for Ericaria amentacea populations along Italian coasts. The Neighbor-Joining phylogenetic analysis of E. amentacea populations along the Italian coasts revealed a distinct structure largely shaped by geographic location (Figure 5), supporting the phylogeographic divergence observed by the median-joining haplotype network (Figure 4). With bootstrap values ranging from 47 to 62, samples from the Ligurian region (Pontetto, Bergeggi, and Bonassola) formed a substantially monophyletic group, suggesting a notable genetic consistency within northern populations. However, the samples from Apulia and Sicily (Capo Milazzo) showed significant genetic divergence, forming a distinct lineage, as reflected by intermediate bootstrap values (65-52%). Two geographically separate sets of Sardinian populations have been identified: one that includes samples from Torre dei Corsari and the other that includes samples from Corona Niedda. The genetic differentiation between northern and southern Sardinian locations (bootstrap 73%) suggests limited connectivity and potential regional divergence. Compared to all other populations, Pantelleria specimens, forming a special and unique clade at the base of the E. amentacea group, showed the highest level of genetic divergence. The Pantelleria population is very isolated, according to this model. The monophyly of E. amentacea was confirmed by the inclusion of E. zosteroides as an outgroup; all intraspecific variations revealed stronger ties between them than with this related species. Figure 5 Neighbor-Joining phylogenetic analysis of E. amentacea populations along the Italian coasts. DISCUSSION Genetics and phylogeography studies on Ericaria amentacea species are important for understanding its evolutionary and ecological dynamics. By investigating genetic variability within and among species, researchers can uncover historical processes such as migration patterns, geographical isolation, or recolonization events that shaped their current distribution. These approaches also enable to identify the discrete genetic lineages, emphasizing significant intraspecific variation, which is critical for resilience and adaptation to environmental changes, particularly those caused by global warming and human influences on coastal ecosystems. Furthermore, phylogeography provides significant insights for population management and conservation by describing major evolutionary units and informing protection methods to ensure the genetic diversity and stability of marine communities, where this species performs an important ecological function. The study of genetic parameters is fundamental for understanding the structure, diversity and evolution of natural populations. These parameters, such as haplotype diversity, nucleotide diversity, the number of polymorphisms or even genetic differences between populations, make it possible to evaluate the level of genetic diversity of a species, a key indicator of its adaptability to environmental changes and anthropogenic pressures. Polymorphism results on E. amentacea populations revealed very low nucleotide diversity (Pi = 0.00597) and high haplotype diversity (Hd = 0.851), indicating a moderate to high intraspecific genetic variation. Thus, these results are coherent with expectations for a species with geographically structured populations that preserved several multiple lineages throughout its repartition areas. Similar levels of COI variability have been declared in other Mediterranean brown algae such as Cystoseira species and Ericaria crinita, where limited dispersal capacity and fragmented coastal habitats contribute to the accumulation of regional haplotypes (Buonomo et al., 2017; Robba et al., 2006; Orellana et al., 2019). The average number of pairwise differences (K = 3.64) suggests the presence of separate haplotype groupings within populations, which could represent both historical isolation and limited contemporaneous gene flow (Rogers and Harpending, 1992; Alberto et al., 1999; Provan and Maggs, 2012). The estimated mutation parameter (θ = 0.0102) and transition / transversion ratio (R = 1.06) are within the typical range for mitochondrial genes in brown algae (Hoarau et al., 2007; Draisma et al., 2010). These numbers indicate that the substitution pattern is balanced and there is no significant bias in the genetic composition. This nearly equal ratio reflects a steady mutation process with a modest compositional bias. Such balanced substitution patterns are indicative of evolutionary stability and minimal selective pressure in the COI region (Hoarau et al., 2007; Saunders and McDevit 2013). Thus, the observed ratio supports the hypothesis that genetic variations among haplotypes are recent and indicates Mediterranean diversification from the late Pleistocene to the Holocene (Hoarau et al., 2007; Provan and Maggs, 2012; Neiva et al., 2012). The combination of a strongly negative Fu Fs, low R² values and unimodality of the mismatch distribution, despite non-significant values for Tajima D and Fu & Li, are usually interpreted as the signal of a recent demographic expansion from a reduced population (bottleneck) and a rapid accumulation of rare alleles during recolonization (Rogers and Harpending, 1992; Fu, 1997). This profile has already been reported in several macroalgae and marine organisms and is compatible with post-glacial scenarios of recolonization and rapid expansion along the Mediterranean coasts (Bermejo R. et al. 2018; Veith et al. 2020). Post-glacial scenarios of recolonization and rapid expansion along Mediterranean coasts are explained by the major climate changes that occurred at the end of the last glaciation, approximately 18,000 to 10,000 years ago (Patarnello et al., 2007; Maggs et al., 2008). The Last Glacial Maximum (LGM) was the period of maximum ice volume during the last ice age, occurring about 20,000 years ago, though the exact timing is now estimated to be between 23,000 and 19,000 years ago. During this time, massive glaciers covered about 8% of Earth’s surface, global sea levels were over 120 meters lower than today, and climate patterns were drastically altered, leading to increased aridity in some regions and expanded deserts. During the Last Glacial Maximum (LGM), the decrease in temperatures and sea levels, profoundly transformed coastal habitats, leading to the reduction or fragmentation of rocky areas favorable to macroalgae and other benthic organisms (Rohling et al., 1998; Patarnello et al., 2007; Lambeck et al., 2011). In this context, several temperate marine species survived in glacial refugia, located mainly in the southern and eastern regions of the Mediterranean, especially in North Africa (Tunisian’ coasts), Sicily, Sardinia and Greece, where environmental conditions remained relatively stable (Maggs et al., 2008). The results of the analysis of molecular variance (AMOVA) clearly highlight a strong genetic structuring within Ericaria amentacea populations. Indeed, the majority of the total genetic variability (79.39%) is attributed to differences between populations (ΦST = 0.79391; p < 0.05), while a lower proportion (20.61%) is observed within populations. This distribution reflects restricted gene flow between populations and marked genetic differentiation, likely related to the low dispersal of propagules and the sedentary nature of spores and gametes in Cystoseira group. These results are consistent with several previous studies on species of the genus Cystoseira / Ericaria , which have often highlighted a very pronounced local genetic structure and limited connectivity between geographically close populations (Susini et al., 2007; Thibaut et al., 2016; Riquet et al., 2021). Comparison between continental and island populations reinforces this observation, revealing even more pronounced structuring (ΦCT = 0.64917; ΦSC = 0.60393; ΦST = 0.86105; p < 0.05). Most of the genetic variation (64.91%) is due to differentiation between the two groups, highlighting the major role of geographic isolation of islands in genetic divergence. Island environments, often characterized by specific ecological conditions and small population sizes, favor genetic drift and significantly reduce gene flow with continental populations (Neiva et al., 2012; Bermejo et al., 2018). This trend is typical of Mediterranean benthic macroalgae, whose dispersal capabilities are severely limited by the low mobility of zygotes and the short lifespan of propagules (Falkowski & Raven, 2007). Studies on other Mediterranean macroalgae, such as Padina pavonica or Dictyota dichotoma , have also observed progressive differentiation along latitudinal gradients, often attributed to a combination of isolation by distance and variable environmental conditions (Wernberg et al., 2018; Assis et al., 2014). Thus, all the AMOVA results confirm that Ericaria amentacea populations present a complex and hierarchical genetic structure, dominated by allopatric differentiation processes (continent / island) but attenuated by a certain regional connectivity between geographically close populations. This populations structure is supported by network analysis and phylogenetics. The specimens from the Ligurian region (Pontetto, Bergeggi, and Bonassola) formed a well-supported monophyletic group, indicating high genetic homogeneity among northern populations and suggesting continuous gene flow along the Ligurian coastline. Within this cluster, minor sub-structuring was observed among sites, which may reflect local adaptation or limited dispersal between nearby coastal habitats. Gene flow analysis confirms these results. The highest values of Nm are observed between Pontetto, Bergeggi, and Bonassola (Nm > 4). The distinct clustering of Sicilian samples (Capo Milazzo) together with those from Apulia into a separate lineage, supported by moderate bootstrap values (65 - 52%), reflects a clear genetic differentiation likely driven by geographical isolation and the biogeographic role of the Strait of Messina as a semi-permeable barrier to gene flow. This pattern is consistent with previous phylogeographic studies on Mediterranean marine taxa showing that the Strait of Messina, separating the Tyrrhenian and Ionian basins, represents a major genetic discontinuity zone due to its complex hydrodynamics, strong currents, and contrasting ecological conditions (Patarnello et al., 2007; Maltagliati et al., 2010; Guglielmo et al., 2015). For species such as Ericaria amentacea , whose propagules have limited dispersal capacity (few meters to tens of meters) (Reynes et al., 2021) such oceanographic discontinuities strongly restrict gene flow. This assists genetic drift and local differentiation (Thibaut et al., 2016; Riquet et al., 2021). Similar phylogeographic ruptures have been documented in other Mediterranean macroalgae, such as Padina pavonica (Bennett et al., 2015) and Dictyota dichotoma (Hwang et al., 2005), where Sicilian and southern Italian populations form separate groups from those in the Tyrrhenian or Ligurian seas. Sardinian populations separated into two geographically coherent clusters: one containing samples from Corona Niedda and the other from Torre dei Corsari. This genetic distinction between northern and southern Sardinian sites (bootstrap 73%) indicates low connectivity and likely regional divergence across the island, which could be caused by hydrodynamic patterns or habitat discontinuities that affect propagule dispersal. The Pantelleria samples, which represent a separate and very different clade at the base of the E. amentacea group, had the highest genetic divergence from all other populations. This pattern provides evidence that the Pantelleria population is strongly isolated, which is quite expected due to its marine island position in the Strait of Sicily and the consequent long-term isolation from mainland and island populations. The gene flow and Fst analyses support this finding: Nm values range from 0 to 0.06, while Fst ranges from 0.081 to 1 in Pantelleria and other Italian regions. The concordance between the Neighbor-Joining phylogeny and the haplotype network and Molecular variance analysis, strongly supports the existence of geographically structured lineages within E. amentacea across the Italian coasts. Both analyses revealed a clear separation between all Italian-sampled coast site as well as Islands-continental Italian populations, with Ligurian samples forming a cohesive genetic group characterized by high haplotype sharing and low mutational divergence, consistent with continuous connectivity along the northern mainland. The observed phylogeographic structure in E. amentacea populations has important implications for the conservation and management of these threatened brown algae. The observed genetic difference among regional populations, particularly between the Ligurian, Sicilian, Sardinian, and Pantelleria groups, suggests limited linkage and gene flow throughout the species’ range. Such genetic variety implies that each regional population may be an evolutionarily significant unit (ESU) that contributes to the species’ adaptive capacity. Certain populations, such as Pantelleria and southern Sardinia, are particularly vulnerable to local extinction since recolonization through migration from other locations is unexpected. This pattern of significant population structure suggests that conservation efforts should use a geographically varied approach rather than a uniform management plan. These findings support the idea that effective conservation of canopy-forming brown algae necessitates the incorporation of phylogeographic data into conservation marine reserves designs Maritime Spatial Panning policies, ensuring that management networks include the entire range of genetic variation across the species’ distribution. ACKNOWLEDGMENTS We would like to thank prof. Luigi MUSCO (Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy) for his valuable support in this research project. We declare that Generative AI (ChatGPT) was used to assist with rephrasing sentences to improve clarity and correct grammatical errors. All scientific content, analyses, and interpretations were performed solely by the authors. CONFLICT OF INTEREST : The authors declare that there is no conflict of interest regarding the publication of this paper. AUTHOR CONTRIBUTIONS Maha MOUSSA: Conceptualization (lead); Data curation (lead); Formal analysis (lead); Methodology (lead); Software (lead); Writing –original draft (lead); Writing –review & editing (lead). Sarra CHOULAK: Formal analysis (lead); Methodology (lead); Software (lead); Writing –original draft (equal); Writing –review & editing (lead). Valentina ASNAGHI: Methodology (equal); Writing –review & editing (equal). Daniele GRECH: Methodology (equal); Writing –review & editing (equal). Khaled SAID: Methodology (equal); Visualization (lead); Writing –review & editing (lead). Mariachiara CHIANTORE: Project administration (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing –review & editing (lead). Sonia SCARFI: Project administration (lead); Supervision (lead); Validation (lead); Visualization (lead); Writing –review & editing (lead). All authors have read and agreed to the published version of the manuscript. 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Figure 5 Neighbor-Joining phylogenetic analysis of E. amentacea populations along the Italian coasts. Information & Authors Information Version history V1 Version 1 27 January 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords genetics laboratory marine molecular genetics plants sequencing statistical Authors Affiliations Maha Moussa University of Genoa View all articles by this author Sarra Choulak 0000-0003-0088-315X [email protected] University of Monastir Higher Institute of Biotechnology of Monastir View all articles by this author Valentina Asnaghi 0000-0003-1659-2613 University of Genoa View all articles by this author Daniele grech National Biodiversity centre Piazza Marina View all articles by this author Khaled Said University of Monastir Higher Institute of Biotechnology of Monastir View all articles by this author Mariachiara Chiantore Università degli Studi di Genova Facoltà di Scienze Matematiche Fisiche e Naturali View all articles by this author Sonia Scarfi 2National Biodiversity Future Center (NBFC) View all articles by this author Metrics & Citations Metrics Article Usage 156 views 82 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Maha Moussa, Sarra Choulak, Valentina Asnaghi, et al. First assessment of genetic diversity, phylogeographic relationships, and population structure of the brown seaweed Ericaria amentacea from Italian coasts using cytochrome oxidase subunit I (COI) gene. Authorea . 27 January 2026. DOI: https://doi.org/10.22541/au.176949737.74841877/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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