Genetic structure and community turnover of botryllid ascidians along the Turkish marine corridor

Genetic structure and community turnover of botryllid ascidians along the Turkish marine corridor | 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 ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 10 March 2026 V1 Latest version Share on Genetic structure and community turnover of botryllid ascidians along the Turkish marine corridor Authors : Begum Ece Tohumcu 0000-0002-5071-0786 [email protected] , Esra Ozturk , and Arzu Karahan Authors Info & Affiliations https://doi.org/10.22541/au.177312459.94681048/v1 179 views 110 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Marine corridors connecting basins with contrasting hydrographic and physicochemical regimes provide natural laboratories to investigate how environmental filtering and historical connectivity shape genetic structure and community composition. The Turkish Straits System links the brackish Black Sea to the hypersaline Mediterranean through a series of semi-enclosed basins, generating sharp gradients in salinity, temperature, and productivity that act as biogeographic filters for benthic organisms with limited dispersal. Here, we present a coast-wide, integrative analysis of the phylogeography and community turnover of botryllid and other colonial aplousobranch ascidians along the entire Turkish coastline, spanning the Black Sea, Sea of Marmara, Aegean Sea, and eastern Mediterranean. Using a mitochondrial gene region (COI) from 321 colonies and environmental data, we reconstructed lineage relationships, delimited operational taxonomic units, and quantified species–environment associations and multivariate community–environment structure. Bayesian phylogenetic and species-delimitation analyses revealed nine colonial ascidian taxa and pronounced hierarchical structuring in Botryllus schlosseri, with distinct Black Sea and Mediterranean sub-lineages within the same mitochondrial clade. Several Botrylloides species formed genetically cohesive lineages with restricted southern distributions. Notably, the occurrence of Botryllus gaiae in the Aegean Sea represents the first genetically confirmed record for the Turkish coastline, extending its known distribution into the northeastern Aegean. Species composition exhibited strong latitudinal turnover, with B. schlosseri dominating low-salinity northern basins, whereas taxonomic richness and assemblage diversity increased toward the Aegean and Mediterranean. Environmental analyses showed that salinity and temperature were the primary drivers of species distributions and community structure, with redundancy analysis indicating that basin-scale hydrographic gradients, rather than local nutrients, explain most variation in assemblage composition. Together, these results show that the Turkish marine corridor functions as a semi-permeable biogeographic filter, promoting lineage divergence and shaping community turnover across connected seas, and highlight the combined roles of historical connectivity and contemporary environmental gradients in structuring benthic marine biodiversity. 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex Genetic structure and community turnover of botryllid ascidians along the Turkish marine corridor Begüm Ece Tohumcu*, Esra Öztürk, Arzu Karahan Institute of Marine Sciences, Middle East Technical University (METU-IMS), 33731 Erdemli, Mersin, Türkiye Running title: Botryllid phylogeography across Turkish seas Corresponding Author: Begüm Ece Tohumcu, Institute of Marine Sciences, Middle East Technical University, 33731 Erdemli, Mersin, Türkiye, E-mail: [email protected] , ORCID: https://orcid.org/0000-0002-5071-0786 Co-author: Esra Öztürk, Institute of Marine Sciences, Middle East Technical University, E-mail: [email protected] , ORCID: https://orcid.org/0000-0002-4373-2362 Co-author: Assoc. Prof. Dr. Arzu Karahan, Institute of Marine Sciences, Middle East Technical University, E-mail: [email protected] , ORCID: https://orcid.org/0000-0002-4096-9372 Funding: This study was supported by the Middle East Technical University Scientific Research Projects Coordination Unit (METU-BAP) under projects ADEP-701-2023-11278 and TEZ-D-701-2023-11019, DEKOSIM laboratory (BAP-08-11-DPT2012K120880). Conflict of Interest: The authors declare no conflict of interest. Ethics and Permits: Field sampling was carried out in non-protected coastal areas, and no specific collection permits were required under national regulations. Abstract Marine corridors connecting basins with contrasting hydrographic and physicochemical regimes provide natural laboratories to investigate how environmental filtering and historical connectivity shape genetic structure and community composition. The Turkish Straits System links the brackish Black Sea to the hypersaline Mediterranean through a series of semi-enclosed basins, generating sharp gradients in salinity, temperature, and productivity that act as biogeographic filters for benthic organisms with limited dispersal. Here, we present a coast-wide, integrative analysis of the phylogeography and community turnover of botryllid and other colonial aplousobranch ascidians along the entire Turkish coastline, spanning the Black Sea, Sea of Marmara, Aegean Sea, and eastern Mediterranean. Using a mitochondrial gene region (COI) from 321 colonies and environmental data, we reconstructed lineage relationships, delimited operational taxonomic units, and quantified species–environment associations and multivariate community–environment structure. Bayesian phylogenetic and species-delimitation analyses revealed nine colonial ascidian taxa and pronounced hierarchical structuring in Botryllus schlosseri , with distinct Black Sea and Mediterranean sub-lineages within the same mitochondrial clade. Several Botrylloides species formed genetically cohesive lineages with restricted southern distributions. Notably, the occurrence of Botryllus gaiae in the Aegean Sea represents the first genetically confirmed record for the Turkish coastline, extending its known distribution into the northeastern Aegean. Species composition exhibited strong latitudinal turnover, with B. schlosseri dominating low-salinity northern basins, whereas taxonomic richness and assemblage diversity increased toward the Aegean and Mediterranean. Environmental analyses showed that salinity and temperature were the primary drivers of species distributions and community structure, with redundancy analysis indicating that basin-scale hydrographic gradients, rather than local nutrients, explain most variation in assemblage composition. Together, these results show that the Turkish marine corridor functions as a semi-permeable biogeographic filter, promoting lineage divergence and shaping community turnover across connected seas, and highlight the combined roles of historical connectivity and contemporary environmental gradients in structuring benthic marine biodiversity. Keywords: Phylogeography, environmental filtering, community turnover, Turkish Straits System, hydrographic gradient, colonial ascidians Introduction Tunicates (Tunicata) represent a key chordate lineage whose evolutionary position and life-history traits make them particularly suitable for investigating population connectivity, local adaptation, and evolutionary responses to environmental gradients in marine systems. Although historically grouped with invertebrates based on adult morphology (Milne-Edwards, 1843; Haeckel, 1866), early developmental studies revealed their chordate affinities through the presence of a notochord and a dorsal hollow nerve cord in the larval stage (Huxley, 1851; Kowalevsky, 1866; Stolfi & Brown, 2015). Molecular phylogenetic analyses have since demonstrated that tunicates are the closest living relatives of vertebrates within the clade Olfactores, rather than a basal chordate lineage (Delsuc et al., 2006; Christiaen et al., 2009). The subphylum was previously divided into three major classes: Appendicularia, Thaliacea, and Ascidiacea. However, recent phylogenomic studies have shown that Thaliacea (salps, doliolids, and pyrosomes) do not constitute a separate class but are instead nested within Ascidiacea, forming a lineage more closely related to the orders Aplousobranchia and Phlebobranchia than to Stolidobranchia (Delsuc et al., 2018; Giribet, 2018; Kocot et al., 2018; Calatayud et al., 2021). Ascidiacea includes the exclusively colonial botryllid ascidians ( Botryllus and Botrylloides ), which are the focal taxa of the present study (Gasparini et al., 2015). Tunicates are sessile suspension feeders whose dispersal is restricted to a short-lived larval stage, whereas adults remain permanently attached to natural and artificial substrates in coastal habitats (Cooper & Albert, 2015). Their distribution and reproductive success are strongly shaped by temperature, salinity, and nutrient availability, with additional influences of hydrodynamics, substrate type, predation, and competition (Millar, 1971). Although many species tolerate broad thermal ranges, salinity constitutes a much stronger physiological constraint, implying that sharp oceanographic gradients can act as effective environmental filters and potential barriers to gene flow in tunicate populations (Lambert, 2005; Gab-Alla, 2008; Monniot & Monniot, 1997; Primo & Vazquez, 2009; Oğul Ünal et al., 2025). At the genomic level, tunicates exhibit unusually high mutation rates and remarkable regenerative capacities mediated by stem-cell-based systems, features that are expected to facilitate rapid evolutionary responses to environmental change (Stachowicz et al., 2002; Bourque et al., 2007; Howes et al., 2007; McKindsey et al., 2007; Reem et al., 2013b). In Botryllus schlosseri , for example, microsatellite mutation rates are among the highest reported for marine invertebrates (2.47 × 10⁻²), indicating a substantial potential for generating genetic variation over short timescales. Together with limited larval dispersal and strong environmental filtering, these characteristics create powerful systems for examining how population structure, connectivity, and local adaptation emerge in spatially heterogeneous seascapes. Within tunicates, botryllid ascidians are particularly informative for population-genetic and evolutionary analyses because of their colonial organization and clonal propagation. Colonies consist of genetically identical zooids connected by a common vascular system, enabling coordinated growth, regeneration, and allorecognition (Brunetti & Burighel, 1969; Saito et al., 2001; Rinkevich et al., 1998; Cima et al., 2006). Asexual reproduction proceeds through a blastogenic cycle, whereas sexual reproduction produces short-lived, lecithotrophic larvae that link local population dynamics to episodic dispersal events (Sabbadin, 1960; Manni & Burighel, 2006; Manni et al., 2007). Reproductive output and recruitment are tightly coupled to seasonal patterns of primary productivity and water-column mixing, particularly in temperate and Mediterranean systems (Goodbody, 1961; Lambert, 1968; Becerro & Turon, 1992; Coma et al., 2000; Lambert & Lambert, 2003). Botryllid ascidians are also classical model organisms for studies of allorecognition, stem-cell competition, vascular development, and whole-body regeneration, with B. schlosseri and Botrylloides leachii among the most intensively investigated species (Sabbadin, 1962; Scofield et al., 1982; Rinkevich et al., 2007; Gasparini et al., 2008; Voskoboynik et al., 2013; Blanchoud et al., 2018). Their key phylogenetic position, absence of whole-genome duplication, and exceptional regenerative capacity make them particularly informative for evolutionary and comparative studies (Lemaire, 2011; Voskoboynik et al., 2013). Despite an estimated global diversity of approximately 154 described botryllid species, only a small fraction has so far been documented from the Turkish coasts, a region encompassing the Black Sea, Sea of Marmara, Aegean Sea, and eastern Mediterranean—one of the most pronounced marine biogeographic transition zones worldwide (Çınar, 2014–2016; Temiz et al., 2019; Öztürk et al., 2019; Miroğlu & Yalçın, 2020; Karahan et al., 2022, 2023; Karahan, 2026). Most available records are based solely on morphology, whereas molecular validation and population-level genetic data remain extremely limited. Given the prevalence of cryptic species complexes in botryllids and the strong physicochemical gradients and hydrographic discontinuities characterizing the Turkish Straits System and adjacent seas, this lack of genetic information represents a major gap in our understanding of regional diversity, species boundaries, and large-scale connectivity. Here, we present the first coast-wide, integrative assessment of botryllid and other colonial aplousobranch ascidians along the entire Turkish coastline, spanning all four surrounding seas. Using a COI gene region in combination with spatially explicit physicochemical data (including temperature, salinity, and nutrient regimes), we aim to compile a genetically validated inventory of species, identify previously unrecognized or cryptic lineages and potential new records for the Turkish fauna, and test how major oceanographic discontinuities and environmental gradient’s structure genetic diversity and population connectivity across this biogeographic transition zone. By establishing a molecular baseline for botryllid diversity at the scale of the whole Turkish seascape, this study provides a reference framework for future ecological, evolutionary, and conservation-genetic investigations of colonial ascidians in the region. 2. Materials and Methods 2.1. Study Areas and sampling design Field surveys were conducted along the entire Turkish coastline between August 2023 and May 2024. The study area spans latitudes around 38.96° N and longitudes around 35.24° E, encompassing the Black Sea, Sea of Marmara, Aegean Sea, and eastern Mediterranean. This region represents one of the most pronounced marine biogeographic transition zones, shaped by strong hydrographic gradients and the connectivity constraints imposed by the Turkish Straits System. The primary coast-wide survey was carried out in August 2023, during which all major coastal sectors were visited from the eastern Black Sea to the western Mediterranean. Additional targeted surveys were conducted in October 2023 and May 2024 in the eastern Mediterranean, focusing on localities with previously reported botryllid occurrences and under-sampled areas. In total, 36 coastal stations were surveyed, of which 26 yielded botryllid ascidian colonies (Fig. 1). One station (AK8, Bozyazı) was sampled exclusively for physicochemical parameters and served as a negative control. The Konacık locality in the eastern Mediterranean was revisited in three different seasons to assess temporal consistency (AK-1, 11, 12). Across all stations, a total of 566 colonies were collected and processed for genetic analyses (Table S1). 2.2 Colony sampling and preservation Samples of ascidian colonies were collected from natural substrates (including rocks, macrophytes, and bivalve shells) or artificial surfaces at the stations listed. Sampling was conducted by hand during surveys in the lower intertidal zone of rocky shores and in the subtidal zone at depths of 0–1.5 m, parallel to the shoreline. In addition, colonies attached to anchor ropes, boat hulls, wooden harbor pilings, and fixed or floating rubber pontoons within ports and marinas were surveyed and sampled. To minimize repeated sampling of clonemates, only one colony per distinct morphotype was collected from a given substrate, following established protocols for colonial ascidians (Grosberg & Quinn, 1986). Colonies were photographed in situ and labelled prior to collection. A small fragment was carefully excised using a sterile razor blade, ensuring that the integrity of the remaining colony was preserved. From each colony, three subsamples were taken: (i) one preserved in 4% formaldehyde in seawater for morphological examination, (ii) one stored at −20°C and subsequently transferred to the laboratory for DNA extraction, and (iii) one preserved in 70% ethanol at room temperature for depository (Karahan et al., 2022). Fixatives and ethanol were replaced every 48 h for three consecutive changes. All samples were transported to the METU Institute of Marine Sciences and stored under controlled conditions until laboratory processing. 2.3 Physicochemical and nutrient measurements In situ measurements of temperature and salinity were obtained using a Hanna HI98319 portable salinity meter (accuracy ±0.5°C; ±2 ppt). Turbidity was measured with a WTW Turb® 355 T portable turbidimeter. Dissolved oxygen and pH were recorded using a HORIBA LAQUA PD210-K multiparameter probe. For chlorophyll-a, 1 L of surface seawater (~1 m depth) was collected at each station in light-proof bottles and filtered through 47 mm GF/F glass-fiber filters using a Bürkle Vampire field peristaltic pump. Filters were wrapped in aluminum foil and frozen until analysis. Chlorophyll-a concentrations were determined fluorometrically using a HITACHI F-2500 spectrofluorometer following standard protocols (Strickland & Parsons, 1972; UNEP/MAP, 2005). For nutrient analyses (NO₃⁻ + NO₂⁻, NO₂⁻, PO₄³⁻, and Si), triplicate seawater samples were collected in acid-washed high-density polyethylene bottles and stored frozen (−20 °C) until analysis. Concentrations were measured at the METU-IMS Chemistry Laboratory using standard colorimetric methods with a Bran Luebbe four-channel auto-analyzer (Grasshoff et al., 1983). 2.4 DNA extraction, PCR amplification and Alignment DNA was extracted from all botryllid colony samples collected during the field surveys conducted in August and October 2023 and May 2024, following a modified phenol–chloroform protocol (Karahan et al., 2022). DNA quality and concentration were assessed using a Nanodrop spectrophotometer and working dilutions (14–60 ng µl⁻¹) were prepared. In total, 529 samples passed quality control and were arrayed into six 96-well plates. Stock DNA extracts were stored at −20°C. The mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified by polymerase chain reaction (PCR) using degenerate primer pairs developed for botryllid ascidians (Iannelli et al. 2007; Brunetti et al., 2017; Reem et al., 2018; Salona et al., 2021; Table S2). PCR reactions were performed using two primer combinations (DCOIF2/DCOIR2, dinF/nux1R and cat1F/ux1R) under cycling conditions detailed in Table S2. Amplification success was verified by agarose gel electrophoresis, and products showing clear bands of the expected size were selected for sequencing. Of the 510 amplified samples, 371 yielded high-quality PCR products and were sent to the Macrogen Inc. (Seoul, South Korea) for Sanger sequencing. After quality control sequences were assembled into consensus sequences using the Tracy Assemble tool (Rausch et al., 2020) implemented on the Galaxy.eu platform. Consensus sequences were manually checked and edited, and multiple-sequence alignment was performed in BioEdit v7.0.9.0 (Hall, 1999). After trimming and quality filtering, 321 COI sequences were retained for downstream analyses. Taxonomic identity of each sequence was verified by similarity searches against the GenBank database using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) engine. 2.5 Model Selection, Bayesian Phylogenetic Inference and Genetic Distances The best model for the MrBayes (Ronquist et al ., 2012) was chosen by using PhyML-SMS v3 software (SMS: Smart Model Selection in PhyML; Lefort et al ., 2017). The best-fit nucleotide substitution model for the dataset was identified as TN93+R based on Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC). The selected model (TN93+R) exhibited the lowest AIC and BIC scores, indicating the best balance between model fit and complexity (Lefort et al., 2017), (Table S3). Because the TN93+Γ model is not directly implemented in MrBayes, Bayesian phylogenetic inference was performed using the GTR model (nst = 6) with rate parameters constrained to reproduce the TN93 substitution scheme, and with among-site rate heterogeneity modeled by a discrete gamma distribution. Specifically, the relative substitution rates were fixed to follow the TN93 pattern by constraining the GTR rate matrix (revmatpr = fixed[1 2 1 1 3 1]), while base frequencies were estimated under a Dirichlet prior (statefreqpr = dirichlet[1,1,1,1]). Rate variation among sites was modeled using four discrete gamma categories (rates = gamma, ngammacat = 4). Bayesian analyses were conducted in MrBayes v3.2 (Ronquist et al., 2012) on a dataset comprising both newly generated sequences (56) and reference sequences retrieved from GenBank (42). Two independent Markov chain Monte Carlo (MCMC) runs (nruns = 2), each consisting of four chains (nchains = 4), were run for 1,000,000 generations, with trees and parameters sampled every 100 generations. Convergence between runs was monitored by examining the average standard deviation of split frequencies, which fell below 0.01 (final value = 0.0068), indicating adequate mixing and stationarity. The first 25% of sampled trees were discarded as burn-in, and a 50% majority-rule consensus tree was constructed from the remaining posterior distribution. Convergence and parameter stationarity were further evaluated using effective sample sizes (ESS). ESS were evaluated after discarding the first 25% of samples as burn-in. The two independent MCMC runs were combined, yielding a total of 1,800,000 post-burn-in samples. All estimated parameters exhibited ESS values greater than 750, indicating adequate sampling of the posterior distribution and satisfactory convergence and mixing of the Markov chains (Table S4). Trace plots and posterior distributions were inspected in Tracer v1.7.2 (Rambaut et al., 2018), confirming that independent runs converged on the same posterior distribution. Posterior probabilities were used as measures of nodal support (Fig. S1). Final trees were visualized with FigTree v.1.4.4 (Rambaut, 2018, http://tree.bio.ed.ac.uk/software/figtree). MEGA12 (Kumar et al., 2024) was used to calculate the Kimura 2 parameter (K2-P) distance model with Uniform rates (Kimura, 1980). 2.6 Species Delimitation Analyses Primary species hypotheses were inferred using the Assemble Species by Automatic Partitioning (ASAP) method (Puillandre et al., 2021), implemented in the standalone ASAPy application (version 2023.01.31). The aligned COI dataset was analysed under three evolutionary distance models (Jukes-Cantor 69 (JC69), K2-P and simple-distance) to evaluate the robustness of clustering patterns to model choice. Default clustering parameters were applied. Whereas three distance matrices produced similar results, due to slightly high intraspecific distance score K2P results were used for further analysis. The partition exhibiting the lowest ASAP score together with the strongest barcode-gap signal was selected as the primary species hypothesis and subsequently compared with Bayesian tree and PTP-based delimitation results (Fig. S2). Species boundaries were further assessed using the Bayesian implementation of the Poisson Tree Processes model (bPTP; Zhang et al., 2013), applied to the unrooted Bayesian consensus tree inferred from the COI haplotype dataset. Analyses were conducted on the bPTP web server (https://species.h-its.org/) with an MCMC run of 100,000 generations, discarding an initial burn-in fraction prior to posterior summarization. Posterior probabilities associated with delimited entities were used as measures of confidence in species boundaries. bPTP clusters were compared with ASAP partitions and Bayesian phylogenetic clades to evaluate cross-method congruence and to discriminate between well-supported species-level lineages and potential intraspecific genetic structuring. 2.7 Species–Environment Relationships Associations between botryllid species occurrence and physicochemical parameters were evaluated using Spearman’s rank-order correlation, a non-parametric approach appropriate for non-normally distributed ecological data (Zar, 2010; Legendre & Legendre, 2012). Species were coded as presence–absence across stations, and species richness was calculated as the total number of species per station. Correlations were computed for temperature, salinity, pH, dissolved oxygen, turbidity, chlorophyll-a, PO₄, NO₃ + NO₂, NO₂, Si, NH₄, total nitrogen (TN) and total phosphorus (TP). Analyses were performed in R using the cor.test() function, with statistical significance assessed at P < 0.05 and P < 0.01. 2.8. Multivariate Community–Environment Analysis To examine how environmental gradient’s structure botryllid assemblages, redundancy analysis (RDA) was conducted using the vegan package in R (R Studio team, 2023; Oksanen et al., 2024; R Core team, 2024), following the framework of constrained ordination in community ecology (Legendre & Legendre, 2012; Borcard et al., 2018). Species abundance data were Hellinger-transformed to reduce the influence of double zeros and to render the data suitable for linear ordination methods (Legendre & Gallagher, 2001). Environmental variables (temperature, salinity, pH, dissolved oxygen, chlorophyll-a, PO₄, NO₃ + NO₂) were standardized (z-scores) prior to analysis. The significance of the overall RDA model and of individual environmental predictors was assessed using permutation-based ANOVA tests with 999 permutations. Only variables showing significant marginal effects were retained as constraining factors in the final ordination and biplot representation. 3. Results 3.1 Species identification A total of nine colonial ascidian species were detected in the study area. Species identification was based on a combination of NCBI nucleotide BLAST sequence similarity and intraspecific thresholds derived from species delimitation analyses. BLAST searches revealed high sequence similarity between samples from the present study and reference sequences in NCBI for Botryllus schlosseri (95.15-100%), B. gaiae (98.01-98.61%), B. humilis (97.28-100%), Botrylloides niger (98-100%), B. israeliensis (98.05-100%), Botrylloides sp. (98.64-99.22%), Symplegma brakenhielmi (93.59-98.64%), and Polyclinum constellatum (98.63-99.42%) (Figs. 2–3; Table 1). Of the 321 COI sequences retained after quality filtering, the majority were assigned to B. schlosseri (n = 257), followed by B. niger (n = 62), B. humilis (n = 19), B. gaiae (n = 14), B. israeliensis (n = 7), P. constellatum (n = 6), Botrylloides sp. (n = 4), and S. brakenhielmi (n = 4). In contrast, one botryllid lineage was represented by a single sequence and showed only 86.43% similarity to its closest NCBI reference ( Botrylloides cf. lentus SMBL5.1 (ON098245)). This lineage was therefore interpreted as either a taxon for which the COI gene region has not yet been deposited in NCBI or a potentially undescribed species ( Botrylloides sp. 2). Spatial patterns of species occurrence showed pronounced heterogeneity along the Turkish coastline. B. schlosseri was the only species exhibiting a nearly continuous distribution, extending from the eastern Black Sea through the Sea of Marmara and the Aegean Sea to the southwestern Aegean coast (Muğla, Bodrum). In the eastern Mediterranean, however, this species was detected only at the Konacık (Hatay) station (Fig. 4). B. gaiae was recorded exclusively from the Cunda (Ayvalık) station during the summer survey. The highest local species richness was observed at Konacık, where multiple botryllid taxa co-occurred. Within the Aegean and Mediterranean regions, B. niger represented the most widely distributed species, followed by B. humilis (Fig. 4). 3.1.1 Bayesian lineage structure and Species delimitation analyses (ASAP and bPTP) Convergence of the Bayesian analyses and the reliability of posterior estimates were evaluated using Tracer. All model parameters exhibited high effective sample sizes (ESS), exceeding 450–3000 after removal of the initial burn-in, and both posterior and likelihood traces showed stable mixing across independent runs, indicating adequate exploration of parameter space and robust convergence (Table S4). The Bayesian phylogenetic reconstruction, based on representative sequences from this study together with the closest matching COI reference sequences retrieved from GenBank, recovered a set of well-supported and clearly separated clades corresponding to B. schlosseri (Clade A), B. niger , B. humilis , B. gaiae , B. israeliensis , Polyclinum constellatum and Symplegma brakenhielmi , Botrylloides sp., Botrylloides sp2. with high posterior probabilities for the major nodes. Species-delimitation results obtained with ASAP and bPTP were subsequently projected onto the Bayesian topology to assess congruence between tree-based and distance-based clustering (Fig. 6). Pairwise K2P distances revealed a pronounced barcode gap between intra- and interspecific variation. The absence of overlap between the upper tail of intraspecific distances and the lower tail of interspecific distances indicates a well-defined barcode gap around 1.5–3.5%, fully consistent with the species partitions recovered by ASAP (Fig. S2). Under the K2P distance model, the ASAP analysis yielded three alternative solutions with identical minimum ASAP scores (ASAP score = 1.0), delimiting 15 OTUs. Partitions exhibited complete barcode-gap recovery, demonstrating a strong and stable signal of genetic discontinuity among lineages, and it showed the lowest associated P-value and the steepest slope (pente), thus representing the most conservative and statistically supported delimitation (Fig. S3). At the specimen level, ASAP consistently resolved sample AK1-28 as a distinct and well-supported OTU across all optimal partitions. In contrast, samples from EG5 (Cunda) clustered with the B. schlosseri Clade E / B. gaiae lineage, in agreement with the taxonomic framework proposed by Brunetti et al. (2020), (Fig. S3). The bPTP analysis applied to the Bayesian COI phylogeny delimited 19 putative OTUs in the highest-probability partition, with generally high posterior support values across the major nodes (Fig. S4). In comparison with the primary ASAP solution (Partition 1; 15 OTUs), the overall lineage structure was largely congruent; however, bPTP tended to infer a higher number of within-clade splits. This pattern was particularly evident within B. schlosseri and several Botrylloides lineages, which were subdivided into multiple OTUs by bPTP rather than forming the more inclusive clusters recovered by ASAP. Despite this tendency toward over-splitting, most bPTP-delimited entities corresponded to well-supported monophyletic groups in the Bayesian phylogeny and to the OTUs defined by ASAP (Fig. 6), indicating a high degree of concordance between tree-based and distance-based species-delimitation approaches. 3.1.2 Genetic Distances Intraspecific genetic distances: Intraspecific genetic distances were calculated using K2P model for the 15 operational taxonomic units (OTUs) delimited by ASAP and bPTP. Levels of intraspecific divergence varied substantially among taxa. The lowest values (~1%) were observed in B. niger , B. israeliensis , and P. constellatum intermediate values of approximately 2% were detected in Botrylloides sp., B. humilis , and B. gaiae , while B. schlosseri Clade A showed a higher intraspecific divergence of about 3%. The highest level of intraspecific divergence was recorded in S. brakenhielmi , reaching approximately 5%. Six OTUs were represented by ≥4 sequences and permitted robust estimation of within-lineage genetic variability, whereas one OTU was represented by a single sequence due to limited sampling and was therefore excluded from intraspecific distance calculations. B. gaiae was represented by two distinct sequences, both of which were included. Interspecific genetic distances: The K2P distance model showed clear genetic differentiation among all examined botryllid and related ascidian taxa. Pairwise interspecific distances among Botrylloides and Botryllus species ranged from 12 to 24%. The lowest distance values were observed among Botrylloides cf. anceps (MT873573), Botrylloides sp., and Botrylloides diegensis (MW579604, MT232722), (K2P ~ 12–16%), (Table S5). Interspecific distances among Botryllus taxa, including B. schlosseri Clade A, B. gaiae , and B. humilis , ranged between 16% and 22%. S. brakenhielmi exhibited higher divergence values relative to botryllid taxa (K2P ~ 20–27%). The highest interspecific distances were observed between P. constellatum and all other taxa (K2P ~ 42–49%). (Table 2). The AK1-28 Botrylloides sp.2 lineage showed clear genetic differentiation from all other examined taxa. Pairwise K2-P distances between AK1-28 and other Botrylloides and Botryllus species ranged from 15 to 22%, with the lowest divergence observed relative to Botrylloides cf. lentus (15%) and Botrylloides sp. (16%), (Table 2). 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex 3.2 Field-based habitat observations Field surveys revealed pronounced regional differences in habitat association and colony characteristics of botryllid ascidians along the Turkish coastline. Along the Black Sea coast and through the Turkish Straits System up to the Dardanelles (Çanakkale Strait), no colonies were detected beneath natural rocky substrates despite systematic searches of undersurface habitats. In this region, botryllid colonies occurred almost exclusively on artificial structures, particularly within small fishing ports and marina environments, where they were attached to pontoons, ropes and pilings. South of the Dardanelles, this pattern progressively changed. In the Aegean and Mediterranean Seas, botryllid colonies were increasingly encountered beneath natural rocky substrates, whereas their occurrence on artificial harbor structures became more localized and restricted to highly impacted sites. A clear example of this habitat contrast was observed in the Kuşadası region, where two closely spaced stations (~1.5 km apart) differed markedly in substrate type and species composition (EG6–EG7; Fig. 3). At Yılancı Burnu (EG6), characterized by a sandy-bottom rocky headland connected to the mainland, colonies were found primarily on the undersurfaces of natural rocks and were dominated by B. schlosseri . In contrast, the nearby Kuşadası fishing port (EG7) is dominated by artificial substrates and subject to strong anthropogenic influence, hosted assemblages attached mainly to anchor ropes and harbor structures. It was dominated by B. niger and P. constellatum . These two stations were therefore treated as paired sites representing natural versus artificial habitat conditions within the same coastal sector (Fig. S5). A gradual latitudinal shift in colony morphology was observed, particularly in B. schlosseri (Fig. 5). Colonies from the Black Sea were characterized by a thick tunic forming pendulous, curtain-like extensions and by a two-sided organization of zooids within the colonial matrix. In the Sea of Marmara, colonies retained a comparatively thick tunic, but the strongly pendulous structures typical of Black Sea populations were absent. In contrast, colonies from the Aegean and Mediterranean Seas displayed a markedly thinner tunic and a more compact and delicate colony structure, with zooids embedded in a flatter and less massive colonial matrix (Fig. 5). These morphological gradient parallels the pronounced environmental transition from low-salinity, high-turbidity conditions in the Black Sea to warmer, more saline and oligotrophic conditions in the southern basins. 0pt 2.5ex plus 1ex minus .2ex 1.5ex plus .2ex 0pt 2ex plus .5ex minus .2ex 1ex plus .2ex 0pt 1.5ex plus .5ex minus .2ex 0.8ex plus .2ex 3.3 Species–Environment Relationships Raw physicochemical measurements for all stations are provided in Tables S6 and S7. Spearman’s rank-order correlation analysis revealed species-specific associations with environmental variables (Table 3). B. schlosseri showed a strong negative correlation with salinity and a significant negative correlation with NO₂ concentrations. In contrast, B. niger exhibited no significant relationships with any measured parameter, indicating broad environmental tolerance. B. humilis displayed a significant negative correlation with NH₄. In addition, overall botryllid species richness was positively correlated with PO₄ and total phosphorus (TP). 3.4 Multivariate Community–Environment Analysis (RDA) Redundancy analysis indicated that among the environmental variables examined, salinity and temperature were the only factors significantly associated with the observed biological patterns. Salinity explained the highest proportion of variance (Variance = 0.1569) and showed a strong and highly significant effect (F = 7.65, p = 0.001). Temperature also exhibited a significant influence, although with a lower explanatory power (Variance = 0.0785; F = 3.83; p = 0.027). All remaining variables, including pH, dissolved oxygen, chlorophyll-a, turbidity, and nutrient concentrations (PO₄, NO₃+NO₂, NO₂, Si, NH₄, TP, TN), were not significant (p > 0.05) and explained only a small proportion of the total variance (Table 4). The overall constrained RDA model was significant (F = 1.61, P = 0.01), and the first two canonical axes together explained 80.1% of the species–environment relationship (RDA1 = 68.8%, RDA2 = 11.3%; Fig. 7). The ordination indicated that salinity and temperature were the main environmental variables associated with the observed patterns, with both vectors aligned predominantly along the negative direction of RDA1. Sampling stations from the Mediterranean and Aegean Seas clustered mainly on the negative side of RDA1 and were closely associated with higher salinity and temperature values. Several botryllid taxa, including Botrylloides sp ., B. humilis , and B. gaiae , were positioned near the center of the ordination space, indicating broad environmental overlap among these taxa. In contrast, B. schlosseri was clearly separated along the positive side of RDA1, indicating a distinct ecological position relative to the other species. B. niger was separated along the negative end of RDA1 and RDA2, whereas P. constellatum , S. brakenhielmi , and B. israeliensis clustered toward the negative side of RDA2 (Fig. 7). 4. Discussion In total, 15 OTUs were recovered based on combined analyses of newly generated sequences and NCBI reference data. Nine OTUs originated from the present study. Among these, one lineage represents a putative new species (AK1-28_ Botrylloides sp.), and one species constitutes a new regional record ( B. gaiae ). Species-level patterns were evaluated together with physicochemical parameters to investigate drivers of botryllid diversity and distribution along the Turkish coastline. Habitat use and spatial patterns Field observations revealed pronounced regional differences in habitat use. In the Black Sea and throughout the Turkish Straits System up to the Dardanelles, B. schlosseri was the only botryllid species recorded. Colonies were predominantly associated with artificial substrates and occurred as fouling organisms on mussel shells and seaweeds, whereas no colonies were found on or beneath natural rocks. This pattern suggests that harbors, pontoons, and ropes provide favorable settlement conditions in northern basins by offering stable hard surfaces, increased organic input, and reduced hydrodynamic stress (Carver et.al., 2006 Cima et al., 2015; Hamar et al., 2025). South of the Dardanelles, where additional tunicate and botryllid species were present, colonies were more frequently encountered beneath natural rocks, indicating that substrate availability and local environmental conditions jointly govern habitat selection along the latitudinal gradient. In contrast, B. niger and P. constellatum dominated the assemblage at the Kuşadası fishing port station (EG7), one of the most polluted sites in the Aegean Sea, where they occurred mainly on artificial substrates. Similar records worldwide indicate that these species are tolerant of pollution. (Lambert& Lambert, 2003; Streit et al., 2021; Della Salla et al., 2022). Species-level genetic and distributional patterns Botrylloides niger: All B. niger samples clustered within a single, well-supported Bayesian lineage and were delimited as one OTU by both ASAP and bPTP, showing low intraspecific divergence. Previously, B. niger was reported only from the Turkish Mediterranean coast; the present study extends its confirmed distribution into the Aegean Sea for the first time. Turkish samples clustered with Mediterranean and Atlantic haplotypes, including material from the Turkish Levantine coast (Karahan et al., 2023; Temiz et al., 2023), Italy (Ramalhosa et al., 2021), and the western Atlantic–Caribbean (Sheets et al., 2016) region. This pattern indicates that Turkish populations are part of a broader Eastern Mediterranean–Atlantic metapopulation rather than a locally isolated genetic unit, consistent with recurrent secondary introductions and high connectivity mediated by shipping and rafting (Ben-Shlomo et al., 2010; Reem et al., 2013b; Sheets et al., 2016; Ramalhosa et al., 2021). B. niger exhibited lack of significant correlations with measured physicochemical parameters in the Spearman rank correlation analysis. Consistently, its position in the RDA ordination showed no strong association with the main environmental gradients, indicating that its distribution was not tightly constrained by the measured variables. Together, these results suggest broad environmental tolerance and high habitat plasticity, in agreement with previous reports of B. niger occurring across a wide range of coastal substrates and ecological conditions worldwide (Della Salla et al., 2022; Temiz et al., 2023). Botryllus humilis: B. humilis formed a single, well-supported clade and was delimited as one OTU by both species-delimitation methods. It was recorded at only three stations (Alanya, Mezitli, and Konacık), indicating specific habitat requirements. The species is considered an introduction from the Red Sea (Brunetti, 2009). Based on its limited distribution within the Mediterranean and the absence of rapid range expansion, B. humilis currently does not exhibit invasive potential. Present study samples clustered with Mexican (Palomino-Alvarez et al., 2022) and previously published Turkish sequences (Karahan et al., 2023), supporting the interpretation of a broadly distributed but genetically cohesive lineage. Although experimental data are lacking for B. humilis , its negative correlation with ammonium suggests sensitivity to organic enrichment, potentially explaining its restricted distribution. This pattern is consistent with observations in other ascidian taxa, where increased ammonium and organic enrichment favor opportunistic species while more sensitive taxa decline (Lambert & Lambert, 1998; Streit et al., 2021). At the community level, positive correlations between species richness and phosphorus concentrations likely reflect a productivity-driven increase in niche availability under moderately enriched conditions, consistent with patterns reported for fouling and benthic filter-feeding communities (Glasby & Connell, 1999; Coma et al., 2001). Botryllus schlosseri: Botryllus schlosseri was the only species detected in all four Turkish seas and dominated the Black Sea and Sea of Marmara, where no other botryllid taxa were recorded. This finding aligns with previous studies identifying B. schlosseri as the most widespread and ecologically tolerant botryllid, capable of persisting across broad gradients of temperature, salinity, and habitat type (Ben-Shlomo et al., 2001, 2010; Reem et al., 2013a, b; Brunetti et al., 2017). All Turkish samples belonged to mitochondrial Clade A and were clearly separated from Clade E with full posterior support. Species-delimitation analyses reflected this structure: ASAP recovered Clade A as a single OTU, whereas bPTP subdivided it into three OTUs corresponding to Mediterranean, Black Sea, and Israeli (Reem et al., 2021; Karahan et al., 2023) samples. Despite its broad northern distribution, B. schlosseri showed a restricted occurrence in the eastern Mediterranean, being detected only at the Konacık (Hatay) station. This pattern suggests that regional environmental constraints—particularly salinity, temperature, hydrodynamic exposure, and food availability—may limit its establishment in this basin (Lambert, 2005; Agius, 2007; Shenkar & Swalla, 2011). B. schlosseri showed strong negative associations with salinity and nitrite concentrations, consistent with its reported dominance in low-salinity and moderately disturbed coastal environments. Previous studies have documented the persistence and proliferation of B. schlosseri in eutrophic habitats, particularly in marinas and harbors characterized by elevated ammonium and organic loads (Lambert & Lambert, 1998; Lambert, 2001; Streit et al., 2021). Its capacity to tolerate wide fluctuations in key abiotic factors and to thrive under nutrient-enriched conditions has led to its characterization as an opportunistic fouling species commonly associated with anthropogenically disturbed coastal habitats (Bak et al., 1996; Rinkevich, 2005). Morphological plasticity in B. schlosseri: A clear latitudinal gradient in colony morphology was observed in B. schlosseri , ranging from thick, pendulous tunics in the Black Sea to thinner, more compact colonies in the Aegean and Mediterranean Seas. The Sea of Marmara displayed intermediate morphotypes, consistent with its transitional hydrographic conditions. These patterns indicate environmentally mediated phenotypic plasticity rather than abrupt morphological shifts. This trend is compatible with previous observations showing that colonies in sheltered, nutrient-enriched harbor environments initially expand isometrically and later develop folded or curtain-like tunic structures as colony size increases, particularly on vertical substrates (Cima et al., 2015). Salinity appears to be a key driver of both distribution and morphology. Low surface salinities in the Black Sea approach the physiological limits for many ascidians, and increased tunic thickness may represent a compensatory response to osmotic stress, consistent with stress-related morphological adjustments reported in other aquatic taxa. Botryllus gaiae: Clade E was represented in this dataset by B. gaiae from the Cunda (Ayvalık) station, clustering with Adriatic (Gissi unpub.; Gulnick, 2024 (MSc Thesis) and Mediterranean coast of Israel (Reem et al., 2021) samples. This study reports B. gaiae from the Turkish coastline for the first time. Its restriction to a single station is consistent with its predominantly European–Mediterranean distribution (Lopez-Legentil et al., 2006; Bock et al., 2012; Nydam et al., 2017; Reem et al., 2021). Botrylloides israeliensis: B. israeliensis formed a well-supported mitochondrial clade and was consistently delimited as a single OUT, with samples from the present study clustering closely with previously published sequences from the same region (Karahan et al., 2023). This genetic coherence, together with its geographically restricted distribution along the eastern Mediterranean coast, is consistent with a Lessepsian (Red Sea) introduction followed by limited secondary spread. In the eastern Mediterranean, B. israeliensis was recorded at only three stations (Tisan, Kızkalesi, and Konacık), which suggests constraints on dispersal and/or establishment success outside a narrow environmental window. Lessepsian migration via the Suez Canal has been established as a major vector for ascidian introductions into the Mediterranean Sea (Brunetti, 2009) and given the proximity of the Israeli and Turkish Levantine coasts to the Red Sea, these records support an eastern origin with restricted westward expansion. B. israeliensis occupied a central position in the RDA space and showed no strong alignment with the main environmental vectors, suggesting that its distribution is not primarily driven by salinity or temperature gradients. That indicates a preference for intermediate environmental conditions, rather than extreme or highly disturbed habitats. This pattern suggests a comparatively narrower, potentially habitat-dependent ecological niche as parallel to observed distribution pattern (Brunetti, 2009; Karahan et al., 2023). Botrylloides sp.: Samples from Mersin-Taşucu and Antalya-Kemer formed a strongly supported monophyletic group with eastern Mediterranean and Red Sea material. Both delimitation methods identified this lineage as a single OTU. Although initially recorded from the Red Sea and later from the Antalya–Kemer region, its distribution remains highly restricted, with no records from adjacent southern stations such as Konacık, suggesting limited dispersal or low establishment success. The present study reports the first record of this species from the Taşucu region, whereas previous studies documented its occurrence only along the Antalya–Kemer coast (Karahan et al., 2023). Botrylloides sp. 2 (AK1-28): A single specimen (AK1-28) formed a deeply divergent and well-supported lineage and was consistently recovered as a distinct OTU. Genetic distances exceeded intraspecific thresholds (15%) to its closest relative ( Botrylloides cf. lentus (ON098245)), supporting its recognition as an independent evolutionary lineage. However, representation by a single individual precludes formal taxonomic description. Additional sampling and integrative morphological and genetic analyses are required to determine whether this lineage represents a rare endemic species or an under-sampled cryptic taxon. Symplegma brakenhielmi: Previous records of Symplegma brakenhielmi from the study region documented only a single colony morphotype characterized by red pigmentation (Karahan et al., 2023). In the present study, two additional and morphologically distinct colony morphotypes were recorded for the first time, representing the first records of these morphs from the Turkish coast. Despite clear morphological differentiation among the three morphotypes, COI-based phylogenetic analyses consistently assigned all specimens to S. brakenhielmi , supporting their conspecific status. However, BLAST similarity values showed considerable variation (98.64–93.59%), and S. brakenhielmi exhibited the highest level of intraspecific genetic divergence among all examined taxa, reaching approximately 5% . Pronounced morphological variability combined with elevated mitochondrial divergence has previously been reported for S. brakenhielmi and other styelid ascidians, particularly in the Mediterranean Sea, where this species is considered non-indigenous and shows heterogeneous patterns of spread (Mastrototaro et al., 2019; Ramos-Esplá et al., 2020). Such patterns are commonly interpreted as reflecting phenotypic plasticity , strong population structuring , or the presence of divergent mitochondrial lineages within nominal species , especially in taxa with broad ecological tolerance and complex dispersal histories (Kott, 2005; Monniot & Monniot, 2001). Together, these findings highlight S. brakenhielmi as a morphological and genetically heterogeneous taxon and emphasize the need for integrative taxonomic approaches, including multilocus genetic data and expanded geographic sampling, to fully resolve intraspecific diversity within this species. Latitudinal diversity gradients: In botryllid ascidians Botryllus schlosseri was the most widespread and diverse species along the Turkish coastline. Botrylloides niger and Botryllus humilis followed in prevalence but were restricted to the Aegean and Mediterranean Seas. Additional species were detected primarily along the southern coasts, with the highest local diversity recorded at Konacık (Hatay). Collectively, these patterns reveal a marked north–south shift in species composition, with B. schlosseri dominating the Black Sea and Sea of Marmara and increasing botryllid diversity characterizing the Aegean–Mediterranean transition. Similar gradients have been attributed to the combined effects of environmental filtering, basin connectivity, habitat availability, and anthropogenic disturbance in semi-enclosed marine systems (Lambert, 2005; Shenkar & Swalla, 2011; Cima et al., 2015). Integrating genetic and environmental data is therefore essential to disentangle the relative roles of colonization history, ecological tolerance, and population structuring in shaping botryllid distributions (Ben-Shlomo et al., 2010; Reem et al., 2013b). 5. Conclusion Species delimitation analyses defined nine species along the coastline. Although only a few samples were obtained for some taxa, most species were represented by multiple specimens, strengthening confidence in OTU assignments. For the first time, physicochemical parameters were collected at the sampling stations and evaluated in relation to species distribution. Species responses to physicochemical gradients were species-specific rather than uniform across taxa. Redundancy analysis demonstrated that salinity and temperature are the primary drivers of community differentiation, outweighing the effects of local nutrient variability. This pattern reflects the strong hydrographic contrasts among the connected basins, particularly the pronounced saline gradient between the Black Sea–Marmara system and the Aegean–Mediterranean, a well-recognized biogeographic filter shaping marine species distributions along the Turkish Straits System. Acknowledgements We thank the laboratories of the Chemistry Department at the Institute of Marine Sciences, Middle East Technical University, and Prof. Dr. Süleyman Tuğrul for assistance with physicochemical analyses. We are grateful to İrem Bekdemir for her contributions to field sampling. We also thank the local fishermen at the fishing harbors along the Black Sea coast for their help and cooperation during sample collection. Sunset Apart Otel in Fethiye, Ölüdeniz for their valuable support. References Agius, B. P. (2007). Spatial and temporal effects of pre-seeding plates with invasive ascidians: Growth, recruitment and community composition. 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(2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics , 29 (22), 2869–2876. https://doi.org/10.1093/bioinformatics/btt499 Data Accessibility Statement: DNA barcode sequences generated in this study are publicly available in the BOLD Systems database under the project “DATC – Botryllid ascidians along the Turkish marine corridor.” (Process IDs: between DATC001-26 and DATC056-26). All other relevant data supporting the findings of this study are included within the article and its supplementary information. Benefit-Sharing Statement: No benefits to report. Author Contributions: Design of the study: B.E.T. and A.K. carried out field sampling, performed laboratory and molecular analyses, and conducted statistical and phylogeographic analyses; B.E.T., E.Ö., and A.K. wrote the first draft of the manuscript. B.E.T. revised the article and approved the final version: B.E.T., E.Ö., A.K. Figure Legends Figure 1. Sampling stations along the Turkish coastline. (A) Inset map showing the geographic location of Türkiye within the Mediterranean and Black Sea region. (B) Detailed distribution of the sampling stations across the Black Sea, Sea of Marmara, Aegean Sea and eastern Mediterranean. Green hexagons indicate stations where both physicochemical parameters and biological samples were collected; orange hexagons indicate stations represented by a single-colony record; yellow hexagons indicate stations with colony sampling only; and the red hexagon denotes the station where only physicochemical measurements were obtained. Figure 2. Collected botryllid samples. (A-G) Botryllus humilis , (H-M) Botrylloides niger , (N) Botrylloides sp2., (O-R) Botrylloides israeliensis , (S-W) Botrylloides sp., (V, X) Botryllus schlosseri Clade A, (Y, Z) Botryllus gaiae Figure 3. Other colonial aplousobranch ascidian samples. (A, B) Symplegma brakenhielmi, (C-E) Polyclinum constellatum Figure 4. Species distribution map showing the relative proportion of colonial ascidian taxa at each sampling station. Pie charts represent species composition at each locality, with colors denoting distinct taxa and circle size proportional to the number of colonies sampled per station. Figure 5. Latitudinal variation in colony morphology of Botryllus schlosseri along the Turkish coastline. Upper panels show ink-style illustrations generated from representative field photographs (lower panels) from (A) the Black Sea, (B) the Sea of Marmara, (C) the Aegean Sea, and (D) the Mediterranean Sea. Illustrations were produced using an AI-based image generation tool (DALL·E) based on photographs taken during this study. Figure 6. Bayesian phylogenetic tree showing COI lineage relationships among botryllid and other colonial aplousobranch ascidians. Species-delimitation results obtained with ASAP and bPTP are projected onto the topology with different colors. Bold red nodes indicate clades that were delimited as distinct OTUs by only bPTP. Figure 7 . RDA biplot showing relationships between botryllid taxa, sampling stations, and environmental variables. RDA1 and RDA2 explain 68.8% and 11.3% of the constrained variance, respectively. Stations are colored by sea region. Species scores are shown in red, and only significant environmental variables (salinity and temperature) are displayed as blue arrows. Table Legends Table 1. List of botryllid and other colonial aplousobranch ascidians detected in the sampling area, showing the number of colonies sequenced and analyzed, along with reference authorities. Table 2. Pairwise K2-P model distance matrix among botryllid and related ascidian taxa based on COI data. Pairwise distances were calculated in MEGA v12.1, with values representing mean divergences between species. Table 3. Spearman’s Rank-order correlations between botryllid species and physicochemical parameters (* Significant relationship at P < 0.05 level, ** Significant relationship at P < 0.01 level). Table 4. Marginal effects of environmental predictors on botryllid species composition based on permutation tests from the RDA (999 permutations; P < 0.05*, P < 0.01**). Supplementary materials Figure S1. LnL value of MrBayes analysis for combined two runs: 1800000 Figure S2. Histogram of pairwise K2-P genetic distances among COI sequences used in the ASAP species-delimitation analysis. The distribution shows a clear separation between low intraspecific and higher interspecific divergence, illustrating the presence of a barcode gap. Figure S3. ASAP species-delimitation results based on the K2-P model. The partition selected for subsequent analyses (ASAP score = 1.0) recovered 15 OTUs. Colors represent different OTUs, and the numbers within each OTU indicate the total number of samples assigned to that unit. The first row above the OTU columns shows the total number of OTUs, while the second row reports the corresponding ASAP scores (Puillandre et al., 2021). Figure S4. Bayesian phylogenetic tree based on COI sequences, with OTUs inferred by the Poisson Tree Processes (PTP) model indicated in blue. Node values represent PTP support scores for delimited entities. Figure S5. Habitat contrast between two closely spaced sampling stations in Kuşadası, Muğla (EG6 and EG7). Photographs illustrate (A) the general habitat characteristics of each station and (B) representative colonies of the dominant ascidian species at each site. The schematic map was prepared using Canva (www.canva.com). The photograph of the fishing port at EG7 was obtained from the official website of the Kuşadası Municipality (https://kusadasi.bel.tr). Table S1. Sampling stations along the Turkish coastline, including station codes, geographic coordinates, sea basin, habitat type, sampling date, and the number of colonial ascidian colonies collected and analysed at each locality. Table S2. Degenerate primer pairs (a) and PCR cycling conditions (b) used for amplification of the mtCOI region in botryllid ascidians (Brunetti et al., 2017; Reem et al., 2018; Karahan et al., 2023). Table S3. Model selection results obtained using the Smart Model Selection (SMS) procedure implemented in PhyML. The best-fit nucleotide substitution model for the dataset (alldatav9) was identified as TN93+R based on Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC). The table shows the comparison of alternative substitution models, including the number of free parameters (K), log-likelihood (Lik), AIC, and BIC values. Table S4. Posterior parameter estimates and effective sample sizes (ESS) obtained from the Bayesian phylogenetic analysis after removal of the first 25% of samples as burn-in. All parameters showed ESS values > 750, indicating adequate sampling of the posterior distribution and satisfactory convergence of the Markov chain Monte Carlo runs. Table S5. COI reference sequences obtained from GenBank, showing accession numbers, taxonomic identity, record titles, and sampling localities used for phylogenetic reconstruction and species delimitation. Table S6. Physicochemical parameters measured at each sampling station, including temperature (°C), salinity (ppt), pH, dissolved oxygen (DO; m/ L −1 ), turbidity, and chlorophyll-a concentration (mg/ L −1 ). Station codes and station IDs are provided for cross-reference with biological and genetic datasets. Table S7. Concentrations (µmol/ L −1 ) of dissolved inorganic nutrients and total nutrients at each sampling station, including phosphate (PO 4 ), nitrate + nitrite (NO 3 + NO 2 ), nitrite (NO 2 ), silicate (Si), ammonium (NH 4 ), total phosphorus (TP), and total nitrogen (TN). Supplementary Material File (tables.docx) Download 25.88 KB Information & Authors Information Version history V1 Version 1 10 March 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords genetics invertebrate marine molecular genetics sequencing Authors Affiliations Begum Ece Tohumcu 0000-0002-5071-0786 [email protected] Middle East Technical University Graduate School of Marine Sciences View all articles by this author Esra Ozturk Middle East Technical University Graduate School of Marine Sciences View all articles by this author Arzu Karahan Middle East Technical University Graduate School of Marine Sciences View all articles by this author Metrics & Citations Metrics Article Usage 179 views 110 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Begum Ece Tohumcu, Esra Ozturk, Arzu Karahan. Genetic structure and community turnover of botryllid ascidians along the Turkish marine corridor. Authorea . 10 March 2026. DOI: https://doi.org/10.22541/au.177312459.94681048/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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