New insights into the Syllis prolifera species complex from the eastern Mediterranean Sea

159 New insights into the Syllis prolifera species complex from the eastern Mediterranean Sea Fernando Belvis1*, M. Rosario Martín-Hervás1* , Irene del Olmo1, Víctor Ruiz1, Patricia Álvarez-Campos1 1 Centro de Investigación en Biodiversidad y Cambio Global (CIBC-UAM) & Departamento de Biología (Zoología), Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain Corresponding authors: M. Rosario Martín-Hervás (maria.mar[email protected]); Patricia Álvarez-Campos (patricia.alv[email protected]) Copyright: © Fernando Belvis et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Reports of cases of cryptic and pseudocryptic speciation have increased in the last decade among marine invertebrates thanks to the use of integrative taxonomy that combines all available data sources to correctly establish species boundaries, now routinely incorporated into classical taxonomical studies. This approach has enhanced our understanding of the real biodiversity of a given area, which has fundamental implications for conservation and management of marine ecosystems. Among polychaete annelids, considered one of the most ubiquitous and abundant groups in our oceans, there are numerous documented cases of cryptic and pseudocryptic speciation, mainly in those highly diverse families as Syllidae Grube, 1850. One of the most recent cases of species complex has been reported in Syllis prolifera Krohn, 1852 – traditionally considered a widespread, cosmopolitan species – and now recognised as a pseudocryptic complex in the western Mediterranean Sea. However, its broader distribution suggests that additional pseudocryptic lineages may exist in other regions of the Mediterranean basin. In this study, newly collected specimens from the Greek islands of Crete and Mykonos are combined with all previously published data on the S. prolifera complex, aiming to assess the existence of additional putative Mediterranean species. Combining morphological, biogeographical, and molecular data (COI, 16S rRNA, 18S rRNA, and 28S rRNA markers) the presence of at least three more novel lineages for the eastern Mediterranean Sea is revealed. However, unlike previous studies, no clear diagnostic morphological characters were found for each lineage, suggesting that the complex includes both pseudocryptic and cryptic potential new species. Our results further reinforce the view that species diversity within the family Syllidae remains underestimated and highlight the necessity of integrative studies to accurately assess marine invertebrate biodiversity. Key words: Cryptic speciation, integrative taxonomy, species complex, species delimitation, Syllis Introduction The Mediterranean Sea harbours a great biodiversity, with approximately 17,000 described species and a high percentage of endemism (Coll et al. 2010), making it an area of high interest for the study of marine ecosystems. However, nearly Academic editor: Greg Rouse Received: 28 August 2025 Accepted: 9 November 2025 Published: 16 December 2025 ZooBank: https://zoobank. org/7F3B588B-6B45-4A41-B9C2B798648ADD72 Citation: Belvis F, Martín-Hervás MR, del Olmo I, Ruiz V, Álvarez-Campos P (2025) New insights into the Syllis prolifera species complex from the eastern Mediterranean Sea. ZooKeys 1264: 159–181. https://doi. org/10.3897/zookeys.1264.170411 ZooKeys 1264: 159–181 (2025) DOI: 10.3897/zookeys.1264.170411 * These authors contributed equally to this work. 160 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean all regions of the Mediterranean basin face threats such as habitat degradation, overexploitation, pollution, and the spread of invasive species, which contribute to biodiversity loss (Cramer et al. 2020). Furthermore, the underestimation of marine species richness poses a challenge in the context of the global biodiversity crisis (Nygren 2014), highlighting the need to implement conservation strategies based on a deep understanding of the most diverse areas, such as the Mediterranean Sea. Among all the species described in this area, nearly 8,000 are macroscopic invertebrates, including crustaceans, molluscs, and annelids (Musco and Giangrande 2005; Coll et al. 2010). Within the latter, polychaete worms represent one of the most diverse communities in Mediterranean benthic fauna and, specifically in the Aegean Sea, with more than 750 recorded species (Chintiroglou et al. 2005; Çinar et al. 2014, 2024). The family Syllidae Grube, 1850 (Annelida: Phyllodocida) usually stands out among polychaetes due to its great diversity and abundance predominantly in coastal areas, and specifically in the eastern Mediterranean Sea, with more than 100 species reported (Çinar and Ergen 2002; Çinar et al. 2003, 2014, 2024; Rousou et al. 2023; Toso et al. 2024). However, the number of recognised species within this group is continually changing, driven by the exploration of previously unsampled areas (Langeneck et al. 2019) and the application of integrative taxonomy–an approach that has uncovered numerous cases of cryptic and pseudocryptic speciation in syllids (Álvarez-Campos et al. 2017a, 2017b, 2025; Langeneck et al. 2020; del Olmo et al. 2024). Within the diverse type genus Syllis Lamarck, 1818 the occurrence of pseudocryptic species complexes was suggested in multiple lineages in nominal taxa with an alleged cosmopolitan distribution (e.g. Aguado et al. 2012; Ba Akdah et al. 2018) and they were studied in detail in two of them, S. gracilis Grube, 1840 (Alvarez-Campos et al. 2017a) and S. prolifera Krohn, 1852 (del Olmo et al. 2024). Notably, in this latter species, multiple Mediterranean lineages exhibit morphological and ecological distinctions that correspond with substantial molecular divergences (del Olmo et al. 2024). Nevertheless, its widespread distribution suggests that additional pseudocryptic lineages may exist in other regions of the Mediterranean basin, and, therefore, including further localities would be essential for establishing the correct species boundaries within this complex. Thus, we combined the information of newly collected specimens from the Greek islands of Crete and Mykonos with all previously published data on the S. prolifera complex to evaluate the potential presence of additional putative species. To test the species status in this region, we combined molecular evidence – using coalescence-based species delimitation methods – with traditional morphological studies. Our work is based on a multilocus dataset comprising two mitochondrial and two nuclear markers, including 31 specimens of S. prolifera collected from four localities in the Aegean Sea, as well as 52 additional specimens from other Mediterranean regions. Materials and methods Sampling and morphological study Specimens were collected in a scientific survey in July 2024 by hand and snorkelling among algae at four different beaches located on the Greek islands of Crete and Mykonos (Fig. 1). Specimens were sorted and fixed in 96% ethanol and stored at 4 °C in the Department of Biology (Zoology) of the Universidad 161 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Figure 1. Geographic distribution of the sampling localities in the Mediterranean Sea. Map showing the localities sampled in this study (coloured dots) and other Mediterranean localities of specimens included in the analyses (uncoloured dots) from a previous study (del Olmo et al. 2024). Image source: Google Earth. Autónoma de Madrid (UAM). Further morphological examination and identification was carried out using an Olympus SZ 30 stereomicroscope and an Olympus CX 43 light microscope. Images of the mid and posterior compound chaetae were taken for each morphotype using an Olympus CX 31 microscope equipped with an Olympus SC 50 camera. For scanning electron microscopy (SEM), selected individuals were critical-point dried using an Emitech K850 device, coated with 15 nm of gold in a Q150T-S Turbo-Pumper and then examined with a Hitachi S-3000N at the Servicio Interdepartamental de Investigación (SIDI) of the UAM. Additionally, unpublished SEM images of posterior compound chaetae from individuals analysed in a previous study (del Olmo et al. 2024) were also included to facilitate morphological comparisons with S. prolifera specimens from all the Mediterranean localities analysed in the present study. All light microscopy and SEM images were edited in Adobe Photoshop CS6. All specimens used for this study were deposited at the Museo Nacional de Ciencias Naturales de Madrid (MNCN). Voucher numbers, collection date, locality, and other relevant information are listed in Table 1. Molecular analyses Genomic DNA was extracted from a 2–3 segment section from 20 specimens, using the Speedtools DNA extraction kit (Biotools), following the manufacturer’s protocol. Molecular markers commonly used in other phylogenetic and species delimitation studies consisted of fragments of the nuclear 28S rRNA (28S, ~531 bp) and 18S rRNA (18S, ~1800 bp) and the mitochondrial markers 16S 162 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Table 1. Specimen collection data. Examined specimens with collection localities, substrates, coordinates, catalogue numbers, and GenBank accession numbers for all specimens sequenced. Phylogenetic lineages defined in this study are indicated in bold. Specimens not assigned to any lineage are marked as Not assigned. Dashes (–) indicate absence of data. Code Lineage Locality Substrate Coordinates (decimal degrees) Voucher numbers 16S COI 18S 28S ELA 1.4 Lineage 1 Elafonisi Beach, Crete, Greece Corallina sp. and unidentified green algae 35.271162, 23.541309 MNCN 19484 − − PV475185 PV422772 MYK 1.4 Megali Ammos Beach, Mykonos, Greece Corallina sp. and unidentified green algae 37.438991, 25.326656 MNCN 19485 −PV422749 PV475194 PV422776 MYK 1.2 MNCN 19486 − PV422753 PV475189 PV422779 MYK 1.1 Lineage 2 Megali Ammos Beach, Mykonos, Greece Corallina sp. and unidentified green algae 37.438991, 25.326656 MNCN 19487 − − PV475186 − MYK 1.3 MNCN 19488 −PV422755 PV475187 − MYK 1.6 − − − − − MYK 1.7 −−−−− MYK 2 MNCN 19489 PV435825 PV422756 PV475190 PV422783 ELA 1.1 Lineage 3 Elafonisi Beach, Crete, Greece Corallina sp. and unidentified green algae 35.271162, 23.541309 MNCN 19490 PV435828 −PV475193 − ELA 1.3 MNCN 19491 PV435826 PV422757 PV475191 − ELA 1.6 − − − − − ELA 1.7 −−−−− ELA 1.8 −−−−− CHA 1.6 Nea Chora Beach, Crete, Greece Corallina sp. and unidentified green algae 35.51374, 24.00602 − − − − − CHA 1.7 −−−−− CHA 1.8 −−−−− KED 1.1 Kedrodasos Beach, Crete, Greece Corallina sp. and unidentified green algae 35.26858, 23.56310 MNCN 19492 −PV422761 PV475188 PV422773 KED 1.2 Lineage 4 Kedrodasos Beach, Crete, Greece Corallina sp. and unidentified green algae 35.26858, 23.56310 MNCN 19493 PV435832 PV422750 PV475181 PV422777 ELA 1.5 Elafonisi Beach, Crete, Greece Corallina sp. and unidentified green algae 35.271162, 23.541309 MNCN 19494 PV435833 PV422751 PV475182 PV422778 ELA 2 MNCN 19495 PV435830 PV422759 PV475198 PV422782 MYK 1.5 Megali Ammos Beach, Mykonos, Greece Corallina sp. and unidentified green algae 37.438991, 25.326656 MNCN 19496 PV435836 PV422762 PV475195 PV422770 CHA 1.2 Nea Chora Beach, Crete, Greece Corallina sp. and unidentified green algae 35.51374, 24.00602 MNCN 19497 PV435829 PV422758 PV475199 − CHA 1.3 MNCN 19498 PV435837 PV422763 PV475196 PV422771 CHA 1.1 Not assigned Nea Chora Beach, Crete, Greece Corallina sp. and unidentified green algae 35.51374, 24.00602 MNCN 19499 PV435835 PV422754 PV475197 PV422781 CHA 1.4 MNCN 19500 PV435831 PV422760 PV475183 PV422775 CHA 1.5 MNCN 19501 PV435827 −PV475192 PV422774 ELA 1.2 Elafonisi Beach, Crete, Greece Corallina sp. and unidentified green algae 35.271162, 23.541309 MNCN 19502 PV435834 PV422752 PV475180 PV422780 163 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean rRNA (16S, ~470 bp) and cytochrome c oxidase subunit I (COI, ~650 bp), were amplified by polymerase chain reaction (PCR). Primers 28Sa and 28Srd5b (Edgecombe and Giribet 2006) were used to amplify the 28S rRNA fragment. For 18S rRNA, three overlapping pairs of primers were used: 18S1F-18S4R, 18S3F-18Sbi, and 18Sa2.018S9R (Giribet et al. 1996; Whiting et al. 1997). Primers 16SarL and 16SbrH (Palumbi 1996) were used to amplify 16S rRNA, and the modified primers with inosine jgLCO1490 and jgHCO2198 (Geller et al. 2013) were employed to amplify in all specimens. The PCR reactions consisted of 1.5 μl of DNA template in 19.5 μl reaction volume containing 12 μl of RED Taq polymerase master mix (VWR), 5 μl of H2O, and 0.5 μl of each of 10μM primers. In some cases, 0.8 μl of 10 mM bovine serum albumin (BSA) was added to stabilise enzymes during the process, bringing the final volume to 20.3 μl. The PCR protocols used were the same as those employed in previous studies on the family Syllidae (Álvarez-Campos et al. 2017b; Moreno-Martín et al. 2023; del Olmo et al. 2024). Sanger sequencing was conducted by Macrogen Spain (Madrid, ES) (https:// dna.macrogen.com/). All sequences were edited using GENEIOUS Prime 2025.0.2 (https://www.geneious.com/), to remove primers, and were deposited in the NCBI database (http://www.ncbi.nlm.nih.gov/genbank/). Phylogenetic analysis The phylogenetic relationships among the collected specimens were studied by combining the newly obtained sequences with 143 additional sequences available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) (Suppl. material 1). A total of 29 species of the genus Syllis were also included to determine the phylogenetic position of the different Mediterranean population of S. prolifera, together with 16 species of some other genera from the subfamily Syllinae (Trypanosyllis Claparède, 1864; Eurysyllis Ehlers, 1864; Ramisyllis Glasby, Schroeder & Aguado, 2012; Haplosyllis Langerhans, 1879; Branchiosyllis Ehlers, 1887; and Plakosyllis Hartmann-Schröder, 1956), used as outgroups. The best evolutionary model for each amplified gene was selected using jModeltest2 (Darriba et al. 2012), following the Akaike Information Criterion (AIC) (Akaike 1974). The best evolutionary model for each gene was identified as the General Time Reversible model with gamma distribution and a proportion of invariable sites (GTR+G+I). The sequences of each gene were aligned using MAFFT (Katoh and Standley 2013) under default parameters and then concatenated using SeaView v. 5.1 (Gouy et al. 2021). Maximum Likelihood (ML) phylogenetic analysis of the concatenated sequence dataset was performed using raxmlGUI 2.0.13 (Edler et al. 2021), employing the GTR+G+I model with 500 bootstrap replicates to estimate support values. Bayesian Inference (BI) analysis was performed using MrBayes v. 3.2.7 (Ronquist et al. 2012), with four Markov chains starting from a random tree, run simultaneously for 50 million generations, sampling one tree every 5,000 generations (samplefreq = 5000). Additionally, the first 25% of the trees were discarded as burn-in (burninfrac = 0.25) after evaluating convergence using Tracer v. 1.7.2 (Rambaut et al. 2018). The resulting phylogenetic trees from both analyses were exported and visualised using FigTree v. 1.4.4 (http://tree. bio.ed.ac.uk/software/figtree/). Final editing of the phylogenetic trees, species delimitation trees and haplotype networks were performed using Inkscape v. 1.4 (https://inkscape.app/es/). 164 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Species delimitation and haplotype networks COI and 16S dataset were analysed separately using three different methods for species delimitation analyses: Automatic Barcode Gap Discovery (ABGD), General Mixed-Yule Coalescent (GMYC), and Bayesian Poisson Tree Process (bPTP), the latter in two variations (bPTP_BI and bPTP_ML). All species delimitation models were applied to simplified data set matrices including only one representative for each haplotype, as recommended by a previous study (Magoga et al. 2021). The ABGD method was run on its web server (https://bioinfo. mnhn.fr/abi/public/abgd/abgdweb.html) using the Kimura 2-parameter model (K2P) (Kimura 1980) and default parameters. For GMYC and bPTP models, ultrametric trees were first generated using BEAST v. 1.10.4 (Suchard et al. 2018) via BEAUti v1.10.4 using the GTR+G+I model, an uncorrelated lognormal relaxed clock, and a coalescent constant size prior. MCMC chains ran for 50 million generations (sampling every 1,000; 25% burn-in), with convergence (ESS > 200) checked in Tracer v. 1.7.2. Resulting trees were summarised to a maximum clade credibility topology in TreeAnnotator v. 1.10.4 (Suchard et al. 2018) (25% burn-in, mean node heights) and subjected to single-threshold GMYC analysis using the web server interface (https://species.h-its.org/gmyc/). Additionally, bPTP analyses was performed using the web server (https://species.h-its.org/ ptp/) with the best ML tree and 200,000 generations (25% burn-in). Genetic distances within and between lineages of S. prolifera sequences identified in the phylogenetic analyses were estimated using the software MEGA 11.0.13 (Tamura et al. 2021). Distances were calculated using the Kimura 2-parameter (K2P) model (Kimura 1980) for the COI and 16S markers separately. Although this model has not been reported as an appropriate model in DNA-barcoding studies (Srivathsan and Meier 2012), and it has not been confirmed as the model that best fits our data, we estimated the distances using it to allow further comparisons with other studies. Haplotype networks – used to illustrate evolutionary relationships between populations – were built using PopART v. 1.7 (Population Analysis with Reticulate Trees) (Leigh and Bryant 2015), applying the TCS algorithm (Clement et al. 2002). Results Phylogenetic reconstruction Final alignments of all concatenated markers had a total length of 3,348 base pairs (bp). From the 163 specimens included in the analyses, 140 of them had sequences for the COI marker (624 bp), 110 for 16S (421 bp), 98 for 18S (1,990 bp), and 47 for 28S (313 bp). ML and BI analyses yielded largely congruent topologies for the four-gene concatenated dataset, with minor differences observed in clade support values and some interspecific relationships (Fig. 2, Suppl. materials 2, 3). Both ML and BI analyses revealed that Syllis does not form a monophyletic group, with species from the genera Haplosyllis and Branchiosyllis clustering within the Syllis clade (Fig. 2, Suppl. materials 2, 3). Our analyses showed topologies with four major clades: Clade A (BS = 100%, PP = 1), which includes S. amica Quatrefages, 1866 and several specimens identified as Syllis cf. prolifera from brackish waters in Sardinia, Italy (del Olmo et al. 2024); Clade 165 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Figure 2. Phylogenetic relationships of Syllis and related genera based on concatenated molecular data. Tree obtained by the Maximum Likelihood method using the GTR+G+I model for the concatenated dataset (COI, 16S, 28S, 18S), showing the phylogenetic relationships of the studied specimens and other closely related species. Numbers above the nodes indicate bootstrap support values equal to or greater than 75% (BS ≥ 75%), and numbers below indicate posterior probability values equal to or greater than 0.95 (PP ≥ 0.95). Dashes indicate lack of support in one of the analyses, and asterisks indicate that those clades were not recovered by the BI analysis (see Suppl. material 3). The names of the newly sequenced individuals are highlighted in blue. Within the S. prolifera complex, the newly defined lineages are shown in black, while the lineages defined in previous studies are outlined in grey. Eurisyllis sp. Trypanosyllis sp. Plakosyllis sp. 100 100 100 1 Ramisyllis sp. 1 1 0.98 76 0.98 0.99 0.98 0.99 1 1 1 1 0.98 0.99 1 0,99 0.99 0.2 0.92 1 1 * * * 1 1 1 1 1 1 1 1 1 1 1 1 1 Syllis amica Clade A Clade B Clade C Clade D Syllis cf. prolifera Syllis armillaris Syllis hyalina _ _ _ 98 87 98 100 100 100 100 100 100 100 99 97 77 Syllis cf. armillaris MYK 3 Syllis hyalina Syllis gerundensis 100 Syllis gracilis Syllis picta Syllis gracilis Syllis gracilis Syllis ypsiloides Syllis lutea & 1 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Syllis garciai Branchiosyllis sp. Syllis ferrani Syllis variegata Syllis prolifera Haplosyllis spongicola * * * * * Syllis crassicirrata Syllis crassicirrata Syllis bella Syllis magdalena Syllis albae Syllis pectinans Syllis patriciae Syllis prolifera species complex Syllis setoensis Syllis vittata Syllis okadai Syllis monilata Syllis marugani Lineage 1, Greece (Crete & Mykonos) Italy lineage I Spain lineage I Spain lineage II Lineage 4, Greece (Crete & Mykonos) Lineage 2, Greece (Mykonos) Lineage 3, Greece (Crete) Syllis ehlersoides Syllis pigmentata Syllis prolifera ELA 1.4 Syllis prolifera MYK 1.4 Syllis prolifera CHA 1.4 83 81 Syllis prolifera MYK 1.2 Syllis prolifera CHA 1.5 Syllis prolifera MYK 2 Syllis prolifera MYK 1.3 Syllis prolifera ELA 1.2 Syllis prolifera ELA 1.1 Syllis prolifera ELA 1.3 Syllis prolifera KED 1.1 Syllis prolifera MYK 1.1 _ 1 Syllis prolifera CHA 1.1 99 80 SproGB02 Spro0506 Spro0405 Spro0407 Spro0402 Spro0505 Spro0401 Spro0403 Spro0404 Spro0507 Spro0504 Spro0509 Syprolif3PI SproGB03 96 96 92 99 1 1 Spro0122 Spro0120 Spro0108 Spro0121 Spro0124 Spro0126 Spro0127 Spro0117 Spro0119 Spro0118 Spro0125 Spro0123 Spro0116 Spro0301 Spro0303 Spro0107 Syllis prolifera CHA 1.2 Syllis prolifera_ KED 1.2 Syllis prolifera CHA 1.3 Syllis prolifera ELA 1.5 Syllis prolifera ELA2 Spro0502 Spro0510 Spro0501 Spro0309 Spro0306 Spro0305 Spro0304 Spro0308 Spro0307 Spro0310 Spro0302 _ _ 1 1 _ _ 1 100 87 99 Italy lineage I Syllis prolifera MYK 1.5 166 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean B (BS = 87%, PP = 1), which includes Syllis species reported within the S. gracilis species complex and related species (Álvarez-Campos et al. 2017a); Clade C (BS = 100%, PP = 1), which contains S. lutea (Hartmann-Schröder, 1960) and S. garciai (Campoy, 1982) together with Haplosyllis and Branchiosyllis species; and Clade D, only supported in the BI analysis (PP = 0.99) comprises the rest of Syllis species (Fig. 2, Suppl. materials 2, 3). Within clade D, there is a well-supported subclade in BI results (PP = 0.99) that contains the S. prolifera species complex (shaded on the tree). This complex contains four well-supported lineages with specimens from Mykonos and Crete together with other four individuals not included in any supported lineage (CHA 1.1, 1.4, 1.5, and ELA 1.2), as well as the five lineages previously identified by del Olmo et al. (2024). Our Greek lineage 1 includes only three individuals from Crete and Mykonos (BS = 83%, PP = 1) and is the sister group to the remaining lineages (BS = 92%, PP = 1). Lineage 2 only includes three individuals from Mykonos (BS = 100%, PP = 1) and appears closely related to lineage 3, which groups three individuals from Crete (BS = 100%, PP = 1). Finally, lineage 4 (PP = 0.98) clearly differentiated from the remaining lineages from Greece, includes six mixed individuals from all studied sites and is the sister group of a lineage from Italy. Species delimitation within the S. prolifera complex Species delimitation analyses of the COI marker using only the 25 haplotype sequences identified six or seven lineages and – 5 singletons (Fig. 3a). GMYC model identified seven lineages – four of them including specimens from Greece– and five singletons, one from Crete (CHA 1.5) and two from Mykonos (Fig. 3a, Suppl. material 4). Greek lineages 1 and 3 comprised the same specimens recovered by the phylogenetic analyses, lineage 2 included two specimens from Mykonos and lineage 4 contained three specimens from the different localities in Crete (Fig. 3a). ABGD model recovered six lineages – three from Greece – and two singletons, one of them being the Cretan CHA 1.5 (Fig. 3a, Suppl. material 5). Greek lineages 1 and 4 included the same individuals than the phylogenetic analyses, and lineages 2 and 3 grouped together as a single lineage (Fig. 3a). Similarly, the ML variant of the bPTP model delimited the same six lineages as the previous model and three singletons, including again CHA 1.5 (Fig. 3a, Suppl. material 6). Once more, Greek lineages 1 and 4 included the same individuals than the phylogenetic analyses, and lineages 2 and 3 grouped together as a single lineage. Finally, the BI variant of the bPTP analysis identified seven lineages –four including specimens from Greece – and four singletons (Fig. 3a, Suppl. materials 6). In this case, Greek lineages 1, 3, and 4 comprised the same individuals as in the phylogenetic analyses. The lineages from Italy and Spain previously identified by del Olmo et al. (2024) were the same in all the delimitation analyses of COI, except for specimen Spro0506 (Italian lineage I), that was considered a singleton in GMYC and both bPTP analyses (Fig. 3a, Suppl. materials 4, 6). On the other hand, the species delimitation analyses for the 16S marker using only the 13 haplotype sequences identified one to four lineages and two singletons (Fig. 3b). The species delimitation results for the 16S haplotypes were exactly the same for the GMYC and ABDG models, both considering a unique lineage except for individuals Spro0302 and MYK 1.2, considered as singletons 167 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean (Fig. 3b, Suppl. materials 7, 8). Likewise, ML and BI variants of bPTP identified the same two singletons and four lineages, two of them containing specimens from Greece (Fig. 3b, Suppl. materials 9). Greek Lineage 4 comprised the same specimens recovered by the phylogenetic analyses, while lineages 2 and 3 appeared together as a single lineage (Fig. 3b). Genetic distances and haplotype networks Pairwise genetic distances (K2P) for the S. prolifera species complex were calculated between the nine supported lineages identified by phylogenetic analysis. The K2P distance for COI ranged from 3.2% ± 0.6 to 25.7% ± 2.6, with the lowest distance between the lineage 2 (Mykonos) and lineage 3 (Crete), and the largest one between lineage 1 (Crete and Mykonos) and lineage I from Italy (Table 2). The genetic distances between the Cretan specimen CHA 1.5 and the rest of lineages, ranged from 15.4% ± 2.0 to 24.7% ± 2.7. Intralineage distances for the COI ranged from 0 to 2.8% ± 0.5, with the lowest in the Spanish lineage II and the highest within the Greek lineage 2 (Table 2). On the other hand, the K2P distances for 16S ranged from 0.8% ± 0.5 to 45.6% ± 6.2, with the lowest again between the Greek lineages 2 and 3, and the largest between the Greek lineage 1 and the Italian lineage II (Table 2). The variation of genetic distances between the Cretan individual ELA 1.2 and the remaining lineages ranged from 6.4% ± 1.3 to 43.7 ± 5.9 (Table 2). The intralineage distances ranged from 0 to 1.2% ± 0.5, with the lowest being within the Greek lineage 3 and the Spanish lineage II, and the highest distance again being within the Greek lineage 2 (Table 2). To explore the genetic differences among sampled S. prolifera populations in the eastern Mediterranean and the previously studied ones from the western MediFigure 3. Ultrametric trees of the different models used for species delimitation in the Syllis prolifera complex using COI (a) and 16S (b) haplotype datasets. Black bars indicate haplotype clusters/potential species supported by each analysis (see Suppl. materials 4–9) while grey bars indicate those from a previous study (del Olmo et al. 2024). Numbers within each bar correspond to the lineages (1–4) resulting from the phylogenetic analysis. MYK 1.1 GMYC ELA 1.1 MYK 2 ELA 1.3 MYK 1.3 Spro0406 Spro0401 Spro0501 Spro0506 CHA 1.5 MYK 1.5 ELA 1.5 ELA 1.4 MYK 1.4 CHA 1.2 KED 1.2 MYK 1.2 Spro0107 Spro0302 Spro0510 Spro0117 Spro0115 Spro0118 Spro0120 Spro0122 Spro0302 Spro0123 Spro0506 Spro0401 Spro0504 Spro0108 SproGB03 SproGB02 ELA 1.5 ELA 1.1 MYK 1.1 MYK 1.2 MYK 1.5 ABGD bPTP_ML bPTP_ML bPTP_BI GMYC ABGD bPTP_BI COI 16S a b 2 2 2 2 4 4 4 4 4 1 1 1 1 2 + 2 + 4 1 1 1 1 2 + 3 3 3 3 3 174 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Funding This work was funded by the Spanish Government througth the project MCIN/ AEI/10.13039/501100011033 and by the European Union “Next Generation EU”/PRTR (CNS2023-145193) to PÁ-C. This research was partially funded by JDC2022-049810-I grant by MICIU/AEI/10.13039/501100011033 and European Union NextGenerationEU/ PRTR to M. Rosario Martin-Hervás. Author contributions Conceptualization: PÁC. Data curation: FB, PÁC, MRMH. Formal analysis: FB, MRMH. Funding acquisition: PÁC. Investigation: VR, FB. Methodology: IO, VR, FB, MRMH. Project administration: PÁC. Resources: PÁC. Supervision: MRMH, PÁC. Validation: VR, PÁC, MRMH, FB, IO. Visualization: PÁC. Writing – original draft: FB. Writing – review and editing: PÁC, VR, MRMH, IO. Author ORCIDs M. Rosario Martín-Hervás https://orcid.org/0000-0002-9673-2673 Irene del Olmo https://orcid.org/0000-0001-5448-1039 Patricia Álvarez-Campos https://orcid.org/0000-0001-9472-2378 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. References Aguado MT, San Martín G, Siddall ME (2012) Systematics and evolution of syllids (Annelida, Syllidae). 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Mediterranean Marine Science 25(3): 715– 731. https://doi.org/10.12681/mms.37998 178 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Whiting MF, Carpenter JC, Wheeler QD, Wheeler WC (1997) The Strepsiptera Problem: Phylogeny of the Holometabolous Insect Orders Inferred from 18S and 28S Ribosomal DNA Sequences and Morphology. Systematic Biology 46(1): 1–68. https://doi. org/10.1093/sysbio/46.1.1 Supplementary material 1 Sequences used in the phylogenetic analysis Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: xlsx Explanation note: List of specimens with species names, collection localities, substrates, geographic coordinates, catalogue numbers, and GenBank accession numbers. Dashes (–) indicate absence of data. Asterisks (*) indicate unpublished sequences. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl1 Supplementary material 2 Phylogenetic tree of the S. prolifera complex (ML, GTR+G+I) Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: svg Explanation note: Most likely phylogenetic tree obtained by the ML method with the GTR+G+I model for all sequenced markers (COI, 16S, 28S, 18S). Posterior probabilities are shown next to each node. The clade containing the specimens used as the outgroup is highlighted in black. In the S. prolifera complex, the new lineages are shown in black, while the lineages defined in previous studies are outlined in grey. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl2 179 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Supplementary material 3 Phylogenetic tree of the S. prolifera complex (BI, GTR+G+I) Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: svg Explanation note: Most likely phylogenetic tree obtained by the BI method with the GTR+G+I model for all sequenced markers (COI, 16S, 28S, 18S). Posterior probabilities are shown next to each node. The clade containing the specimens used as the outgroup is highlighted in black. In the S. prolifera complex, the new lineages are shown in black, while the lineages defined in previous studies are outlined in grey. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl3 Supplementary material 4 Ultrametric tree resulting from the species delimitation analysis under the GMYC model for COI haplotypes of S. prolifera individuals Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: svg Explanation note: Posterior probabilities are shown next to each node. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl4 Supplementary material 5 Species delimitation results for ABGD model for the COI haplotypes from S. prolifera individuals Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: tif Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl5 180 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Supplementary material 6 Trees resulting from species delimitation under the bPTP model for COI haplotypes of S. prolifera specimens Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: tif Explanation note: The tree on the left corresponds to the bPTP_ML analysis, while the tree on the right corresponds to the bPTP_BI analysis. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl6 Supplementary material 7 Ultrametric tree resulting from the species delimitation analysis under the GMYC model for 16S haplotypes of S. prolifera individuals Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: svg Explanation note: Posterior probabilities are shown next to each node. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl7 Supplementary material 8 Species delimitation results for ABGD model for the 16S haplotypes from S. prolifera individuals Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: tif Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl8 181 ZooKeys 1264: 159–181 (2025), DOI: 10.3897/zookeys.1264.170411 Fernando Belvis et al.: Syllis prolifera complex eastern Mediterraean Supplementary material 9 Trees resulting from species delimitation under the bPTP model for 16S haplotypes of S. prolifera specimens Authors: Fernando Belvis, M. Rosario Martín-Hervás, Irene del Olmo, Víctor Ruiz, Patricia Álvarez-Campos Data type: tif Explanation note: The tree on the top corresponds to the bPTP_ML analysis, while the tree on the bottom corresponds to the bPTP_BI analysis. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1264.170411.suppl9