﻿Three new species of the genus Perinereis (Annelida, Nereididae) from Egyptian coasts

﻿Abstract Despite being one of the most common groups of polychaetes on intertidal shores, the genus Perinereis (Nereididae) is comparatively poorly known taxonomically, with confusion still existing due to the lack of comprehensive systematic studies. The systematics of Perinereis species from the intertidal Egyptian coasts of the Red Sea, Gulf of Suez and Suez Canal have been investigated using morphology and the mitochondrial barcoding marker cytochrome oxidase subunit I (COI). New sequence data was obtained for 102 Perinereis specimens and analysis included all publicly available COI data from other Perinereis species. The COI data indicate that monophyly of the P.nuntia species group is doubtful, as specimens identified in this species group from south-eastern Asia and Australia form a monophyletic group exclusive of the three new species described in this study from the Red Sea region. A morphometric character set (26 characters) was used to identify and characterize each specimen in the study. Three distinct morphospecies belonging to the P.nuntia species group were found, each differentiated by the number and type of paragnaths on pharyngeal areas V and VI, relative sizes of parapodial lobes, type of notochaetae and neurochaetae, and form of the neurochaetal falciger blades. The three morphospecies were well supported by COI data: two of the three new species, Perinereissuezensissp. nov. and Perinereisfayedensissp. nov., are closely similar to P.nuntia sensu stricto, while the other, Perinereisdamiettasp. nov., is similar to P.heterodonta. The new species are described and illustrated, and bring the number of species in Perinereis to 97. The new species are compared and contrasted to the closely similar P.heterodonta, P.nuntia and other congeners from the region.


Introduction
The family Nereididae includes several highly variable species characterized by high intra-specific morphological variation especially associated with the pharynx (e.g., number and arrangement of paragnaths) or associated with the parapodia (relative proportions of lobes/ligules and form of chaetae) and coloration. Often these morphologically variable species also show differences in reproductive biology (Yoshida 1984;Hardege and Bartels-Hardege 1995) and have widespread distributions. Such species were recognized in the old taxonomic literature as 'forms' or 'varieties' of a species (e.g., Fauvel 1932), but today most are recognized as full species (Read and Fauchald 2022). More than other nereidid genera, Perinereis contains a number of 'species-groups' or 'species complexes' (e.g. Wilson 1993;Glasby et al. 2013), which have served to group like forms, although none have been shown to be monophyletic.
Perinereis Kinberg, 1865 is the second most species-rich genus in the family. It includes approximately 94 worldwide-distributed valid species (Villalobos-Guerrero 2019, Villalobos-Guerrero et al. 2021;Bakken et al. 2022;Conde-Vela 2022). Bakken and Wilson (2005) found that the genus was likely to be polyphyletic based on morphology; specifically, that P. nuntia Lamarck, 1818 together with P. variodentata Augener, 1913, andP. vallata Grube, 1857 were more closely related to Neanthes/Nereis species than to the type species of the genus. Subsequent molecular studies have either supported non-monophyly (Glasby et al. 2013), or refuted it (Alves et al. 2020), but both studies lacked in-depth taxon sampling as they did not specifically set out to test Perinereis monophyly. Tosuji et al. (2019) found support for a clade among western Pacific members of the P. nuntia species group, suggesting biogeographic support for splitting the species group, although as P. nuntia s. s. was not included in the analysis, the question of whether the species group is monophyletic remained open.
Members of Perinereis have been long recognized based primarily on the number and type of paragnaths on areas V and VI (e.g., Kinberg 1865;Grube 1878;Horst 1889). Specifically, Perinereis have well-separated, mostly conical paragnaths on both pharyngeal rings and bar-shaped (which are shield-shaped) paragnaths on area VI (Villalobos-Guerrero et al. 2021). Other important characters are the number of paragnath bands on area VII-VIII, the presence of merged paragnaths on area IV, the presence of isolated paragnaths on area III, type and relative sizes of parapodial lobes (particularly dorsal ligule), type and form of the neurochaetal spinigers and falcigers, and presence of teeth on the jaws (Hutchings et al. 1991;Bakken and Wilson 2005;Santos et al. 2005;Sampértegui et al. 2013). Those species having more than two bars (often many more) on area VI have been considered traditionally as varieties of P. nuntia (Savigny in Lamarck, 1818) (e.g., Fauvel 1919Fauvel , 1921Fauvel , 1932Fauvel , 1953Augener 1931). Nowadays, they are all recognized as species belonging to the Perinereis nuntia species complex (Wilson 1993;Wilson and Glasby 1993;Glasby and Hsieh 2006;Villalobos-Guerrero 2019).
Until recently, the Perinereis nuntia species group comprised 15 valid species (Wilson and Glasby 1993;Glasby and Hsieh 2006;Tosuji et al. 2019). It is characterized by the presence of an arc of bar-shaped (including shield-shaped) paragnaths (or a mixture of bars/shields and cones) on area VI (Tosuji et al. 2019). Subsequent revision involving a broader re-examination of the P. nuntia species group has revealed a further two members, historically referred to under Neanthes Kinberg, 1865 [Nereis (Nereis) latipalpa Schmarda, 1861 from Cape Town, South Africa, and Nereis (Neanthes) larentukana Grube in Peters, 1881from Larantuka, Flores, Indonesia (Villalobos-Guerrero 2019]. In total, Villalobos-Guerrero recognized five new combinations, bringing to 20 the number of valid species in the P. nuntia species group. The present study investigates the taxonomy of three putative species belonging to the P. nuntia species group sampled from the Gulf of Suez, Suez Canal, and the northern Red Sea using a detailed morphological study and the mitochondrial barcoding marker cytochrome oxidase subunit I (COI). We compare our material with other members of the species group originally described from the region, including Perinereis nuntia (type locality: Gulf of Suez) and P. heterodonta Gravier, 1899 (type locality: Red Sea, Obock, Gulf of Aden, Djibouti,). Our results show that all three species are new to science: two of them, P. suezensis sp. nov. and P. fayedensis sp. nov. from Gulf of Suez (part of Red Sea), are closely similar to P. nuntia, while the other one, P. damietta sp. nov., from Hurghada (northern Red Sea), is more similar to P. heterodonta. The three species are described below.

Data collection and preservation
Sampling was carried out during the period of January 2015 to July 2017 from four localities along the intertidal zone of Egyptian coasts of the Red Sea, Gulf of Suez and Suez Canal (Fig. 1).
A section of the mid body was removed from the Red Sea specimens and stored in 96% ethanol for molecular analysis. The rest of the animal was fixed in 4% seawater formalin and stored in 70% ethanol for morphological studies.

Morphological examination
Specimens were studied and photographed using a stereo microscope, Leica MZ16, with a Planapo 1.0X and Canon EOS 500D, as well as a compound microscope, Leica DFC420 connected to a Leica Computer CTR600 and a DM600B camera. For morphological characterization, we grouped specimens into three putative species, and recorded 26 morphometric characters for a subset of 45 of the 102 specimens in total (Suppl. material 1, Table 1). Measurements included number of chaetigers, total body length (cm), jaw length (mm), width at chaetiger 10 excluding parapodia (cm), number of paragnaths on area I, area II (left), area II (right), area III, area IV (left), area IV (right), area IV, area V, area VI (left), area VI (right), and area VII-VIII, length of dorsal cirri (DC; along its ventral edge from the proximal junction with the DNL to the distal extremity) at chaetiger 10 (mm), length of dorsal notopodial ligule (DNL; along its dorsal edge from the proximal junction with the DC to the distal extremity, as per Villalobos-Guerrero (2019: fig. 4e)) at chaetiger 10 (mm), ratio DC/DNL, length of dorsal cirri of one chaetiger between chaetigers 75-90 (mm), length of dorsal notopodial ligule of one chaetiger between chaetigers 75-90 (mm), ratio DC/DNL, length of postero-dorsal tentacular cirrus (as indicated by chaetiger reached when posteriorly extended), occurrence of subacicular heterogomph spiniger at chaetiger 10, occurrence of subacicular heterogomph spiniger at chaetigers 75-90, chaetiger of first occurrence of subacicular heterogomph spiniger. We also recorded the form of the notopodial glands in posterior parapodia, and the color pattern in preserved species. Observation of features on the non-everted pharynx required a longitudinal dissection in the mid-ventral oral region. Characters reported in the taxonomic descriptions are those of the holotype, with those of the paratypes in parentheses. Terminology for nereidid features followed Hylleberg et al. (1986), Bakken and Wilson (2005), Bakken et al. (2009), Villalobos-Guerrero andBakken (2018), and Villalobos-Guerrero (2019).

Molecular study
Genomic DNA was extracted from three to four segments of the middle section of each worm using the DNeasy Tissue Kit (Qiagen) according to manufacturers' instructions with at least two elution steps to increase the amount of DNA. For each individual, the nucleotide sequences of the mitochondrial COI were amplified using the primer pair LCO1490JJ (forward, 5'-CHA CWA AYC ATA AAG ATA RYG G-3') and HCO2198JJ (reverse, 5'-AWA CTT CVG GRT GVC CAA ARA ATC A-3') (Astrin and Stuben 2008). The PCR was carried out in a reaction volume of a 20 µl solution each with 3.8 µl water, 2 µl Q solution, 10 µl Qiagen Multiplex-Solution, 1.6 µl 10 pmol/µl LCO1490JJ, 1.6 µl 10 pmol/µl HCO2198JJ and 1µl template DNA. PCR parameters were 95 °C for 15min, 15 cycles of (94 °C for 35s, 55 °C for 90s with "-1 °C decrease per cycle", 72 °C for 90s), 25 cycle of (94 °C for 35s, 50 °C for 90s, 72 °C for 90s) and 72 °C for 10min. The PCR product was purified using ExoProStar (Qiagen, Hilden, Germany). Both strands were sequenced using Sanger sequencing at Macrogen Inc. (South Korea). Sequences were assembled into contigs using Codon-Code Aligner v. 6.0.2 (Centerville, MA). The 102 new COI sequences were deposited at National Center for Biotechnology Information (NCBI) ( Table 1). For the phylogenetic analyses, we included all publicly available COI data from other specimens of Perinereis as well as five nereidid species, who have a complete mitochondrial genome sequenced, as outgroup taxon (Table 1). The sequences were aligned using the multiple sequence alignment software MAFFT v. 7.310 (Katoh and Standley 2013) with an automatic selection of the best alignment method and the option 'globalpair'. The selected alignment strategy was FFT-NS-i plus an iterative refinement method of two cycles. The 5' and 3' prime ends of the resulting aligned, where trimmed until the first position at each having at least 90% consensus (i.e., < 10% of taxa with missing data at the ends). The final dataset had 267 sequences and 583 nucleotide positions. A maximum likelihood (ML) analysis was conducted with IQ-TREE v. 1.6.12 using the automatically selecting the best-fitting substitution model and an ultrafast bootstrap analysis with 1000 pseudoreplicates (Nguyen et al. 2015;Kalyaanamoorthy et al. 2017;Thi Hoang et al. 2018). The selected model was GTR+F+I+G4 (GTR substitution model with ML estimated frequencies, a proportion of invariant sites and a Gamma distribution with four categories).

Phylogenetic analysis
The ML tree (logL = -11297.8710) showed that the genus Perinereis is probably not monophyletic as the outgroup Hediste japonica Izuka, 1908 grouped within the genus (Fig. 2), but bootstrap support is low for most basal nodes with values below 95. While the monophyly of several Perinereis species is strongly supported with values equal to or more than 95, the monophyly of the P. nuntia species group and of several other species seem to be doubtful or, alternatively, specimens have been wrongly assigned to species. For example, the specimen JX644015 of P. nuntia is placed within P. brevicirris (Grube, 1866) (see Remarks for Perinereis suezensis sp. nov.). Other cases comprise species, for example, of P. aibuhitensis (Grube, 1878), P. suluana (Horst,1924), P. brevicirris, P. wilsoni (Glasby & Hsieh, 2006), P. cultrifera (Grube, 1840), or P. euiini (Park & Kim, 2017). Hence, the requirement for a thorough taxonomic revision of the genus is further supported. The specimens collected for this study were grouped into three strongly supported clades, which were supported with bootstrap values of 99, 99 and 100, respectively (boxes in Fig. 2A, C). Herein, we describe them as new species, P. suezensis sp. nov., P. fayedensis sp. nov. and P. damietta sp. nov. Moreover, P. suezensis sp. nov. and P. fayedensis sp. nov. are sister groups to each other with a maximal bootstrap of 100.

Diagnosis.
Perinereis species having conical paragnaths on all areas (rarely absent on area V), except area VI with long bars, which can be shield-shaped or pyramidal paragnaths, arranged in a single-arched row; area V with paragnaths displaced posteriorly (on everted proboscis) to those on area VI; area IV rarely with merged paragnaths.
Remarks. We have restricted the diagnosis of the species complex to include only unique diagnostic features. Some new characters introduced by Villalobos-Guerrero (2019) describing the faint ridges and furrows of the dorsal oral ring may prove to be useful when broader comparisons can be made. However, at this stage we consider that the form of the pharyngeal ridges and furrows is too closely allied to underlying musculature, and therefore could be unduly influenced by the fixation process and length of time in preservative. Similarly, the form (and length) of the deeply embedded paired nuchal organs may prove to be useful when more comparative data are available. However, observation of that character depends heavily on state of preservation (e.g., they are seen more clearly in specimens relaxed before preservation); in the present specimens the nuchal organs were hidden under the anterior edge of the apodous segment and thus not visible externally. Pharyngeal morphologies are reported herein by describing the form and arrangement of paragnaths on the ridges and in the furrows of the pharynx. The form and arrangement of paragnaths on area VI is unique to the genus (and family) and serves as the easiest way to recognize a member of the species complex. However, Tosuji et al. (2019) have demonstrated that in at least two East Asian species of the complex, the number of bars increases with the growth of individuals (fragmentation of the long bars produces multiple shorter bars (= shield-shaped paragnaths)). Therefore, this character should be used cautiously for species identification across the group, and comparisons are best made between individuals of similar size until we have a better understanding of the processes involved.
Prostomium with entire anterior margin; as wide as long. Antennae closely set, as long as ~ 1/3 length of prostomium. Eyes black, anterior pair set slightly further apart than posterior pair; lenses not obvious.
Notochaetae with homogomph spinigers throughout, blades long; teeth short. Neurochaetae in upper fascicle with homogomph spinigers with long blades; one heterogomph falciger with short blades throughout, blades serrated. Neurochaetae in lower fascicle with heterogomph falcigers, blades short and thick, teeth long; and two or three heterogomph spinigers, median long blades, teeth short present throughout body. Aciculae black, single in each ramus (Fig. 3G-I).
Variation (non-type material). Two specimens: one complete with 105 chaetigers, 57 mm long and 2.8 mm wide, and another with regenerating tail, 107 chaetigers, 71 mm long and 4.3 mm wide. Apodous segment ~ 1.3-1.8× longer than chaetiger 1. Posterodorsal tentacular cirri extending back to chaetigers 5 and 6. Jaws with 4-7 teeth. Paragnaths count: area I with 2; area II with 8-17 on left and 9-17 on right; area III with 11 or 12 in two or three rows; area IV with 17 or 18 on both sides, in two or three rows; area V with three or four; area VI with 8-12 on left and 8-11 on right, shieldshaped bars with pointed tips and cones arranged in one row with the right and left side rows almost touching each other; area VII-VIII with 47 or 48, arranged in a single band of two rows laterally to three or four rows deep medially. Dorsal cirrus length ~ 0.8× length of dorsal notopodial ligule anteriorly and 0.7-0.9× length of dorsal notopodial ligule posteriorly. Ventral cirri extending laterally to reach tip or half-length of ventral neuropodial ligule anteriorly. Neurochaetae in upper fascicle with 1-3 heterogomph falcigers. Neurochaetae in lower fascicle with 1-4 heterogomph spinigers, rarely absent.
Distribution and habitat. Gulf of Suez, Suez Canal including Great Bitter Lake, northern Red Sea; intertidal sand and mud, under stones.
Etymology. The new species is named after the port city of Suez (Egyptian Arabic pronunciation: ‫)سيوسلا(‬ located on the north coast of the Gulf of Suez. Remarks. The molecular data place P. suezensis sp. nov. clearly apart from all other species and the monophyly of the species is very well supported by a bootstrap value of 99 ( Fig. 2A). Not considering identical sequences between specimens within each species, the average genetic distance based on the branch length in the tree to its sister-taxon, P. fayedensis sp. nov., is 6.65% (± 0.60%), while the average genetic distance within P. suezensis is only 0.24% (± 0.37%). Hence, there is a clear gap in the genetic distances.
In addition to our sequences, only three additional COI sequences for P. nuntia have been published: JX420257 (Indonesia), JX644015 (South Korea), and MH337359 (Andaman and Nicobar Islands). JX420257 and MH337359 are identical (boot -Table 2. Comparison of key characters between forms resembling P. heterodonta (pale grey) and Perinereis nuntia (dark grey) in the Red Sea, the Gulf of Aden, and the Arabian (= Persian) Gulf. Abbreviations: AIII = Area III; AV = Area V; AVI Area VI; AVII-VIII = Areas VII and VIII; p-dTC = posterior extension of posterodorsal tentacular cirri (chaetiger); ratio of lengths of dorsal cirri v dorsal notopodial lobe in posterior chaetigers; HS, presence (p) or absence (a) of heterogomph spinigers in anterior neuropodia; NA = data not available.  Fig. 2A), however, they are distantly related to P. suezensis sp. nov. (Fig. 2A). Glasby et al. (2013) found that P. nuntia JX420257 clustered with P. helleri (Grube, 1878), and together was the sister group of P. suluana, both relationships with a high Bayesian posterior probability (> 0.95). This confirms the distant relationship between material identified as P. nuntia from the Australasian region. JX644015 nested within a group comprising otherwise only P. brevicirris with a bootstrap value of 100 (Fig. 2B). Together they clustered with the East Asian-restricted P. wilsoni. Hence, it is also dubious whether JX644015 is a P. nuntia specimen and perhaps should be considered to belong to a species related to other East Asian Perinereis based on the molecular data. Reports of P. brevicirris, which was considered a synonym of P. vallata by Wilson and Glasby (1993) but is now accepted as valid (see key in Villalobos-Guerrero 2019), are widespread throughout the Indo-Pacific but most tropical and northern hemisphere records are unlikely to represent this species, which was originally described from Ile Saint Paul, Southern Ocean. The new species is most similar to P. nuntia, which was also described from the Gulf of Suez. Although the exact location of Savigny's specimens has never been established, it is very likely to be from shallow waters of the port city of Suez, as for Savigny's other polychaetes (see Villalobos-Guerrero 2019 and references therein). Perinereis nuntia was recently redescribed by Villalobos-Guerrero (2019), and based on his redescription and Lamarck's type description, we have found two key differences between the two species (values in parentheses those of Villalobos-Guerrero). The number and shape of paragnaths in area VI: 14-21shield-shaped paragnaths in the new species, compared to 8-10 (10-12) short bars in P. nuntia; and the relative length of the posterior dorsal cirri, which are 1.0-1.3× the DNL in the new species and 4-5 (3-4) × the DNL in P. nuntia. The new species also shows similarities with P. heterodonta from Djibouti in having a high number of paragnaths on area VI and short dorsal cirri in the posterior end; however, the new species can be differentiated from P. heterodonta by the greater number of paragnaths on areas V (24 vs. 0-1) and VII-VIII (37-44 vs. 18-35) ( Table 2).

Species
The larger-sized, non-type specimens generally had more paragnaths in each area compared to the type material, except for area VI. The fewer paragnaths in area VI in the non-type specimens is most likely due to loss, as the ones present were irregularly spaced, with some gaps large enough to accommodate a lost shield-shaped bar or two cones. Another reflection on the condition of the non-type specimens is the unusually short dorsal and ventral cirri; on this point, the cirri appeared withered and many were missing, which we attribute to damage or a fixation artifact.
Prostomium with entire anterior margin; wide as long. Antennae closely set, as long as ~ 1/3 length of prostomium. Eyes black, anterior pair set slightly further apart than posterior pair; lenses not obvious.
Remarks. The molecular data place the new species, P. fayedensis, clearly apart from all other species and as sister to P. suezensis ( Fig. 2A). The monophyly is well supported by a bootstrap value of 99. Not considering identical sequences between specimens within each species, the average genetic distance to its sister-taxon, P. suezensis, is 6.65% (± 0.60%), while the average genetic distance within P. fayedensis is substantially lower with a value of 0.01% (± 0.00%). Hence, there is again a clear gap in the genetic distances.
Distribution and habitat. Gulf of Suez, Suez Canal including Great Bitter Lake; intertidal sand and mud, under stones.

Etymology.
The new species is named after the Egyptian city of Fayed on the western shore of Great Bitter Lake approximately halfway along the Suez Canal.
Prostomium with entire anterior margin; relatively large, longer than wide, two pairs of eyes, dark green with black lenses, and two large palps longer than antennae, palpostyles conical. Antennae closely set, as long as ~ 1/3 length of prostomium. Lenses not obvious.
Etymology. The new species is named after the university of the first author, Damietta University, a noun in apposition. Damietta (Egyptian Arabic: Dumyāț ‫)طايمد(‬ is also a port city located on an eastern distributary of the Nile Delta, ~ 15 km from the Mediterranean Sea. Remarks. Perinereis damietta sp. nov. is well supported by the highest bootstrap value of 100 (Fig. 2C) and clearly set apart from the other Perinereis species in the tree. According to the present molecular phylogeny, the sister group to P. damietta is P. vallata, which is also a former variety of P. nuntia (Wilson and Glasby 1993;Glasby and Hsieh 2006). Not considering identical sequences between specimens within each species, the average genetic distance to its sister group is 42.57% (± 6.72%), while the average genetic distance within P. damietta is 1.12% (± 0.74%) and hence substantially lower. Hence, there is again a clear gap in the genetic distances.
Herein, P. vallata also includes one specimen (JX966314) assigned to P. brevicirris (Fig. 2C). This is probably a misidentification given the very strong bootstrap support values of 100 for both the monophyly of P. vallata and the group of P. brevicirris specimens mentioned above ( Fig. 2A, C).
Perinereis damietta is morphologically most similar to P. heterodonta (type locality: Obock, Djibouti, Gulf of Aden). Both species belong to the group of the P. nuntia complex that lack heterogomph spinigers in anterior parapodia, which is unlike P. nuntia.
Other key differences between P. damietta /P. heterodonta and P. nuntia are the shorter tentacular cirri and the fewer paragnaths in area V (0-1) ( Table 2). Perinereis damietta differs from P. heterodonta most notably in having 24-40 pyramidal paragnaths in area VI (vs. 10-16 in P. heterodonta). In this regard, it has the highest number of area VI paragnaths of any species in the P. nuntia species complex, exceeding the next highest (12-16 bars) found in P. vallata (Wilson and Glasby 1993).
Another species originally described from Djibouti, Perinereis djiboutiensis, is unfortunately poorly known, especially in respect to the presence or absence of heterogomph spinigers in anterior parapodia and numbers of paragnaths in areas III and VII-VIII (Table 2). Although it resembles the new species in having one, or no, paragnaths in area V, it may be differentiated from the new species in having only six or seven short bars (may also include cone-shaped paragnaths) in area VI, which is the lowest of all species of the Perinereis nuntia species group in the region (Table 2), and in this regard it is closer to material described as Perinereis nuntia from the Red Sea by Wilson and Glasby (1993).
A novel character introduced by Villalobos-Guerrero (2019), the size of the gap between areas VI and VII-VIII, may also set this new species (and others in this study) apart from other members of the P. nuntia complex. The gap in all three species described here is about 'as wide as palpophore', which is similar to P. nuntia according to Villalobos-Guerrero (2019), but differs from the Southern Ocean species P. latipalpa (Schmarda, 1861) from South Africa and P. vallata from Chile in which the gap is only as wide as the palpostyle (Wilson and Glasby 1993;Villalobos-Guerrero 2019).

Discussion
The present study supports the finding of Bakken and Wilson (2005) of the nonmonophyly of Perinereis and the 'P. nuntia' species complex. Bakken and Wilson (2005) found that the clade containing the type species, P. novaehollandiae Schmarda, 1861 (a junior synonym P. amblyodonta Schmarda, 1861) does not group with the clade P. nuntia+P. vallata, suggesting that the characteristic arc of bars on area VI, may not be homologous between the two groups. Nevertheless, the presence of a large number (> 10) of uniform, very short bars in area VI may be found as an autapomorphy for some subgroups within the species group, for example, in the sister grouping of P. damietta and the Southern Ocean species, P. vallata. Possibly, fine details of paragnath form and pattern may be found to delineate natural groups within Perinereis, which would lend support to Villalobos-Guerrero's (2019) recognition of the taxonomic importance of faint ridges and furrows of the dorsal oral ring. Microstructures of the pharyngeal surface probably reflect underlying muscular and therefore may play a role in the form and function of paragnaths.
Despite recent advancements in integrative studies in many groups of polychaetes, taxonomic confusion still exists in many groups of Nereididae. Perinereis species are especially problematic due to difficult morphological species differentiation and a lack of detailed systematic studies. This has led to informal denomination of the species complex and recognition of geographic morphs and varieties such as P. cultrifera (Scaps et al. 2000) and the P. nuntia species group (Wilson and Glasby 1993;Glasby and Hsieh 2006;Sampértegui et al. 2013). Today, genetic assessment in combination with morphology is considered an effective tool for redescription of several species principally focused on population differentiation (Rouabah and Scaps 2003) and species delimitation (Chen et al. 2002;Park and Kim 2007;Sampértegui et al. 2013;Villalobos-Guerrero et al. 2021). The present study confirms the utility of such an approach, and moreover demonstrates that the specific combination of the barcoding gene and selected morphometric characters is an effective way to delineate cryptic species.
Finally, this study has uncovered further examples of sympatry among polychaetes. All three new species described here were found in the same habitat, viz., intertidal sand and mud, under stones, at the same location. Perinereis damietta appears to have a slightly wider habitat preference as it also occurs sub-tidally, but more intense sampling including exploration of potential microhabitat differences, is required to confirm our observations. Assuming sympatry, identification of the specific isolation mechanism(s) would be interesting. Several studies have suggested the importance of reproductive isolation as an important speciation mechanism in the species group (e.g., Yoshida 1984;Hardege and Bartels-Hardege 1995). This idea merits further investigation as an explanation for the phenotypic similarity of the three cohabiting nereidid species described in this study.