Introduction

The eel catfish genus Plotosus Lacepède 1803 is widely distributed in the tropical Indo-West Pacific, with species occurring in estuarine, coastal shallow waters and offshore deeper waters habitats (e.g., Gomon and Taylor 1982; Burgess 1989; Ferraris 1999; Ng and Sparks 2002). The number of valid species in this genus is highly contentious, but it includes at least nine recognized species: the dog-tooth eel catfish Plotosus canius Hamilton 1822, the short-tail eel catfish Plotosus abbreviatus (Boulenger 1895), Plotosus fisadoha Ng and Sparks 2002, the Japanese eel catfish Plotosus japonicus Yoshino and Kishimoto 2008, the spiny eel catfish Plotosus limbatus Valenciennes in Cuvier and Valenciennes 1840, the striped eel catfish Plotosus lineatus (Thurnberg 1787), Plotosus nhatrangensis Prokofiev 2008, the pond spiny eel catfish Plotosus nkunga Gomon and Taylor 1982 and Plotosus papuensis Weber 1910. The latter is the only species to be found in freshwater habitats (Fricke and Eschmeyer 2023; Froese and Pauly 2023).

The type species of the genus, Plotosus lineatus, commonly known as striped eel catfish or lined catfish, is eel-shaped, with a brown body and two white longitudinal stripes and was described from the Indo-Pacific region, in the East Indian Ocean, more than 200 years ago (Plotosus lineatus Thunberg 1787). It has the widest distribution range of all species of Plotosus. Geographically, it has been collected in Japan, southern Korea, the Ogasawara Islands, Australia, Lord Howe Island, Palau and Yap in Micronesia, East Africa to Samoa, Madagascar, Red Sea and the Persian Gulf (Taylor and Gomon 1986; Myers 1999; Wakwabi and Mees 1999; Ali et al. 2007; Ketabi and Jamily 2016; Froese and Pauly 2023).

Since the opening of the Suez Canal in 1869, about 100 fish species have migrated from the Red Sea to the Mediterranean Sea (Bentur et al. 2018). Plotosus lineatus was recorded for the first time in the Mediterranean Sea in 2002 (in Israel), as the second introduced catfish species (Golani 2002). Since then, several other occurrences in the Mediterranean followed, with large schools being observed and bycatch being present in fishing nets in Egypt (Temraz and Souissi 2013), Lebanon (Bitar 2013), Syria (Ali et al. 2015), Tunisia (Ounifi-Ben Amor et al. 2016), and Turkey (Bayhan and Ergüden 2022). Despite its relatively slow rate of spread in the area, it has been alleged to displace native fishes through competition (Edelist et al. 2012) and is included in the list of the 100 worst invasive species in the Mediterranean (Streftaris and Zenetos 2006). Plotosus lineatus was assessed as a high-risk marine invasive alien species for the EU and included in the list of invasive alien species of Union concern (the Union list) due to its potential to threaten native ecosystems and human well-being throughout the Mediterranean and within EU marine waters (Galanidi et al. 2019; http://data.europa.eu/eli/reg_impl/2016/1141/oj).

To date, the taxonomic status of P. lineatus remains unclear. This taxonomic clarification obstacle is worrisome since the species is important for both the aquarium trade and human consumption (Situ and Sadovy 2004; Asriyana et al. 2021, 2022). Moreover, it is also an insightful model organism to study osmoregulation, given its uniqueness among teleosts by possessing a dendritic organ (e.g., Lanzing 1967; Malakpour Kolbadinezhad et al. 2018a, b) and ammonia excretion (Malakpour Kolbadinezhad et al. 2020). Additionally, P. lineatus has venomous spines that cause painful injuries, sometimes accompanied by hypertension and tachycardia and a risk of secondary infection (Dogdu et al. 2016; Bentur et al. 2018), which has serious socio-economic effects in the area and, for example, forces shrimp fishermen to change the time and place of fishing (Edelist et al. 2012; Galanidi et al. 2018). Moreover, it has been widely used as a model organism to study the potential use of its venom in biomedical applications (Shiomi et al. 1988; Fahim et al. 1996). Recently, a reference nuclear genome assembly has been released (Shao et al. 2023). Given the complexity of the taxonomic status of Plotosus, a comprehensive phylogenetic study of the various lineages and diversity will have wide impacts on future research avenues of this genus.

Few attempts have been made to clarify the genetic diversity within the species, but some studies have suggested the existence of several evolutionary lineages within P. lineatus (Bariche et al. 2015; Kundu et al. 2019; Goren et al. 2020). Nevertheless, these studies were limited either in geographic or phylogenetic scope. No study has ever included P. lineatus individuals from across its full (native and introduced) distributional range, and using all the available mtDNA Cytochrome c oxidase subunit 1 (COI) sequences, which is a standardized molecular identification system used in fishes (Hebert et al. 2003). Although is a single mtDNA marker, the effectiveness and benefits of barcoding fishes have been demonstrated in flagging previously overlooked species and enabling identifications where traditional methods cannot be applied, or are insufficient (Ward et al. 2009).

In this study, all available Plotosus sp. mitochondrial COI sequences (NCBI and BOLD; n = 204) were analysed, including all P. lineatus from specimens covering the current known distribution range, in an attempt to clarify the diversity of lineages in this species. We discuss several aspects that have added to P. lineatus taxonomic uncertainty. Finally, to prevent the recurrence of the major problems observed, we created a comprehensive list of all information accessible for each sequence, including morphology-based misidentifications and the place where the samples were collected, both of which are important details that are not always (easy) directly related to the sequence resources.

Materials and methods

Phylogenetic analyses. For the phylogenetic analysis, all sequences labelled as Plotosus were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/genbank/) and the Barcode of Life Data System (BOLD) (accessed in January of 2023) [Electronic Supplementary Material (ESM) Table S1]. Redundant entries and sequences with less than 500 bp were manually removed (i.e., sequences KP296155.1 and MH331830.1). Several Gymnotiformes and one representative of each Siluriformes family (according to Betancur-R et al. 2017) were also retrieved from NCBI and used as outgroup (see ESM File S1 for the Accession numbers).

Alignments were produced using MAFFT v7.453 (Katoh and Standley 2013) (parameters: default) and trimmed after with trimAL v.1.2rev59 (Capella-Gutiérrez et al. 2009) (parameters: -gt 0.5). The final alignment consisted of 237 sequences and a total length of 645 bp. The resulting alignment was translated to infer putative indels and unexpected stop codons. The best-fit model of nucleotide substitution evolution and Maximum Likelihood (ML) phylogenetic inference were produced in IQ-TREE v.1.6.12 (Nguyen et al. 2015; Kalyaanamoorthy et al. 2017).

Genetic diversity and haplotype network. For each of the major clades identified in the phylogeny, genetic distances (Kimura 2-parameter distance model, K2P) were assessed using MEGA11 (Kumar et al. 2018). For haplotype network construction, all sequences placed in the P. lineatus cluster (depicted in the phylogeny, see Results) were retrieved from the alignment and trimmed with trimAL v.1.2rev59 (Capella-Gutiérrez et al. 2009) (parameters: -gt 0.95) to a length of 564 bp. The haplotype network was inferred using TCS 2.1 (Clement et al. 2000) implemented through PopART (Leigh and Bryant 2015), considering sequences with the same length (407 bp).

Plotosus lineatus lineage distribution. Based on published and unpublished data, sampling locations for all the sequences used to construct the haplotype network were gathered and assembled (ESM Table S2). The acquired coordinates were loaded into QGis 3.22, whereas for the remaining sequences/haplotypes for which only approximations of places were obtained (e.g., Country and/or Province name), an approximation of location was represented using the same software. The acquired P. lineatus lineages distributions were then depicted using the same haplotype network's colour scheme (and phylogeny).

Results

Plotosus spp. phylogenetic relationships. In the COI alignment used for the phylogenetic inference, no stop codon, insertions or deletions were found in any of the sequences. The ML phylogeny retrieves one highly supported clade, Plotosidae, which includes all Plotosus spp. sequences except for two that cluster with the Nematogenyidae representative, very likely resulting from morphology-based misidentifications (Plotosus sp. UKFBJ138-08 and KF268174.1 in Fig. 1; ESM Table S1).

Fig. 1
figure 1

DOWN: maximum likelihood (ML) phylogenetic tree inferred from the cytochrome c oxidase subunit I (COI) gene fragment for one representative of each Siluriformes family, all Plotosidae sequences available (collapsed for easier visualization) and several Gymnotiformes; Asterisk indicate unknown clustering of two sequences deposited as Plotosus lineatus. For details see ESM Table S1 and ESM File 1. TOP: Uncolapsed Plotosidae Clade. Only bootstrap values > 60% are depicted. TOP-RIGHT: photographs of all available individuals retrieved from the Barcode of Life Data System (BOLD) with reference codes linked to each photo. Plotosus lineatus Lineages I–IX colours follow Figs. 2 and 3. For details see ESM Table S1

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The Plotosidae clade is further divided into two highly supported clades; one containing all the P. lineatus, P. japonicus and P. limbatus sequences, and the other all the remaining available species of Plotosus (Fig. 1). Plotosus lineatus sequences cluster into nine distinct highly supported lineages, here named Lineages I–IX (Fig. 1). The genetic distances among the nine P. lineatus Lineages ranged from 2% to 16% (Table 1). The highest genetic diversity was seen within Lineage VI (Table 1). The minimum genetic distance observed between P. japonicus and P. lineatus Lineages I–III was 3% while a maximum of 14% was observed between P. japonicus and P. lineatus Lineage IX (Table 1).

Table 1 Mean genetic divergence for the COI data set, between and within all the Plotosus spp. Lineages depicted in the phylogeny (Fig. 1)
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Sister to P. lineatus Lineages is a clade containing all P. canius and P. nkunga individuals whose first split corresponds to one highly divergent individual, Plotosus sp., with a minimum of 14% to P. nkunga (Fig. 1; Table 1; ESM Table S1). A unique assembly with the majority of the P. nkunga individuals is sister to five P. canius lineages (P. canius Lineages I–V; Fig. 2; ESM Table S1) and a group with high within-genetic diversity that included several individuals labelled as both species (Fig. 2; ESM Table S1).

Fig. 2
figure 2

Haplotype (TCS) network showing the relationships of all published Plotosus lineatus sequences (ESM Table S2). Circle size is proportional to the observed haplotype frequencies; numbers indicate nucleotide substitutions. Mean genetic divergence (COI) between Lineages (dash lines) is indicated in grey (see Table 1 for details). Plotosus lineatus Lineages I–IX colour scheme follows Figs. 1 and 3. n = Number of individuals; h = haplotypes

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All the available individual photos in BOLD were downloaded and linked to each Lineage in the phylogeny (Fig. 1).

Plotosus lineatus genetic diversity and phylogeographic patterns. The resulting haplotype network allows a more detailed display of spatial patterns according to the distribution of haplotypes (Fig. 2) obtained from the 133 samples of the nine P. lineatus Lineages depicted in the phylogeny. A total of 31 distinct haplotypes were recovered, 15 of which occurred only once with the most frequent haplotype (H5) occurring in 43 individuals across a wide geographical range (ESM Table S2). The geographic mapping of P. lineatus lineages is presented in Fig. 3. We were only unable to link one haplotype (H28; ESM Table S2) with its sampling site. Plotosus lineatus Lineage I displayed a star-like topology, with one high-frequency central haplotype (H5) and several additional haplotypes connected by few-step mutations (Fig. 2). It has the highest number of haplotypes (n = 12) sampled across the widest geographical area (Fig. 3). Haplotype 11 was present in India and also in the Red Sea and the Gulf of Aqaba (Figs. 2, 3; ESM Table S2). Plotosus japonicus single haplotype was placed in an intermediate position, with a minimum of 11 mutations to P. lineatus Lineage I and a minimum of 10 mutations to P. lineatus Lineage II (Fig. 2). Plotosus lineatus Lineage I was further linked by a minimum of 25 mutations to P. limbatus (Fig. 2). Plotosus lineatus Lineages III, IV, VII, VIII and IX were represented by a single haplotype (and are all restricted to a single location each; Figs. 2, 3). Plotosus lineatus Lineage V was only found in the Mediterranean with three haplotypes (Figs. 2, 3; ESM Table S2).

Fig. 3
figure 3

Plotosus lineatus COI Lineages I–IX geographic mapping distribution. Colour scheme follows the phylogeny (Fig. 1) and the haplotype network (Fig. 2). For details see ESM Table S1

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Discussion

Here, we demonstrate that Plotosus spp., and in particular P. lineatus (and P. canius) represent a ‘classic’ case of marine fish cryptic diversity. Cryptic diversity refers to the occurrence of numerous genetically separate populations or species that are morphologically similar (Walker 1964; Struck et al. 2018 supplemental table S4 online for a list of definitions). These populations or species may have diverged from a common ancestor as a result of geographic isolation, reproductive strategies, limited dispersal, habitat adaptation, convergent evolution, or environmental diversity, and may now exhibit distinct behaviours, physiological adaptations, or ecological roles (Fišer et al. 2018). Several species examined in this study share similar morphological features; therefore, they may be mistakenly classified as a single species or lumped into a larger taxonomic group, as is demonstrated here to be the case of striped eel catfish P. lineatus. This may help explain the reason why, despite its long history, the taxonomic status of the P. lineatus species complex remains understudied and uncertain. However, we have uncovered several issues that add complexity to the taxonomic confusion of P. lineatus and therefore, to avoid perpetuating this concern, we have compiled all available information for Plotosus spp. for future use (ESM Table S1).

Lack of adequate data on Plotosus. In our dataset, several incidents of inaccurate data in Plotosus spp. were detected. In some cases, sequences with both BOLD and NCBI entries had different associated manuscripts (e.g., EF607324.1 and EF607323.1 attributed to Zhang 2011 whereas the BOLD IDs, FSCS284-06 and FSCS285-06, are linked to Zhang and Hanner 2012). Others, where the accession number provided has different descriptions (e.g., MF601472 in NCBI is attributed to P. canius whereas in the associated manuscript, Habib et al. 2017, the same accession number is associated with Lepturacanthus savala). In the end, some informed assumptions were made, based on our extensive revision. For example, we have linked sequences EU148553, EU148554 and FJ918911 to the Lakra et al. (2011) information provided in NCBI, although we were unable to find these sequences or any other sequence in the manuscript.

Further aspects add to P. lineatus taxonomic confusion, one being the lack of the exact sampling collection site information. Based on an exhaustive literature review, we detected several situations where this information was missing, i.e., not linked to the analysed individual, neither to the sequence deposited (in either database) nor to the morphological data (e.g., species descriptions). In the majority of the cases, some “wider geographic” information is provided (e.g., Country or Province), although this information is not always directly linked to the data making its discovery challenging. Sometimes, this information is unpublished, others resulted from mistakes [e.g., sequences JF494186.1–JF494188.1 from South Africa in Steinke et al. (2016), but erroneously labelled as being from Lebanon, Mediterranean, in Kundu et al. (2019)]; and sometimes is not available at all (e.g., FJ918911, MZ329579, MW649911 and MW649912). Therefore, we linked all P. lineatus sequences used to their collection locality (ESM Tables S1, S2). We were able to obtain a phylogeographic insight into the nine P. lineatus Lineages (I–IX) for the first time as a result of this valuable data which is covered in more detail below.

Morphology-based misidentifications, sequence similarity search and Plotosus lineatus phylogeny. Lack of adequate data will lead the BLAST searches to ambiguous results. Misidentification of the voucher specimen, contamination during sample processing, inadequate taxonomic identification, or issues with synonyms and syntax could all contribute to this issue. (e.g., Tautz et al. 2003). Morphological confusion between P. lineatus and other (described) species has been reported, e.g., P. lineatus has been mistaken for P. limbatus, frequently seen as the adult of the smaller, striped eel catfish, because both have relatively large eyes and short barbells (Gomon and Taylor 1982). Thus, it is not surprising that our finding of several sequences in both databases (NCBI and BOLD) resulted from morphology-based misidentifications. This problem can mislead the identification of new samples in both databases, due to the sequence similarity search and although previously discussed (e.g., Kundu et al. 2019) has been maintained.

Thus, to avoid the perpetuation of this concern, we compiled all available information for Plotosus spp. for future use (ESM Table S1). The two highly distinct sequences that cluster together in our phylogeny (Unknown species UKFBJ138-08 and KF268174.1 in Fig. 1; ESM Table S1) deposited on NCBI/BOLD as P. lineatus are a clear result of morphology-based misidentifications. On the other hand, the phylogenetic position of the other sequence deposited as Plotosus sp. (MZ606227; Fig. 1; ESM Table S1) does fall within the Plotosidae Clade but is very distantly related to the existent Plotosus sp. sequences available, not allowing for now to draw further comments.

Regarding P. lineatus, we have also found one sequence that was deposited as P. lineatus in both databases (JF952819.1/ABFJ031-06; Fig. 2; ESM Tables S1, S2), even though no morphology-based identification was provided (Zhang and Hanner 2011). This sequence is the same P. japonicus haplotype that was seen in our haplotype network. Another sequence, deposited as P. canius (MN747967.2, unpublished; ESM Table S1) corresponds to haplotype H5 of P. lineatus Lineage I (Fig. 2; ESM Table S2). Finally, three sequences deposited as Plotosus sp. in both databases, correspond to haplotype H30 of P. lineatus Lineage III (Fig. 2; ESM Tables S1, S2). This careful analysis and compilation have also allowed us to increase the number of COI-validated P. lineatus sequences.

Accurate species identification is essential for understanding the nature and extent of marine fish cryptic diversity. By accurately identifying and distinguishing between closely related but genetically distinct species, researchers can improve conservation efforts, understand ecological relationships, and gain insights into the evolutionary history of marine fish communities (e.g., see Ward et al. 2009, for a review). The effectiveness of the barcoding system has been repeatedly demonstrated by the identification of marine fish species with a 2% difference in the DNA barcode as a divergence threshold and a standard cut-off value for species delimitation in fishes (Mabragaña et al. 2011). The present COI Plotosus spp. phylogeny reveals the extent of the hidden complexity of cryptic diversity in species of Plotosus, e.g., nine P. lineatus lineages, ranging from 2% to 15% (Table 1) with two species, P. japonicus and P. limbatus clustering in between them; five P. canius lineages and one group of both species mixed (Fig. 1). The phylogenetic relationships among the nine revealed P. lineatus lineages are generally well established, except for the position of P. lineatus Lineage VII and P. limbatus. This is most likely due to missing taxa/genetic information, and while we analysed 132 P. lineatus spp. individuals, P. lineatus Lineage VII only has one sequence available. Additionally, two other P. lineatus Lineages, i.e., P. lineatus Lineages IV and VIII are also only composed of one sequence each and five P. lineatus Lineages are confined to single geographic areas (Figs. 2, 3; ESM Table S2).

Altogether, our phylogenetic results highlight the crucial need and the urgency to sequence all species of Plotosus described since at the moment only a small fraction of these have molecular data. There is a clear need to increase the sampling within all the identified P. lineatus Lineages to clarify their genetic diversity, followed by in-depth taxonomic analysis, which might allow revisions to describe them as species.

Unravelling the complexity of cryptic diversity and phylogeographic patterns of Plotosus lineatus. Plotosus lineatus was described from the East Indian Ocean in the Indo-Pacific, a region known to be a hotspot of marine fish cryptic diversity, with numerous examples of genetically distinct populations or species that are difficult to distinguish based on their external appearance (e.g., Lutjanus kasmira complex, Miller and Cribb 2007; surgeonfishes (Acanthuridae), DiBattista et al. 2016). The assessment of genetic diversity within P. lineatus has been previously attempted (e.g., Bariche et al. 2015; Kundu et al. 2019; Goren et al. 2020). However, without including individuals from across P. lineatus wide distributional range (natural and introduced/invasive) and using all available mtDNA COI sequences linked to their geographic sampling locations, those results were inconclusive. Here, the phylogeographic patterns of all P. lineatus lineages are provided (Figs. 2, 3). Only P. lineatus and P. canius are known to have cosmopolitan distributions, while the other species have narrow distribution ranges. However, in this study, we show that within P. lineatus, several monophyletic and highly divergent lineages seem to be restricted to small ranges, e.g., P. lineatus Lineages VII, VIII and VIX (Fig. 3; ESM Table S1). Some caution is needed when extrapolating these findings since P. lineatus Lineages VII and VIII are only represented by single sequences and, although the haplotype of P. lineatus Lineage IX has been retrieved in six individuals, they were all, very likely, caught at very close locations (ESM Table S2).

Nevertheless, clear and interesting phylogeographic patterns can be observed. Plotosus lineatus Lineage I is the most geographically widespread, with the highest number of haplotypes and individuals sequenced (Figs. 2, 3; ESM Table S2). This lineage exhibits a star-like topology, with one high-frequency central haplotype and several additional haplotypes connected by few-step mutations, a pattern of connectivity (between haplotypes) that provides support for a recent population expansion. Two haplotypes are geographically widespread; H5, the highly frequent central haplotype, that is present from Indonesia in the East, to India in the West; and H11, sequenced in only seven individuals but present from India in the East, to the Red Sea in the West (Fig. 3; ESM Table S2). Some private haplotypes are also found in P. lineatus Lineage I, e.g., H31 is present only in Seychelles (Fig. 3; ESM Table S2). Plotosus lineatus Lineage II is formed by two haplotypes present in Eastern South Africa, Lineage III seems to be restricted to Madagascar, and Lineage IV is composed of a single sequence from the Réunion Island (Fig. 3; ESM Table S2).

Plotosus lineatus Lineage V is the most puzzling. The geographic mapping of P. lineatus lineages (Fig. 3) reveals that P. lineatus Lineage I is present in the Red Sea and Gulf of Aqaba, so one might expect this lineage to be present in the Mediterranean basin. However, only three haplotypes (retrieved from 29 individuals) of P. lineatus Lineage V are present in the Mediterranean Basin. The first record of P. lineatus in the Mediterranean was in 2002, in Israel (Golani 2002), and since then the species has been recorded in several other coastal locations of the basin, i.e., Lebanon, Syria, Turkey, Egypt, Tunisia and Iran (Bitar 2013; Temraz and Souissi 2013; Ali et al. 2015; Ketabi and Jamily 2016; Ounifi-Ben Amor et al. 2016; Bayhan and Ergüden 2022). Unfortunately, none of these records includes molecular data. Our results reveal P. lineatus Lineage V with a minimum genetic distance of 4% to Lineage VI and a maximum of 13% to Lineage IX (Table 1). Lineage VI is present from Vietnam in the East, to Saudi Arabia Gulf in the West, with no records found in the Red Sea (Fig. 3; ESM Table S2). Several hypotheses could explain the exclusive presence of P. lineatus Lineage V in the Mediterranean. First, this lineage could be endemic in the Mediterranean. This hypothesis seems unlikely because striped eel-catfish is a very charismatic species, morphologically very easy to identify and it has only been recorded for the first time in the Mediterranean in 2002. A more likely explanation would be that P. lineatus Lineage V migrated from the Red Sea to the Mediterranean Sea, through the Suez Canal, a pattern described for several other fish species (Bentur et al. 2018). However, no other haplotypes were found outside the Mediterranean and this lineage has a minimum genetic distance of 9% to Lineage I (Table 1), the only lineage found in the Red Sea so far (Fig. 3). There should be yet, other hypotheses to consider. For example, the presence of P. lineatus Lineage V in the Mediterranean could result from the release of striped eel catfish individuals from aquarium-imported and/or ship-ballast water, whose natural geographic range has not yet been sampled. This species is commercially important for both the aquarium trade and human consumption (Situ and Sadovy 2004; Scandol and Rowling 2007; Asriyana et al. 2021, 2022) and the “escaped/released” individuals would have been able to survive and establish in the Mediterranean. However, an important question remains unanswered: is this P. lineatus Lineage V the one that has been continuously recorded in the Mediterranean since 2002? Is it the genetic pool that has been assessed as a high-risk marine invasive alien species for the EU and included in the list of invasive alien species of EU concern? Or is there another P. lineatus lineage, most likely P. lineatus Lineage I, already present in the Red Sea, in the Mediterranean? There is, therefore, an urgent need to investigate the complete molecular diversity of Plotosus spp. in the Mediterranean.

Finally, our extensive compilation and haplotype geographic mapping revealed the existence of several pairs of species/Lineages living in sympatry, e.g., P. limbatus and P. lineatus Lineage I, P. lineatus Lineage I and P. lineatus Lineage VI (at two different locations), P. nkunga and P. canius, P. canius and P. lineatus Lineage I (Fig. 3; ESM Tables S1, S2). These findings raise an array of additional questions, which remain unanswered for the time being.

Final remarks. Overall, the Plotosus genus, and P. lineatus in particular, represents a fascinating example of marine fish cryptic diversity in the Indo-Pacific region, with numerous genetically distinct but morphologically similar species that are extremely difficult to distinguish based on external characteristics alone. The current study identified numerous significant roadblocks to the clarification of P. lineatus taxonomy and revealed the existence of nine P. lineatus Lineages for the first time, along with their known geographic range. We highlighted the urgent need for taxonomic revisions to describe them as species.