A phylogenetic assessment of nematodes (Oxyuroidea: Pharyngodonidae) infecting Moroccan lizards

Defining, cataloguing, mapping and preserving biodiversity is generally accepted to be one of the key challenges for the 21^st Century. The Kingdom of Morocco possesses the richest and most varied herpetofauna of the Maghreb and the western Mediterranean, is characterized by high richness of reptiles, endemism and European relict species (del Mármol et al., 2019). Phylogenetic analyses performed over recent decades have identified notable evolutionary lineages in the Moroccan herpetofauna, several of which represent new (cryptic) species or species complexes (e.g. Barata et al., 2012; Salvi et al., 2018), demonstrating the value of incorporating molecular tools into diversity assessments. Still, despite the well-known biodiversity of reptiles, parasite diversity associated with these hosts remains poorly known. Part of the problem in estimating parasite diversity has historically been that, especially for endoparasites, identification based on morphological characters and life-cycle traits is often difficult. Parasites often present a simplified morphology (Jorge et al., 2011), and while DNA sequencing approaches should help overcome these problems, molecular studies have lagged behind those of free-ranging organisms (Criscione et al., 2005). However, application of molecular tools is starting to show that some parasites currently considered as a single species actually consist of genetically different lineages or cryptic species (e.g. Jorge et al., 2012, 2013a).

Reptiles are parasitized by various helminth species, typically occurring in depauperate communities (Aho, 1990), possibly due to characteristics of the hosts such as the simplicity of the alimentary canal, their low vagility, a nonspecialized diet, and characteristics of the parasites such as direct life cycles (Roca & Hornero, 1994). Various studies have suggested that the typical helminth infection pattern in reptiles is that few species occur frequently, while many species are rare (Birlik et al., 2015). In studies of reptiles from the Iberian Peninsula and the North of Africa, helminth fauna of assessed lizards was poor, and mainly composed by members of the family Pharyngodonidae that are often detected in insectivorous reptiles (Chabaud & Golvan, 1957; Ibrahim et al., 2005; Carretero et al., 2011; Roca et al., 2020). These are usually identified on the basis of male morphology, since females are generally similar among species (Jorge et al., 2014). Although this family includes 21 genera, only a few of these are typically found in insectivorous lizards, with Spauligodon the most commonly reported (Roca et al., 2020).

Recent nematode surveys of reptiles in the Mediterranean region resulted in several new host records and descriptions of new species (Jorge et al., 2011, 2013a, 2014). Despite the high diversity of reptiles in Morocco, parasites from these hosts remain poorly studied, with a few incidental assessments as part of surveys at the Canary Islands (e.g. Jorge et al., 2011, 2018). In this sense, no survey including molecular data has been performed for nematodes of reptiles from Morocco. Indeed, with one or two exceptions (e.g. Carretero et al., 2011), there have been few molecular studies in nematodes from reptiles across the whole of the Maghreb. The aim of this study was to fill this gap, by assessing diversity of Pharyngodonidae nematodes from six lizard species from Morocco. By sequencing exemplars for two molecular markers (fragments of the 18S rRNA and Cytochrome Oxidase 1 gene), we aim to place these in a phylogenetic framework, to potentially identify cryptic forms, and to examine patterns of host-specificity.

The research related to animal handling complied with all the relevant national regulations and institutional policies for the care and use of animals. The authorization for sampling of wild animals was granted by Cadi Ayyad University, Marrakech, Morocco. A field-work permit was issued by “Haut-Commissariat aux Eaux et Forêts et à la Lutte Contre la Désertification (HCEFLCD)”.

Three different genera were identified using microscopy. Identification of Spauligodon was based on 125 adult specimens (81 females and 44 males). These were identified as small cylindrical nematodes, sexually dimorphic, with males approximately one quarter the length of female. Lateral alae present in male, absent in female. Anterior extremity tapered, mouth surrounded by 3 small lips, each with shallow midline indentation; dorsal lip with 2 sessile papillae, ventrolateral lips with 2 sessile papillae and 1 prominent lateral amphid. Identification of Paraphyngodon was based on 6 adult specimens (4 females and 2 males). These were identified as small nematodes, whitish in colour, males smaller than females, sexual dimorphism moderate. Body fusiform, cuticle with distinct transverse striations beginning just behind the cephalic extremity and continuing to the anus. In males, lateral alae well developed, initiating anteriorly somewhere at the level of bulbus and extending posteriorly to last third of body. Oral opening subtriangular, surrounded by three lips, in female separated into six parts. Identification of Thelandros was based on 104 adult specimens (80 females and 24 males). These were identified as robust nematodes with prominent annulations beginning just behind cephalic extremity and continuing to anus. Cuticle with distinct longitudinal striations approximately 4 apart. Moderate sexual dimorphism. Triangular oral opening surrounded by 3 bilobed lips, 1 small pedunculate amphid on each ventrolateral lobe. Lateral alae absent. Males without caudal alae; caudal filament terminal, directed posteriorly. Females with vulva post equatorial. We followed Hering-Hagenbeck et al. (2002), considering Parapharyngodon distinct from Thelandros for the following reasons: Males of Thelandros have a genital cone, pendulant papillae outside the genital cone, lateral alae are absent, and the tail is terminal and directed posteriorly; males of Parapharyngodon lack a genital cone, mammiliform papillae surround a more-or-less terminal anus, lateral alae are present, and the tail is subterminal and directed dorsally.

Following previous descriptions (Lucker, 1952; Skrjabin et al., 1967; Ashour et al., 1992; Amer & Bursey, 2008; Mašová et al., 2009; Pereira et al., 2017; Er-Rguibi et al., 2022) samples from hosts of the genus Chalcides were identified as Thelandros alatus or Parapharyngodon micipsae. All other samples were identified as belonging to the genus Spauligodon. Of these, only Spauligodon auziensis from Tarentola mauritanica hosts could be identified to the species level. Sixteen of 17 nematode specimens had their 18S rRNA successfully sequenced. Based on the estimate of phylogeny derived from 18S rRNA sequences (Fig. 2), the species of Spauligodon formed a highly distinct clade. Within this, some forms could be distinguished, including S. nicolauensis from the Cape Verde islands, S. auziensis from the hosts T. mauritanica, and a group from hosts of the genus Chalcides (C. montanus, C. ocellatus and C. polylepis). However, several specimens from the geckos Quedenfeldtia moerens and Quedenfedtia trachyblepharus were identical for this region, and also identical to sequences from GenBank of both Spauligodon saxicolae and Spauligodon carbonelli. The sample identified as P. micipsae was sister taxa to P. micipsae from Gallotia caesaris from the Canary Islands. Thelandros was not a monophyletic group, although some estimates of relationships were poorly supported. Considering the CO1 marker, only 4 samples (all Spauligodon) were successfully amplified. Based on this more variable marker (Fig. 3) two samples from Tarentola mauritania hosts were apparently S. auziensis, related to other specimens from this host species. A specimen from Quedenfeldtia was distinct from all comparative sequences, but with poorly supported relationships. The remaining sample from a Chalcides polylepis host formed a distinct lineage with another Spauligodon sp. from a Chalcides sp. host, also from Morocco. These together were sister taxa to S. nicolauensis, from the Cape Verde islands.

In principle, the geographic distribution of parasite diversity is predicted to match that of host diversity (Jorge & Poulin, 2018). Under this expectation, Morocco with its high diversity of reptile species should also harbor extensive nematode diversity within these hosts. However, this diversity remains essentially unassessed. In this study, we made a preliminary assessment of nematodes in pellets from six reptile host species. While it is known that there is a significantly lower detectability of nematodes from pellets compared to studies of intestines (Jorge et al., 2013b), this noninvasive approach can be used to give some baseline data on the parasites occurring in reptiles in this region.

As expected, the nematodes identified all belonged to three genera, Parapharyngodon, Thelandros and Spauligodon, which are typically identified within insectivorous reptiles (e.g. Mašová et al., 2009; Carretero et al., 2011; De Sousa et al., 2018). Exact relationships between these, and even the monophyly of these genera, has been widely debated (e.g. De Sousa et al., 2018; Pereira et al., 2018).

Fig. 2

Fig. 3

Using universal 18S rRNA primers, we were able to amplify and sequence a region of this gene for all but one of our samples. The specimens of Spauligodon from Morocco fell into three distinct lineages, reflecting the reptile hosts they were recovered from. Seven specimens of Spauligodon from the geckos Quedenfeldtia moerens and Quedenfedtia trachyblepharus were identical for the 18S rRNA region, and also identical to sequences from Gen-Bank of Spauligodon saxicolae and Spauligodon carbonelli, that had been recovered from lacertid lizards in the Iberian Peninsula and Turkey (Jorge et al., 2011, 2014). Three samples of Spauligodon from the gecko Tarentola mauritanica were also identical, to Spauligodon auziensis previously recovered from T. mauritanica from central Morocco (Jorge et al., 2011, 2018). The two samples of Spauligodon from the skinks Chalcides polylepis and Chalcides montanus were related to Spauligodon sp., also from skinks Chalcides ocellatus, from Sardinia (Jorge et al., 2018).

While the distinction of Spauligodon was evident in our analysis of 18S rRNA, the separation of Parapharyngodon was more complex. Although molecular data does support distinction of this genus relative to Thelandros, many species have not been included in analyses. Pereira et al. (2018), using an integrative approach, proposed a clear separation between Thelandros and Parapharyngodon, but included only one representative species of Thelandros in the molecular phylogeny. De Sousa et al. (2018) also indicated that Parapharyngodon was distinct from Thelandros collected from the Canary Islands and Cape Verde islands, but that from Gallotia lizards previously considered Thelandros galloti should be reassigned to Parapharyngodon, as P. galloti. They further suggested that preliminary morphological assessments supported this reassignment, for example since this species lacks caudal alae and has long and wide lateral alae, both typical characteristics of Parapharyngodon (Astasio-Arbiza et al., 1988). On the other hand, P. galloti males had wider alae and longer oesophagus than representatives of Parapharyngodon, highlighting the difficulties of assigning these species to the appropriate genus without molecular data. Abdel-Ghaffar et al. (2020) recently described a new species as Thelandros chalcidiae from Chalcides ocellatus in Egypt. Based on 28S rRNA sequence data, they showed it was closely related to P. galloti (still considered as a member of Thelandros) and Parapharyngodon micipsae. In our analysis, we identified a Parapharyngodon sp. from Chalcides mionecton. Based on morphological aspects, it was considered to be P. micipsae, a species found in many different reptile hosts including lacertids (Martin & Roca, 2004), skinks (Ibrahim et al., 2005), geckos (Mašová et al., 2009) and agamas (Elmahy & Harras, 2019). Following the 18S rRNA analysis, this was closely related to P. micipsae from Gallotia lizards from the Canary Islands (De Sousa et al., 2018), and then P. galloti and P. echinatus. That T. chalcidiae is also closely related to P. galloti shows how difficult it is to assign species to the different genera based on morphological characters. Highlighting the diversity of nematodes found in Chalcides skinks, in another C. mionecton and Chalcides polylepis, representatives of Thelandros were identified, which we considered as T. alatus based on morphological characters, the type species of Thelandros. Based on the 18S rRNA analysis, these were distinct from Parapharynogodon, but also from Thelandros tinerfensis from Tarentola gomerensis hosts in the Canary Islands, thus making Thelandros paraphyletic. Overall, representatives of Spauligodon, Thelandros and Parapharyngodon were identified in Chalcides skinks in Morocco, but which species of Spauligodon is still unclear.

Although we only obtained 4 sequences from Spauligodon species using the CO1 primers, the estimate of phylogenetic relationships derived from this gene is still highly informative (Fig. 3). Unlike with the more conservative 18S rRNA sequences, all Spauligodon species could be distinguished with this marker, and intra-specific variation identified. Two samples from T. mauritanica again grouped with samples identified as S. auziensis. Intraspecific variation within S. auziensis was high, up to 12.6 % uncorrected sequence divergence, although all the samples were collected from the same host species. One specimen from Quedenfeldtia moerens was highly distinct from all other comparative sequences, but relationships of this from were poorly supported. Specimens of Spauligodon were recently identified in Quedenfedtia trachyblepharus (Er-Rguibi et al., 2021), and the results of this study demonstrate that they are genetically distinct from other known Spauligodon species. The fourth specimen, from C. polylepis, was found to be sister taxa to another Spauligodon sp., also from a Chalcides sp., and these in turn were related to Spauligodon nicolauensis from the Cape Verde islands.

To conclude, our new data clarifies several aspects regarding the presence of nematodes in reptiles from Morocco. Each lineage of parasite is quite specific to a related group of hosts, with S. auziensis in Tarentola mauritanica, another lineage in both species of Quedenfeldtia, another only in species of Chalcides, and another previously identified lineage from Podarcis vaucheri. While the universal 18S primers employed successfully amplified almost all samples, variation was too low to distinguish between some accepted species, while the CO1 region showed much greater variation. While classification of species to Spauligodon is relatively straightforward, some species considered as Thelandros may be better reassigned to Parapharyngodon, highlighting the difficulty of distinguishing between these genera using morphological characters. Other markers, such as 28S rRNA, and improved primers for amplifying CO1 across additional species, may help resolve some of these taxonomic issues. Several lineages of Spauligodon, particularly those from Chalcides and possibly also from Quedenfeldtia hosts form divergent lineages and probably warrant consideration as full species, pending detailed morphological assessments. This highlights the undescribed diversity within Morocco and the Maghreb region, and the need for additional surveys of this poorly known parasite fauna.