In this study, we aim to compare haplotypes of monogenean parasites from introduced populations of Nile tilapia from three regions of the Congo Basin and one from Madagascar. In addition, we generated haplotypes of the parasites from a native population of Nile tilapia in Burundi. We expect the introduced parasite populations from Congo and Madagascar to be low in genetic variation. Additionally, if they share many of the same haplotypes they might have a common introduction origin. Our second goal is to evaluate the species status of co-introduced monogeneans of Nile tilapia through four molecular markers. Lastly, this study aims to add sequences of Congolese native and introduced species (Table 1, addendum 1) and evaluate the position of these previously unsequenced species in phylogenetic analyses.
Co-introduced parasites of Nile tilapia
From the COI haplotype networks it can be inferred that the variation within countries is sometimes higher than between countries. For example, C. thurstonae specimens from Upper Congo and Madagascar are identical but they are different from specimens collected in the Lower and Middle Congo (Fig. 2b). This lack of isolation by distance (IBD) typically reflects recent introduction events, which blurs geographic signals (Hayward et al. 2001). For example, Gyrodactylus anguillae Ergens, 1960 had identical rDNA sequences (ITS-1, 5.8S, ITS-2) in North America, Europe and Australia as a result of live eel trade that started forty to fifty years ago (Hayward et al. 2001). Similarly, Gyrodactylus cichlidarum Paperna, 1968 was co-introduced into Mexico with Nile tilapia after fish introductions started in the 1940s (García-Vásquez et al. 2017). The ITS-1 sequences of G. cichlidarum specimens that spilled over to Mexican poeciliids were almost completely identical to G. cichlidarum from Nile tilapia in Ghana (García-Vásquez et al. 2017). Our results, therefore, strongly point to an identical or geographically similar source of introduction of Nile tilapia in the Upper Congo and Madagascar, and a different source (or sources) for the other Congolese regions.
The high haplotype diversity of C. sclerosus within the Upper Congo (Fig. 2a) can partly be explained by the sampling bias (relatively more specimens were sequenced from this locality), but it also strongly suggests that multiple introductions have taken place in this area, from different geographic source populations. Indeed, the variation is higher compared to that of the population of C. sclerosus on native Nile tilapia in Burundi. Aquaculture of Nile tilapia in the DRC started in the Lubumbashi area, Upper Congo, in the late 1940s (Micha 2013). However, there has also been a period of very low aquaculture activity until 1996 (Toguyeni 2004) and it is not known whether Nile tilapia or its parasites from before 1996 still persist in the basin. In any case, our results refute our initial hypothesis that introduced parasite populations suffer bottlenecks. Similar scenarios have been described for other biological invasions, where introduced populations could maintain a high diversity because of multiple introductions from different source populations (Kolbe et al., 2004; Genton et al., 2005), sometimes followed by intraspecific hybridization (Rosenthal et al., 2008).
Finally, the C. tilapiae haplotypes shared by the Lower and Middle Congo suggest that natural gene flow is possible at this scale, or it could point to a shared introduction source (Fig. 2c).
Marker performance and barcoding gap
Based on our histograms (Fig. 4), we find a significant (P<0.05) barcoding gap for COI at 15% (Fig. 4a) but none for 28S, 18S or ITS-1 (Fig. 4b–d). Visually there is a second gap between 7–11% for COI (Fig. 4a) but this was not found significant by ASAP. It should be noted that our fragment of the COI gene covers just less than a quarter of the total COI gene and constitutes the most variable part (unpublished data). Additionally, the COI dataset itself was the smallest of the four markers because hardly any references were available on Genbank and the amplification success of COI was lower than that of the rDNA markers. Whether 15% is representative for Cichlidogyrus/Scutogyrus should be investigated in the future by including more species.
As for the other markers we can find some clues in the literature to a possible barcoding gap. Within Cichlidogyrus, Rahmouni et al. (2021) found intraspecific variation of ITS-1 in C. nshomboi Muterezi Bukinga, Vanhove, Van Steenberge and Pariselle 2012, up to 1.1% and interspecific variation starting at 3.5%. The 18S sequences of C. nshomboi and Cichlidogyrus casuarinus Pariselle, Muterezi Bukinga, Van Steenberge, Vanhove 2015, were identical and the 28S sequences differed 0.13% between the species. COI intraspecific variation within C. nshomboi amounted to 12.2%. This distance for COI roughly corresponds with what we find in our dataset, but the observed distances for the three rDNA fragments are higher in our study. Representatives of Trinigyrus (Dactylogyridae: Monogenea), which infect siluriforms, have interspecific variation <6% for COI and around 1% for 28S (Franceschini et al. 2020). In Dactylogyrus (Dactylogyridae: Monogenea), which infect European cyprinids, the cut-off value was set at 1.4% for a fragment consisting of partial 18S, complete ITS-1 and partial 5.8S (Šimková et al. 2004). Rahmouni et al. (2017b) found 1% for 28S; 0.4% for 18S and 4.3% for ITS-1 of Dactylogyrus parasitizing North-African congeneric cyprinids. From all these we learn that the barcoding gap for 28S may be around 1% and for 18S below 1%. For ITS-1, this is likely higher than 1% but probably lower than the 15%, which we found for COI of the included species of Cichlidogyrus.
Furthermore, 28S sequences can be identical over large geographic distances (C. thurstonae, C. sclerosus and C. falcifer) between introduced and native populations (C. tilapiae) and even between species (C. berradae and C. yanni; Scutogyrus gravivaginus (Paperna & Thurston 1969), S. bailloni Pariselle & Euzet 1995 and S. longicornis (Paperna & Thurston, 1969)) (Addendum 4). However, whether 28S can be identical between species of Cichlidogyrus/Scutogyrus is uncertain as the obtained references from Genbank can be morphologically misidentified. New sequences of C. berradae, C. yanni, S. gravivaginus, S. longicornis and S. bailloni are needed to verify this. Conversely, rDNA fragments can appear conserved in other closely related monogeneans. Kmentová et al. 2016a found identical 28S and 18S+ITS-1 sequences of Cichlidogyrus casuarinus from Lake Tanganyika from different hosts (Bathybates and Hemibates Regan 1920), although intraspecific morphological variation was observed. The COI fragments, on the other hand, were highly differentiated, with distances between haplotypes reaching 4.7% (Kmentová et al. 2016a). For Kapentagyrus tanganicanus Kmentová, Gelnar and Vanhove 2018 infecting Lake Tanganyika sardines, morphological intraspecific variation was found based on host species (phenotypic adaptation for attachment), COI fragment and possibly seasonality, but this was not reflected in the 28S or 18S+ITS-1 sequences (Kmentová et al. 2018). Benovics et al. (2017) observed identical 18S+ITS-1 sequences between Dactylogyrus vastator Nybelin 1924, from the Po River, Italy and Šuica River, Bosnia and Herzegovina. Marchiori et al. (2015) found identical 18S sequences for different, but closely related species of Ligophorus infecting Mugilidae in Brazil (Dactylogyridae: Monogenea), whilst 28S differed 0.2% and ITS-1 0.4%. In conclusion, rDNA sequences might be conserved in conspecific monogeneans over large geographic distances and between conspecifics from different host species also, but it is unlikely for different species to have identical rDNA sequences. Therefore, there is potential for the rDNA fragments to be used for species delineation in Cichlidogyrus/Scutogyrus, as shown for Gyrodactylus spp. (Matejusová et al. 2001, Mendoza-Palmero et al. 2019) and Dactylogyrus spp. (Benovics et al. 2018). However, at this time, we need to include other methods such as bPTP to interpret results from the rDNA fragments about species status. What the rDNA fragments are good at right now are constructing higher-level phylogenies and the COI-fragment is suited for population level studies and species delineation.
Phylogenetic relationships among native and introduced tilapia parasites and evaluation of species status
Previous phylogenetic studies showed that Scutogyrus forms a monophyletic group within Cichlidogyrus, rendering Cichlidogyrus paraphyletic (Pouyaud et al. 2006; Wu et al. 2007; Mendlová et al. 2012; Mendlová and Šimková 2014). Representatives of Cichlidogyrus formed several well-supported monophyletic groups, but the relation between some of these groups were unresolved (Pouyaud et al. 2006; Wu et al. 2007; Mendlová et al. 2012; Mendlová and Šimková 2014). We focus on and add species from the Congo Basin, which were until now sparsely represented in phylogenetic studies of Cichlidogyrus. Our results largely correspond with the previously published analyses on Cichlidogyrus/Scutogyrus (Pouyaud et al. 2006; Wu et al. 2007; Mendlová et al. 2012; Mendlová and Šimková 2014).
Cichlidogyrus halli forms a species complex (see Jorissen et al. 2018a) supported by molecular data in the present study. ASAP suggests the “halli” group to consist of at least three species; firstly, the native specimen from Burundi, secondly the native specimens from Mweru tilapia from Upper Congo together with the specimen from the O. niloticus x mweruensis hybrid from Upper Congo, and thirdly all introduced specimens of C. halli. The genetic distances within the “halli” group are indeed large (2.1% for 28S; 3.2% for 18S; 10.1% for ITS-1 and 36% for COI). This variation is higher than all other intra/interspecific boundaries stated above. The bPTP method is inconclusive for the rDNA markers, where C. halli is divided in six species, including morphotype 2 and C. cf. halli ‘Burundi’, but the support for these divisions is very low. The divisions in COI are better supported and correspond with ASAP.
Morphotype 2 of C. halli (sensu Jorissen et al. 2018a) from Upper Congo, as drawn and discussed by Jorissen et al. (2018a), corresponds in locality and host species with C. halli ex O. mweruensis on the tree (Fig. 3). Therefore, we suggest that morphotype 2 should be elevated to species level. Similarly, the specimens of C. cf. halli ‘Burundi’ from Lake Tanganyika, Burundi belong to a third species within C. halli, as suggested by ASAP. From the same Burundese population we found a specimen of C. cf. halli ‘Burundi’ with elongated and thickened hooklets pair I compared to C. halli (Fig. 5). However, we strongly feel that the species delineation within the “halli” group should be based on morphology and genetics together. Therefore, new morphological material is needed to resolve this. For species within Cichlidogyrus/Scutogyrus, the genital sclerites are important for species identification (see diagnosis in Pariselle & Euzet 2009). Therefore, for our study, we decided to only keep the body part with the genital sclerites and use the body part with the haptor for genetic analysis (Jorissen et al. 2018a). However, recent work on Kapentagyrus and Cichlidogyrus shows that closely related species might first diverge in haptor morphology before genital sclerites (Messu Mandeng et al. 2015, Kmentová et al. 2016a). This implicates the evolution of these parasites is related strongly to microhabitat (attachment site) and host species (Messu Mandeng et al. 2015, Gobbin et al. 2020). We conclude that morphological features of the haptor are important in this complex for species delimitation.
Cichlidogyrus zambezensis and Cichlidogyrus papernastrema together form a clade. However, both species belong to different groups within the genus based on the morphology of the haptoral hooklets. Cichlidogyrus zambezensis has seven pairs of small hooklets (group A sensu Vignon et al. 2011), whilst in C. papernastrema the first pair is thick and elongated (group B sensu Vignon et al. 2011). Pariselle and Euzet (2003) suggested a division of species of Cichlidogyrus in three groups based on the morphology of haptoral hooklets (uncinuli in the source). Additionally, Vignon et al. (2011) found a high congruence between these morphological groups and the molecular phylogeny, meaning that hooklet morphology is phylogenetically constrained. However, the well-supported clade including C. papernastrema and C. zambezensis has a representative of group A and group B. This suggests this group division might not be supported by phylogenies. Pariselle and Euzet (2003) and Vignon et al. (2011) included a subset of species in their analyses. Vignon et al. (2011) even omitted C. arthracanthus from their analysis because it did not fit any of the three groupings. In conclusion, this could mean that firstly, haptor morphology is not as phylogenetically constrained as previously thought (see the ‘halli’ group above) and secondly, that the division in three groupings - whilst useful for morphological identification, see Pariselle and Euzet 2009 – does not cover the morphological evolution of the haptor within Cichlidogyrus fully.
Both C. papernastrema and C. zambezensis have a copulatory tube with a bulbous thickening in the middle and this could be a synapomorphy for the “papernastrema” group instead of characters of haptor morphology. Other species with a bulbous thickening of the copulatory tube and thus possibly belonging to this group are Cichlidogyrus halinus Paperna 1969, Cichlidogyrus sanjeani Pariselle and Euzet 1997, Cichlidogyrus philander Douëllou 1993, Cichlidogyrus bulbophallus Geraerts and Muterezi Bukinga 2020, Cichlidogyrus pseudozambezensis Geraerts and Muterezi Bukinga 2020 and Cichlidogyrus ranula Geraerts and Muterezi Bukinga 2020. Within these candidate species are several representatives from Haplochromine cichlids and others from southern Africa. Cichlidogyrus zambezensis is monophyletic, but we observe variation between specimens of different host species. Douëllou (1993) reports intraspecific morphological variation between different hosts in C. zambezensis. Cichlidogyrus zambezensis is known from four cichlid hosts, belonging to three cichlid lineages (Douëllou 1993, Vanhove et al. 2013, Jorissen et al. 2018a). Additionally, the bPTP analysis of COI splits our samples of C. zambezensis and the reference as different species. Therefore, C. zambezensis is in need of further study and might consist of multiple species. The monophyly of C. papernastrema is not supported. Even more, the genetic distance between the two specimens of C. papernastrema is larger than between this species and C. zambezensis and above 1% for 28S and 15% for COI. Therefore, it is likely that both specimens belong to different species. Jorissen et al. (2018) redescribed C. papernastrema and noted large variation in thickness of the copulatory tube between specimens. It would be worthwhile to check whether this variation is a good diagnostic character to delineate species in tandem with genetic distances.
In the “tiberianus” group, C. tiberianus from Bangweulu-Mweru does not cluster with the reference sequence from Senegal (Mendlová et al. 2012). This species infects representatives of Coptodon ranging from Senegal to Zimbabwe (Douëllou 1993, Pariselle and Euzet 1995, 1996, 2009, Mendlová et al. 2012, Jorissen et al. 2018a). This is a native range of over 7000 km, including different ichthyographic provinces and bassins and it is, therefore, plausible that C. tiberianus might consist of multiple species within this range. Additionally, the genetic distances between C. tiberianus of Senegal and Upper Congo is above 1% for 28S which also point to multiple species; however the distance in 18S is well below 1%. Fannes et al. (2017) used SEM to investigate the sclerotized parts of C. dossoui and C. tiberianus from Upper Congo because both species are morphologically quite similar and share hosts. The COI genetic distances are smaller between C. tiberianus and C. dossoui from Upper Congo than within C. tiberianus. Therefore, it is not surprising that C. dossoui from Bangweulu-Mweru appears as the sister species to C. tiberianus from Bangweulu-Mweru. Cichlidogyrus tiberianus requires a species status re-evaluation backed by genetic data from across its native range and different host species.
Furthermore, in the “tiberianus” group, C. ergensi is situated within C. thurstonae, but with low support (53 posterior probability and not supported in the ML analysis), thus we do not make inferences to this result. All species in the “tiberianus” group belong to group C based on the morphology of the haptoral hooklets (Pariselle and Euzet 2003, Vignon et al. 2011), except for C. arthracanthus and C. sp. 2, which fall outside of the classification in three main groups. Here again, the division by Pariselle and Euzet (2003) is not completely supported.
Cichlidogyrus cirratus was found to be monophyletic (100 posterior probability and 90 bootstrap support value, Fig. 3). However, the branch lengths within C. cirratus are much longer than for example between the different species of Scutogyrus. The genetic distance between C. cirratus from Bangweulu-Mweru and Senegal is 0.5% 18S, 9.8% ITS-1 and 2.8% for 28S (Addenda 2, 4, 5). This indicates that our samples might represent two separate species. It is also debated whether C. cirratus and C. mbirizei Muterezi Bukinga, Vanhove, Van Steenberge, Pariselle, 2012, are conspecific (Zhang et al. 2019). Scanning electron microscopy revealed that the distinguishing characters between C. cirratus and C. mbirizei (Muterezi Bukinga et al. 2012) on specimens of C. cirratus from China (introduced) could be transformed by turning the specimens over (Zhang et al. 2019). In conclusion, we deem it likely that C. cirratus and possibly C. mbirizei consist of multiple species and that this should be investigated further genetically. Subsequently, an evaluation of the morphological characters within C. cirratus and C. mbirizei is needed.
In Cichlidogyrus tilapiae the reference sequence from Senegal appears basal to all other specimens of native and introduced hosts in the Congo Basin (Fig. 3). We do not find any evidence to contest the species status of C. tilapiae as opposed to Pouyaud et al. 2006 who suggested it is a species complex based on ribosomal DNA.