Diversity of myxozoans (Cnidaria) infecting Neotropical fishes in southern Mexico

Myxozoans are a unique group of microscopic parasites that infect mainly fishes. These extremely reduced cnidarians are highly diverse and globally distributed in freshwater and marine habitats. Myxozoan diversity dimension is unknown in Mexico, a territory of an extraordinary biological diversity. This study aimed to explore, for the first time, myxozoan parasite diversity from fishes of the Neotropical region of Mexico. We performed a large morphological and molecular screening using host tissues of 22 ornamental and food fish species captured from different localities of Veracruz, Oaxaca and Chiapas. Myxozoan infections were detected in 90% of the fish species, 65% of them had 1 or 2 and 35% had 3 and up to 8 myxozoan species. Forty-one putative new species were identified using SSU rDNA phylogenetic analyses, belonging to two main lineages: polychaete-infecting (5 species) and oligochaete-infecting (36 species) myxozoans; from those we describe 4 new species: Myxidium zapotecus sp. n., Zschokkella guelaguetza sp. n., Ellipsomyxa papantla sp. n. and Myxobolus zoqueus sp. n. Myxozoan detection increased up to 6 × using molecular screening, which represents 3.7 × more species detected than by microscopy. This study demonstrated that Neotropical fishes from Mexico are hosts of a multitude of myxozoans, representing a source of emerging diseases with large implications for economic and conservation reasons.


Phylogenetic analyses.
To determine the phylogenetic relationships of identified myxozoans, two sequence alignments were performed, one for myxozoans clustering in the oligochaete-infecting lineage and a second for myxozoans clustering in the polychaete-infecting lineage (see 28 ). Each alignment included the newly generated SSU rDNA sequences as well as sequences of closely related myxozoans available in GenBank, which were retrieved using BLASTN. Nucleotide sequences were aligned using MAFFT version 7.490 ( 29 ) 33 was used to set the length of the burn-in period and to identify potential convergence issues. Speciesspecific divergences were identified from proportional distances (in %) calculated in Geneious Prime based on the dataset used for the ML analysis.

Results
Prevalence and detection. Microscopic examination showed that 16% (17/106) of the gall bladders were positive for myxosporean infection (Table 1). Myxozoan prevalence based on microscopic observations ranged from 14.3 to 100% among fish species. Eleven different species/morphotypes could be detected (Tables 1 and  2, Figs. 2 and 3), four of which are described below. Molecular screening revealed that 49% (50/102) of the gall bladder samples were positive for myxozoans, with prevalence of infection ranging from 25 to 100% among the analyzed fish species. None of the intestine and kidney samples were microscopically positive. Molecularly, 33.3% (3/9) of intestine and 11.1% (1/9) kidney samples were positive. The most frequently collected fish host species were three cyprinodontiforms, Profundulus punctatus (Profundulidae) (n = 23), Profundulus oaxacae (n = 14) and Poecilia mexicana (Poeciliidae) (n = 8); one mugiliform, Dajaus monticola (Mugilidae) (n = 7); and one perciform, Dormitator maculatus (Eleotridae) (n = 11). Myxozoan prevalence in the gall bladder of these hosts ranged from no detection to 27.3% microscopically, and from 25 to 85.7% by PCR detection (Table 1). Congruent prevalence of infection between microscopic examination and molecular screening was only observed in Awaous banana (Gobiidae) (100%) i.e., all three specimens analyzed showed myxospores in the bile. In some cases, microscopy did not reveal infection by myxospores, but PCR detected the presence of myxozoans up to 66% in some fish host species (e.g., 66.7% in Mayaheros urophthalmus (Cichlidae), 50% in Poecilia mexicana, see Table 1). Overall, molecular screening increased myxozoan detection rates by 1.7 × to 6×. The only two myxozoan negative fish species were Tlaloc labialis (Profundulidae) (n = 1) and Cichlasoma trimaculatum (Cichlidae) (n = 1).  Table 2. Morphological and molecular data of mixed infections and undescribed species from gall bladder of fish hosts collected from Mexico. Species codes in comments column are according to Table 3. Abbreviations: L, length, W, width, T, thickness, PTL -polar tubule length, PTC polar tubule coils, FS -fresh spores; FFSformalin fixed spores. All measurements are in µm.     Remarks. Myxospore measurements of Myxidium zapotecus sp. n. from another specimen of Awaous banana (N19) are provided in Table 2 (Fig. 3a). In this host, a mixed infection was detected with a Zschokkella sp. (Fig. 3b). Myxidium zapotecus sp. n. has overall myxospore morphology characteristic of the genus Myxidium. For differential diagnosis we selected species of Myxidium reported from North America, from other gobiids and other species that showed sequence similarity to our new species 34    Myxidium whippsi (9-10 vs. 5) and Myxidium glossogobi (9-10 vs. no striations).
The new species overlaps in myxospore size with Myxidium phyllium, Myxidium truttae and Myxidium eminentis. However, Myxidium zapotecus sp. n. has longer and wider myxospores and longer polar capsules than Myxidium phyllium and Myxidium truttae and differs in fish host (river goby Awaous banana vs mosquitofish Gambusia affinis and brown trout Salmo trutta, respectively) and in geographic location (Mexico vs USA and France, respectively). Myxidium zapotecus sp. n. differs from Myxidium eminentis in myxospore width, fish host (river goby Awaous banana vs flat-headed goby Luciogobius guttatus) and geographic location (Mexico vs Japan).
Etymology: The species is named after the colorful festivity of La Guelaguetza, an annual indigenous cultural dancing event that takes place in Oaxaca, Mexico.

Taxonomic summary.
Etymology: The species is named after one of the localities where the ancient Mesoamerican ritual ceremony, "Danza de los Voladores" takes place -Papantla, Veracruz, Mexico.

Taxonomic summary.
Myxobolus zoqueus sp. n. overlaps in myxospore size to Myxobolus mexicanus Yoshino & Noble, 1973. However, the new species has shorter and narrower myxospores, as well as shorter polar capsules. Myxobolus zoqueus sp. n. also differs in polar capsule positions, which was described as 'anteriorly located and shifted to one side of the myxospore's valvular midline' for Myxobolus mexicanus 36

Molecular and phylogenetic results.
A total of 79 novel partial SSU rDNA sequences were generated in this study (Supplementary Data 3). Sixty-seven sequences were representatives of the oligochaete-infecting lineage (mostly freshwater fish infecting species), belonging to three clades: urinary tract clade (11 sequences), biliary tract clade (19 sequences) and Myxobolus clade (37 sequences). Sequences falling in the Myxobolus clade were found to group in subclade I (6 sequences) and VII (31 sequences) (Fig. 4) (subclades according to 37 ). Twelve sequences belonged to the polychaete-infecting lineage (mostly marine fish infecting species), specifically to the biliary tract clade and most likely represented species of Ellipsomyxa Køie, 2003 (Fig. 5). None of the newly generated sequences were conspecific to any species or sequence data available in GenBank.
Using the phylogenetic position (topology, branch length) and a threshold of > 2% genetic distances of the 79 novel sequences and their closest relatives, a total of 41 putative new species were identified (37 putative + 4 new species described above): five species in the oligochaete-infecting urinary tract clade, nine species in the oligochaete-infecting biliary tract clade, including Myxidium zapotecus sp. n. and Zschokkella guelaguetza sp. n. and 22 species in the Myxobolus clade, with three species belonging to the subclade I and 19 species to the subclade VII, including Myxobolus zoqueus sp. n. In the polychaete-infecting biliary tract clade, we identified five species, including the newly described Ellipsomyxa papantla sp. n. (Figs. 4, 5; Table 3).
All five newly identified species from the oligochaete-infecting urinary tract clade clustered together with the closest relative Hoferellus azevedoi Matos, Silva, Hamoy & Matos, 2018 (MF162297) from the Chaetobranchus azevedoi from Marajo Island in northern Brazil. All nine species in the oligochaete-infecting biliary tract clade are closely related species forming two distinct phylogenetic groups with only one sequence of a described species available in GenBank, Myxidium phyllium (MW589654), which was found in gall bladder of Gambussia affinis, a cyprinodontiformid fish with a wide distribution range from North America to Central America.
Myxobolus zoqueus sp. n. OQ888253 Myxobolus zoqueus sp. n. OQ888227 Infection occurrence. Seven myxospore morphotypes detected in fishes are not formally described as new species herein due to an insufficient amount of data or to their occurrence as mixed infections. We report them with all available morphological data ( Table 2; Fig. 3), as well as their sequence data (Supplementary Data 3) as a resource for future species investigations.
Apart from the four described species, 37 potential new taxa were inferred from the molecular and phylogenetic data. Most of the myxozoan species were detected in one single host species (31/41), while ten species showed no host specificity. Eight myxozoan species were detected in two different fish host species (OUTC_spe-cies_1-3, Zschokkella guelaguetza sp. n., OMsCVII_species_5, OMsCVII_species_8, OMsCVII_species_17 and PBTC_species_1) and two myxozoans, Myxidium zapotecus sp. n. and a marine representative PBTC_species_2 were detected in three host species (Table 3).
The fish host species harboring the highest number of myxozoans was the mountain mullet Dajaus monticola, with eight species: six from oligochaete-infecting clade, including Myxidium zapotecus sp. n. in the biliary tract  www.nature.com/scientificreports/ Table 3. Novel and putative myxozoan species of Neotropical fishes from Mexico according to their SSU rDNA molecular data, phylogenetic position (topology, branch length) and genetic distances (> 2%). All species were found in gall bladder, except for four species detected in the intestine (*) and/or kidney ( +).  Detection of mixed infections from gall bladder, meaning more than one species of myxozoan detected in a single fish specimen, occurred in 33.3% of the infected hosts (17 of 51). From those, 16 hosts harbor two taxa/ species and only in one specimen of an unidentified Paraneetroplus sp. (Cichlidae), three species were detected from gall bladder. The most common combination was a representative of the polychaete-infecting biliary tract clade and one representative of the Myxobolus subclade VII (n = 5) or two representatives of the oligochaeteinfecting biliary tract clade (n = 3) (Supplementary Data 3).

Discussion
A large-scale evolutionary diversification of myxozoan species occurred after they acquired fishes as secondary hosts in their life cycles 28 . The highest concentration of freshwater fish diversity in the world occurs in Neotropical regions, with > 6200 species described and estimates of around 9000 38 . This suggests that high diversity of myxozoan species in this region could be expected. The parasite diversity detected in this study is probably just a scratch in the surface of the myxozoan fauna in the Neotropical region of Mexico. Myxozoan infections were detected in 90% of the freshwater fish species (17/19). From those, 65% had 1 or 2 myxozoan species and the remaining 35% had 3 or more species. This aligns with previous speculations of at least two novel myxozoan species per freshwater fish species in the Neotropical region 39 . We speculate that the number of species may be higher, as our study was limited to few organs, and screening of a wider range of organs and tissues may reveal a larger diversity in this region.
Geographically, several localities in Oaxaca had higher myxozoan prevalence than other localities in Veracruz and Chiapas, while overall, all localities in Veracruz had a higher myxozoan discovery rate per fish. However, neither locality or host species were sampled consistently, and the material collected was dependent on the catch success in each locality. Our results unfold lots of exciting questions about the distribution and diversity of these parasites, as for example, if certain river basins are more diverse than others or if differences exist between basins draining to the Pacific vs to the Atlantic. From the 536 species of freshwater fish reported in Mexico, more than half are found in the Nearctic region, of larger extension than the Neotropical area 40,41 . Exploring the diversity of myxozoans in this region and their comparison to Neotropical areas can provide useful information for diversity and conservation purposes; nearly 35% of the fish species are threatened or nearly threatened in Mexico 41 . Parasites play fundamental roles in the ecosystem dynamics and influence host evolutionary trajectories 42 . Populations of Profundulus oaxacae and Rhamdia guatemalensis, two fish hosts analyzed in this study, are a matter of concern and measurements of protection are being placed 43 . We detected three myxozoan species in these hosts. The risk of extinction of the host, is not only a risk for the fish but also for their associated parasites, which remain, in many cases, unknown.
The molecular screening allowed detection of myxozoans from fish tissue without a laborious microscopy evaluation, proving to be a useful approach for biodiversity exploration. This method increased the myxozoan prevalence detection up to 6 ×, representing 3.7 × more species detected by PCR screening than by microscopy; this represents nearly 1.3 × higher than a recent similar screening of fish tissues from the Czech Republic (2.8 ×). In this same study, a newly developed eDNA metabarcoding assay was shown to capture up to 2.5 × more diversity than PCR and Sanger sequencing in fish tissue 44 . The molecular screening carried in this study represents a useful tool for myxozoan diversity exploration and can be used for preliminary screening of fish tissue, representing a less intensive labor method than microscopy screening and a more accessible/less complex methodology than eDNA metabarcoding. However, eDNA metabarcoding assays of myxozoans may provide a closer to reality biodiversity estimation, especially when using fish tissues. Lisnerová et al. 44 also demonstrated the effectivity of these assays when applied to sediment samples, as a non-invasive (no fish sacrifice) technique. This could represent an excellent alternative for future myxozoan diversity estimation in Mexico, where many fish species are rare, threatened or endangered.
Myxozoan proliferative stages and early sporogonic development can be easily detected using molecular techniques. Thus, myxozoan DNA detection does not imply that the parasites develops specifically in its tissues, as positive detection may result from the presence of proliferative stages in the vascular system 45 , or tissue contamination by species sporulating in the body cavity 46 . This may explain the high detection of Myxobolus spp. in this study. Their detection by PCR may suggest their presence as early blood stages or as sporulating in adjacent organs to the gall bladder. Future studies should determine the tissue specificity of these species in their hosts. www.nature.com/scientificreports/ Several of the fish hosts in this study inhabit brackish waters or have migratory behavior, offering a high encounter opportunity to a wider range of invertebrates-infecting myxozoans. That is the case of the Mountain mullet Dajaus monticola who seems to have remarkable diversity of myxozoans (8 species). Mugilids are known hosts for myxozoans, with high diversity reported in other regions of the world 47 . The Mountain mullet is a catadromous species, a characteristic that could explain the abundance of myxozoan species and their assembly composition, with oligochaete and polychaete-infecting myxozoan representatives of three different clades. This species migration and its presence in brackish water could represent a great opportunity for myxozoan dispersion, colonization and transitioning to other environments.
Most of the host species analyzed in this study are of commercial economic importance for fisheries, gaming and/or aquaculture in Mexico 48 (FishBase-Supplementary Data 1). We detected several myxozoan infections in commercially valuable ornamental fishes for aquarium trade like poecilids: shortfin molly Poecilia mexicana (4 species), molly Poecilia sphenops (1 species), the Chiapas swordtail Xiphophorus alvarezi (2 species), or as fish food for aquaculture, like the Mexican mojarra Mayaheros urophtalmus (3 species) and the South American catfish Rhamdia quelen (2 species). The myxozoan diversity unraveled in this study represents a source of emerging disease agents, that could arise under environmental change or under farming conditions 49 . Most of the commercial trade of these species is still heavily dependent on wild populations 50 , which may be carriers of myxozoan infections into culture/rearing facilities. These infections may not be harmful in natural conditions but can become a health issue under stressful culture conditions, as either primary pathogens, like Myxobolus spp. 51 or opportunistic ones, like the ones found in the hepatic-biliary system of fishes 52 . Our results suggest that fish facilities should include myxozoans in their parasitological screenings and monitor fish health as these parasites could become a cause for concern.
Aquarium fishes are a large reservoir of invasive species. The exportation of native fishes as pets have resulted in biological introductions in other regions of the world. For example, Poecilia mexicana can been found in Oceania, Poecilia sphenops in Asia and Europe or Mayaheros urophtalmus in Asia (FishBase), which are some of the hosts analysed in this study. This not only represents a biological invasion that threatens native fish populations but also a biosecurity risk by the co-introduction of their native myxozoan infections 49 . Recently, myxozoan infections were detected in ornamental fish imported from Asia to Australia, which were previously undetected in sanitary inspections 53 . This lack of detection seems to be a common situation, as witnessed by the few published surveys about myxozoans in aquatic pet animals 49 .
In contrast, establishment of non-native fish farms in Mexico is widespread due to the lack of regulations 54 . Some of these species are already invasive, while others were identified as high-risk invasiveness. These exotic fishes can represent new hosts for the native parasites, amongst them myxozoans. While myxozoans have complex life cycles and certain requisites have to be accomplished for the establishment of a myxozoan infection i.e., presence of adequate vertebrate and invertebrate host, temperature-dependent development, longevity of waterborne stages, and others 49 , introductions of myxozoans have occurred in some cases with devastating consequences (e.g. Myxobolus cerebralis 55 , Kudoa iwatai 56 , Myxobolus dechtiari 57 ).

Conclusions
Myxozoans represent a neglected and understudied group of parasites in Mexico. This study demonstrates that the highly diverse group of Neotropical fishes are hosts of a multitude of myxozoan species and that they represent an interesting research area for conservation and economic reasons in Mexico. Fish in this country are commercialized for food or for aquarium trade, including international trade; several are, due to human activities, under risk or already threatened species. In order to design strategies to limit myxozoan introductions and outbreaks, it is essential to increase our knowledge on the baseline biodiversity and taxonomic data to identify emerging myxozoans diseases. Future studies should include non-invasive environmental DNA metabarcoding to fully comprehend the diversity of myxozoan species in Mexico. We hope this manuscript will promote research on this fascinating group of parasitic cnidarians in the megadiverse country of Mexico.