New species of Protomicrocotyle (Monogenea: Protomicrocotylidae), and new information on P. mirabilis, parasites of Caranx spp. from Veracruz, México

Abstract During a study of the helminth parasites of carangid fish of the Gulf of Mexico, Protomicrocotyle mirabilis and a new member of that genus were found. The aim of the present study is to provide new morphological and sequences of 28s rDNA and CO1 mtDNA for P. mirabilis and describe the new species. Between 2005–2022, 73 specimens of Caranx spp. were purchased from local fishermen of the littoral waters of the Gulf of Mexico. Protomicrocotyle veracruzensis sp. nov. is most similar to P. mirabilis than to P. ivoriensis, the only members of the genus known from the Greater Atlantic Ocean Basin. Protomicrocotyle veracruzensis sp. nov. can be distinguished from those two species by the arrangement and number of testes. Measurement data on the haptoral armature for the new species is provided and the potential value and need for comparative data from these structures of other members of the genus is discussed. The results of the molecular analysis and the morphometric analysis of 91 characters confirmed that this new species belongs to Protomicrocotyle.


Introduction
The Carangidae is one of the most morphologically diverse families of fishes (Nelson, 2006).The family is comprised of approximately 140 species, that are assigned to four tribes of 32 genera (Jacobina et al., 2014).Systematic analyses of the genus Caranx Lacépède, 1801 has shown that some recognized species have wide geographic distribution and regional populations with cryptic taxonomic features indicating that they constitute species complexes (Jacobina et al., 2014).In the Gulf of Mexico, the most common species of Caranx are Caranx hippos, Caranx crysos, and Caranx latus (López-Herrera et al., 2021).However, there are some registers of Caranx ruber, Caranx bartholomaei, and Caranx lugubris (Froese & Pauly, 2022).
Protomicrocotyle mirabilis has been reported from the Atlantic Ocean basin [as defined by Craig (2019)] from C. hippos from Ebrié Lagoon, Côte d'Ivoire, Western Africa; a description of that material was provided by Wahl (1972).The species also was reported from the Caribbean waters of Quintana Roo, Mexico by Bravo-Hollis (1989) who found it in C. crysos, C. latus, and Caranx sp.. Protomicrocotyle mirabilis has been reported several times in the Gulf of Mexico; Caballero y Caballero & Bravo-Hollis (1965) reported the species from C. latus collected from the waters off Tuxpan, Northern Veracruz; Bravo-Hollis (1989) reported the species in C. hippos and Trachinotus carolinus, collected from the Laguna de Sontecomapan and Las Cabañas, respectively, in Southern Veracruz, and Caballero y Caballero & Bravo-Hollis (1967) reported it from Campeche, Campeche, in C. hippos.Protomicrocotyle ivoriensis Wahl, 1972, the only other species known from the Atlantic Ocean basin, was described from specimens collected from C. hippos from Ebrié Lagoon, Côte d'Ivoire, Western Africa; Protomicrocotyle manteri Bravo-Hollis, 1966 and Protomicrocotyle nayaritensis Bravo-Hollis, 1979 have been described from the Pacific Coast of Mexico (Bravo-Hollis, 1966;Bravo-Hollis, 1979), and five other species have been described as parasites of fishes collected from the Pacific Ocean and the Indian Ocean basins (Protomicrocotyle carangis Pillai & Pillai, 1978, Protomicrocotyle celebesensis Yamaguti, 1953, Protomicrocotyle madrasensis Ramalingam, 1960, Protomicrocotyle mannarensis Ramalingam, 1960, and Protomicrocotyle minutum Ramalingam, 1960 (Yamaguti, 1953;Ramalingam, 1960;Pillai & Pillai, 1978).
In recent years, molecular approaches have been widely used for the exact identification of monogeneans and treatment of some unsolved taxonomical questions when only morphological methods were used (i.e.diagnosis, the discovery of cryptic species, delimitation of phenotypic variation, phylogeny) (Mendoza-Palmero et al., 2015;Tambireddy et al., 2016;Razo-Mendivil et al., 2016;Ondračková et al., 2020;Azizi et al., 2021;Ayadi et al., 2022).However, our present knowledge of molecular identification of the protomicrocotylids remains very limited due to the lack of genetic data.Currently, only four species of Protomicrocotylidae have been characterized with 28S, and two with cytochrome c oxidase subunit I (CO1) genes (Olson & Littlewood, 2002;Justine et al., 2013;Tambireddy et al., 2016) and genetic data for these species are available in GenBank® (Sayers et al., 2020) database.However, molecular data for many members of Mazocraeidea Bychowsky, 1937 are still lacking.
As part of the ongoing study of the helminths of marine fishes, individuals of C. hippos, and C. latus were collected from the coastal waters of Veracruz, Mexico (2005-2022) (Figure 1; Table 1) and necropsied for helminths.This study presents updated information on P. mirabilis [sensu stricto Kritsky et al. (2011)] and the description of a new species of Protomicrocotyle, a gill parasite of C. latus from Casitas and Puerto de Veracruz, Veracruz, Mexico.An integrative approach, including morphometrical categorization and molecular analyses of the cytochrome c oxidase subunit I and 28S rDNA was used to characterize these monogeneans.

Collection localities
The present study was carried out using fish collected from four localities (port cities) with the following formal names: Tuxpan de Rodriguez Cano, Tecolutla, Casitas, and Veracruz; all in the state of Veracruz de Ignacio de la Llave (INEGI, 2023).To avoid confusion, they will be referred to using the commonly used names: Tuxpan, Tecolutla, Casitas, and Puerto de Veracruz, respectively.The state will be referred to simply as Veracruz or as the State of Veracruz.The number in the parenthesis is represented in Figure 1.

Specimen collection
During the period from 2005-2022, 73 specimens of Caranx were purchased from local fishermen, who caught them in littoral waters of the Gulf of Mexico, offshore of the four localities in the State of Veracruz, Mexico: 67 specimens of C. hippos, crevalle jack (jurel amarillo-Mexican common name) and six of C. latus, horse-eye jack (jurel blanco) (Figure 1; Table 1).The external body surface of each fish was examined for helminths using a magnifying glass and gill arches were excised, placed in a Petri dish with seawater, and examined using a stereomicroscope (Leica Zoom 2000).Members of C. hippos were found to be infected with P. mirabilis and members of C. latus were infected with an undescribed species of that genus (Table 1).Each fish was infected with only a single species of monogenean.
Monogeneans, dead at the time of collection, were removed from gill filaments and transferred temporarily to dishes containing seawater.When all worms had been collected, they were fixed with Alcohol-Formalin-Acetic Acid (AFA) at room temperature for at least 12 h and then transferred for storage to 70% ethyl alcohol for morphological studies [following Pulido-Flores & Monks (2005)]; other specimens were fixed and stored in 96-100% ethyl alcohol for molecular studies.

Morphological and morphometric analysis
Specimens were stained using Gomori's trichrome, Mayer's carmalum, or Delafield's hematoxylin, dehydrated in an ethanol series, cleared in methyl salicylate, and mounted individually as whole-mounts on slides in Canada balsam.Morphometric comparisons were made using the information available in the literature for species of Protomicrocotyle reported in the Pacific and Atlantic Oceans and specimens borrowed from two collections (cited below).Specimens were examined using a compound optical microscope equipped with differential interference contrast (DIC) optics and drawings were made with the aid of a drawing tube.Measurements were made using an ocular micrometer; all measurements are given in micrometers as the mean followed in parentheses by the range and the number of structures (n) measured.For comparison with the shape of the haptoral lappet of other species, the length of the lappet was measured at three positions: right side lateral haptoral lappet (rt), opposite to the clamps; the middle of the lappet (mid); and the left side of the lappet (lf), the side closest to the clamps (Figure 2A).
In order to corroborate preliminary assignment of specimens collected found infecting fish from the localities of Tuxpan, Tecolutla, Casitas, and Puerto de Veracruz, Veracruz to P. mirabilis, the measurements provided by Kritsky et al. (2011) and those of the specimens of this study were compared to determine if there was a significant difference using a two-sample independent Mann-Whitney test.This test can be used to determine if two independent groups are from the same group (MacFarland & Yates, 2016), this case, the same species.
For the morphometric analyses, 91 morphological variables from 150 specimens of Protomicrocotyle were analyzed (88 continuous, and three meristic).The measurements were obtained from specimens that had been identified previously as members of P. mirabilis 317;341;348;349;111),P. manteri (CNHE: 346;348;3114;3115),P. nayaritensis (CNHE: 158;159), Neomicrocotyle pacifica (Meserve, 1938Yamaguti, 1968) (CNHE: 371;372;3116) and the specimens of P. mirabilis and the new species collected as part of this study were used for the construction of a Principal Component Analysis (PCA) and Discriminant Analyses (DA).Individual values were transformed from micrometers to log for statistical analyses.The PCA was used to order and visualize possible clustering among the morphometric characters by evaluating the PCA with the objective of reducing the number of variables introduced (Palacio et al., 2020).The DA determines to what degree the analyzed variables, measurements of objects or individuals, best explain the attribution of the difference of the groups to which said objects or individuals belong (Torrado Fonseca & Berlanga Silvente, 2013;Palacio et al., 2020).The analyzes were performed using Past 4.06b (Hammer et al., 2001).
The cycling conditions included initial denaturation at 94°C for 5 min, followed by 38 cycles of 94°C for 30 s, 45°C for 30 s, and 72°C for 1:10 min, and a final extension of 7:00 min at 72°C.PCR products were visualized in an electrophoresis agarose gel and were purified using polyethylene glycol (PEG) protocol and were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit in 10 µL reactions and 3730xl DNA Analyzer-Thermo Fisher Scientific using the JB3-F and LSU5-F primers, respectively, performed at the Instituto de Biología, UNAM, Mexico.Sequence data and electropherograms were inspected and edited using Pregap4 and Gap4 modules by Staden software V.1.6(Staden, 1996).
Sequences obtained in the present study for CO1 and 28S regions were aligned with sequences from other protomicrocotylid retrieved from GenBank and Allodiscocotyla diacanthi Unnithan, 1962(Allodiscocotylidae Tripathi, 1959 was used as the outgroup, based on the phylogenetic analysis by Tambireddy et al. (2016) (Table 2).Average p-distance between conspecific sequences from GenBank and collected samples (Table 2) were calculated in MEGA 11 (Tamura et al., 2021).A distance matrix was used for clustering analysis and the presentation of tree topology.The Neighbor-Joining (NJ) method was used to builds a tree from a matrix of pairwise evolutionary distances relating to the set of taxa being studied, therefore, the algorithm of the method finds the pairs of sequences that minimize the total length of the topology of the tree in each iteration (Gascuel & Steel, 2006;Saitou & Nei, 1987).The NJ analyses from CO1 and 28S was performed in MEGA 11 with bootstrap analysis based on 1000 resampling of each data set.The trees were edited in Adobe ® Photoshop ® .

Male reproductive structures (Figures
Female reproductive structures (Figure 4C, 4E).Germarium intercecal, post-testicular, comprised of germarial bulb with immature oöcytes, 116 (36-199, n = 75) long, 69 (17-124, n = 75) wide, with a wide ascending duct and an irregularly descending duct that form loops that contain mature oöcytes.The oviduct ends in the oötype; uterus ascends from oötype to the genital atrium (Figure 4E).Vaginal pore ventral, anterior to vaginal vestibule.Vaginal vestibule 69 (28-94, n = 80) long, 51 (19-77, n = 80) wide, located 464 (175-740, n = 80) from anterior end of body and 112 (40-180, n = 80) from the genital pore, lateral to midline on the side opposite to that having the haptoral clamps (Figure 4C).Vaginal vestibule armed with approximately 45 (31-65, n = 63) spines 34 (13-50, n = 76) long, 3 (1-5, n = 76) wide; the spines of the anterior region of the vestibule are larger and those of the middle and basal region smaller.These spines have small spine-like extensions on the distal region of each spine (Figure 4D).The vaginal duct descends from the vaginal vestibule to the germarium and connects to the oötype in the ventral region of the germarium between descending duct and germarium bulb.Seminal receptacle not observed.Vitelline glands in two lateral fields, starting just posterior to vaginal vestibule, overlapping ceca anteriorly and posteriorly and the lateral margin of the testes, uniting posterior to germarium, extending to posterior region of body but not reaching the haptoral lappet (Figure 3A).

Site of infection:
Gill filaments.

Remarks
Members of Protomicrocotyle can be characterized by a suite of characters that include: having an asymmetrical haptor with four Gastrocotylidae-type clamps in a longitudinal row on the side opposite to the vaginal vestibule; clamps with accessory sclerites; the haptoral lappet is transversely elongate (wider than long), armed with two pairs of anchors (larger anchors positioned laterally with smaller anchors between the larger ones), and one pair of hooks (located between the smaller paired anchors); the esophagus and ceca have diverticula; vitellaria and ceca may or may not extend into the haptor; the testes are relatively numerous, with variations in shape, size, and arrangement, but always are anterior to the female complex; the MCO bulbous, may be muscular, and is provided with a crown of numerous spines; genital pore ventral to esophagus; ovary consisting of a germarium and a tubular duct, winding or not, post-testicular; genitointestinal canal present, crossing ovary or not; eggs with filament at each pole; vagina opening ventrally to the right or left, posterior to genital pore, armed or not, with numerous spines of different shapes; vitellaria extends lateral and dorsal to ceca (Yamaguti, 1963).They are parasites of marine teleost's, principally of fishes of Carangidae (Yamaguti, 1963).The material described herein has the diagnostic morphological characteristics of this genus.The reason for reporting this information in detail is the necessity to corroborate the identity of the specimens collected from Tuxpan so that the molecular data could be linked to P. mirabilis, so it could be compared to that of the new species.The results of the Mann-Whitney test indicated that there are no significant differences [Z (U) = 0.729; P = 0.465] in the measurements between the specimens of Protomicrocotyle mirabilis from Florida, as described by Kritsky et al. (2011), and those specimens collected from Veracruz (Figure 1; Table 1).In accordance with the redescription by Kritsky et al. (2011) and the specimens collected as part of this study, P. mirabilis is characterized by having the characters of the genus and of the species, as detailed above.Measurements for P. mirabilis presented in this work are shown in comparison with that reported by Kritsky et al. (2011) in Table 3. Comparing the measurements in Table 3, it is possible to observe variation with respect to some variables, but as demonstrated by the Mann-Whitney test, there are no significant differences.However, it is worth mentioning that there are some characters that are outside the ranges established by Kritsky et al. (2011) in their redescription of P. mirabilis, such as body length, average testes size, the number of spines in the male reproductive organ, the vaginal vestibule, and the number of testes, among other variables (Table 3) of the specimens in this study are larger than what was mentioned by Kritsky et al. (2011).Therefore, it is necessary to carry out studies of specimens from other locations within the different biogeographical provinces that make up the Gulf of Mexico (Carolina Province and Caribbean Province) (Briggs & Bowen, 2012) and of the different ecoregions of which these provinces are pertain (Lara-Lara et al., 2008;Mendelssohn et al., 2017) that include the analysis of morphological and molecular characteristics.In this manner, existing variation in morphology can be attributed to intraspecific variation or if it reveals a complex of cryptic species.However, the current information is interpreted as a confirmation that P. mirabilis (sensu stricto Kritsky et (1967)].Previous records from other localities in the Gulf of Mexico (Bravo-Hollis, 1989;Mendoza-Garfias et al., 2017;Montoya-Mendoza et al., 2017), Caribbean Sea (Bravo-Hollis, 1989), South America (Boada et al., 2012;Vianna et al., 2020) and the Republic of Côte d'Ivoire (Wahl, 1972) could not be corroborated from existing material.Additional material that includes specimens for comparative morphological and molecular analyses must be collect in order to establish the limits of the distribution of P. mirabilis.

Etymology:
The specific epithet is derived from the name of the state of Veracruz, Mexico, where these specimens were collected.
Of the known species of Protomicrocotyle, the new species is most similar to the species from the Atlantic Ocean basin, P. mirabilis and P. ivoriensis.Using the structures mentioned above, the new species can be distinguished from P. mirabilis by having more testes (36-69 vs. 23-33, respectively); there is overlap in the number of testes of P. ivoriensis (36-69 vs. 50, respectively) by having less testes, although, if the number of testes reported for the latter species was invariant, or an average, is not known.The new species also can be distinguished from these two species by the arrangement of the testes; the new species has 1-2 adjacent testes in the lateral rows vs. P. mirabilis, which has testes in a single-file row in each field and P. ivoriensis has groups of 1-3 adjacent testes in each row.The relation between the length and width (L: W) of the testes in the new species is L: W: 1:3 and in P. mirabilis is L: W: 1:1.1, showing that the testes of the new species are larger than those of P. mirabilis.The relationship between the length and width of the testes of the new species is also greater than in P. ivoriensis (1:3 vs. 1:1.6),P. madrasensis (1:3 vs. 1:1.7-2.1),P. manteri (1:3 vs. 1:1.8),P. nayaritensis (1:3 vs. 1:1.4),smaller than P. mannarensis (1:3 vs. 1:3-5.41)and in range with P. minutum (1:3 vs. 1:2.4-3.1).Complete detailed comparative data for the new species and the other nine valid species is given in Table 4.

Morphometric analyses
Results of the principal components analysis (PCA) show an accumulated variance of 52.75 (Table 6A) of the first two components and the morphological variables that most influence the multidimensional arrangement of the first component.They are, in order of importance: the width of the body, the width of the ovary, the width of the haptor, the average width of the testes, the distance from the vaginal vestibule to the genital pore and the total longitude of the body.The variables that most influenced the second component are, in order of importance: the number of spines of the vaginal vestibule and the length of the spines of the vaginal vestibule.Eigenvalues and accumulated variance for first two principal components are presented in Table 6A.
In the Discriminant Analysis (DA), the first two factor axes explained 88.52% of the total variation (Table 6B).The first factor (eigenvalue 55.73, 46.88 of variation) alone was the main discriminant function, which arranged the specimens of Protomicrocotyle and Neomicrocotyle with the morphological variables, in order of importance: the number of spines in the male copulatory organ and the length of the spines of the vaginal vestibule.The second factor (eigenvalue 49.49, 41.64 of variation) has a value almost equal to the first factor, and separated the specimens of Protomicrocotyle and Neomicrocotyle with the morphological variables, in order of importance: the width of the body, number of spines of the vaginal vestibule, number of testes, longitude of the male copulatory organ and the width of the ovary.The DA, like the PCA, in the multidimensional plane, separated the five groups that correspond to the specimens of P. mirabilis, P. manteri, P. nayaritensis, Neomicrocotyle pacifica and the new species, P. veracruzensis sp.nov.(Figure 8A, 8B).An important finding was that Factor 1, of the DA, separated the specimens of the Atlantic species (P.mirabilis and P. veracruzensis sp.nov.) from the specimens of the Pacific species (P.manteri, P. nayaritensis, and Neomicrocotyle pacifica).Finally, Factor 2, separated the specimens of Protomicrocotyle from Neomicrocotyle pacifica (Figure 8B).
The intraspecific genetic variation between specimens of P. mirabilis was 0% to 2.01% for CO1 and 0% to 0.14% for 28S.Between specimens of P. veracruzensis sp.nov., the variation was 0.25% to 0.75% for CO1 and 0% for 28S.The interspecific genetic variation between specimens of P. mirabilis and P. veracruzensis sp.nov.was 8.48% to 10.53% with CO1, and 0.81% to 0.95% with 28S (Figure 9A, 9B; Table 7A, 7B).The NJ analysis using CO1 and 28S sequences of all species of Protomicrocotylidae currently in GenBank and Allodiscocotyla diacanthi from GenBank as outgroup (Table 2) resulted in the identification of a single group of Protomicrocotyle with support of 99% with CO1 and 95% with 28S (Figure 9A, 9B).Protomicrocotyle mirabilis and P. veracruzensis sp.nov.form each one an independent group with the support of 100% with CO1 (Figure 9A), and 92% and 99% with 28S (Figure 9B), respectively.Protomicrocotyle nested within members of the family Protomicrocotylidae with high support with both molecular markers (Figure 9A, 9B).

Discussion
As with many groups of helminths, the time span since the first member of the group was described by MacCallum (1918) and today's descriptions encompass many advances in the development of our understanding of the characteristics that reveal the limits between species.For groups that are not well-studied, like the genus Protomicrocotyle, a suite of characters are most useful for the identification of members on a group level instead of a single diagnostic feature.This has resulted in descriptions which mention only the characters that are known at that time in order to distinguish the new species from a particular congener.The same features, as the number of testes, the number of spines in the MCO, and the number of spines in the vagina were used to distinguish the new species from others of the genus.However, these and other values often overlapped with particular species in such a way that no single character could distinguish the new species from the other species of Protomicrocotyle.For better understanding of the intra-and interspecific variation, Table 4, with the measurements of the 10 known species, was developed and included herein.Most of the measurements of the previously known species were taken from published descriptions (see Table 4 for citations of the works that were consulted).Possibly because some authors were only reporting a new locality or host record, many of those articles did not provide a full description, only mentioning those characters that helped them assign their material.The limitations of that material and those descriptions were not sufficient to confirm the assignment of those specimens to a particular species (Table 3).In those cases, new material and molecular studies will be needed for confirmation of their identity.
One area of interest is in the use of the measurements of the hard parts (hooks, anchors, spines, etc.) as informative characters in descriptions of new taxa; a few examples of the use of the measurements of these structures in recent studies of monogeneans are mentioned herein.Vaughan & Christison (2012), proposed a measurement scheme for the haptoral armament of members of Callorhynchocotyle Suriano & Incorvaia, 1982 and used the measurements in a systematic study of the members of that genus.Most recently, Vaughan et al. (2021) used the same scheme and provided the measurements of the haptoral armament of Scyliorhinocotyle narvaezae Vaughn, Christison & Hansen, 2021.These works prompted us to provide the measurements for the haptoral armament in this study (Table 5).It is hoped that similar measurement data will be provided by authors in future descriptions of new species of Protomicrocotyle.
In the present study, the seminal receptacle of P. veracruzensis sp.nov.was observed and described, although it could not be distinguished in all specimens.This structure often is not mentioned in descriptions of other members of the genus.In most cases, whether the structure is present or not, or whether it was just not mention, cannot be determined.However, Ramalingam (1960) described and illustrated the seminal receptacles of P. madrasensis, P. mannarensis, and P. minutum.A review detailed of the material of the remaining members of the genus is necessary in order to verify the presence or absence of the seminal receptacle in those species.In most cases, collections of new material will be required.
In a similar manner, many authors have not mentioned whether the clamps are sessile or pedunculate.Because the data is incomplete or conflicting, this feature was not used in the comparisons.Of the nine previously-known species, four authors have reported that three species of Protomicrocotyle have clamps with peduncles: P. nayaritensis, P. mirabilis, and P. manteri [see Bravo-Hollis (1979), Caballero y Caballero & Bravo-Hollis (1965), MacCallum (1918), and Bravo-Hollis (1966), respectively].Three authors have reported species that have clamps that are sessile: P. mirabilis, by Kritsky et al. (2011), contrary to previous reports by Caballero y Caballero & Bravo-Hollis (1965) and MacCallum (1918); P. madrasensis, P. mannarensis, and P. minutum by Ramalingam (1960); and P. carangis by Pillai & Pillai (1978).The presence or absence of peduncles for P. mirabilis was not mentioned by Johnston & Tiegs (1922), Koratha (1955), Wahl (1972), andBravo-Hollis (1989), and for P. celebesensis by Yamaguti (1953), and Barton et al. (2009).Wahl (1972) did not mention peduncles for P. ivoriensis; however, in the drawing of P. ivoriensis the clamps are shown to have peduncles [Wahl (1972), his Figure 2a].The clamps of the new species, P. veracruzensis sp.nov., have peduncles similar to those of P. ivoriensis, and specimens of P. mirabilis reported as part of this study had clamps with peduncles.In all these species, the peduncles are short [as mentioned by MacCallum (1918)], which might explain why they were not given emphasis by some authors.
Finally, the orientation of the clamps has been mentioned only by Kritsky et al. (2011), who said that they could be dextral or sinistral, but they did not mention their relationship to other structures.The clamps of specimens of P. mirabilis and P. veracruzensis sp.nov., were observed to be on the right in some worms and on the left side of other worms, but always they were opposite to the vaginal vestibule.The male copulatory organ also always was opposite to the vaginal vestibule, and always on the same side as the clamps.This has not been mentioned by previous author.
The use of morphological and molecular information has been a useful tool in the description of new species of Monogenea (Aguiar et al., 2017;Camargo & Santos, 2020;Torres-Carrera et al., 2020;Zago et al., 2021;Ayadi et al., 2022;Dmitrieva et al., 2022), and the relevance of the integrative taxonomy approach in the description and redescription of species of helminth has recently been highlighted.This study, with the molecular characterization of the CO1 and 28S genes of P. mirabilis and P. veracruzensis sp.nov.can be added to this list.
The molecular analysis revealed that the level of interspecific genetic variation between P. mirabilis and P. veracruzensis sp.nov.with CO1 sequence data is distinctly higher than that of the interspecific genetic variation with 28S (Table 7), as one would expect; other studies show the same pattern of high interspecific genetic variation between members of species of the same genus (Ayadi et al., 2017;Camargo & Santos, 2020;Torres-Carrera et al., 2020).In the genus, Microcotyle Van Beneden & Hesse, 1863, intraspecific nucleotide divergence of 0% to 1.4% has been reported in Microcotyle visa Bouguerche, Gey, Justine & Tazerouti, 2019, and an interspecific difference of 9.5% to 10.7% between Microcotyle isyebi Bouguerche, Gey, Justine &Tazerouti, 2019 andM. visa (Bouguerche et al., 2019).However, percentages of genetic divergence within and among taxa cannot be interpreted until a more complete molecular database is available (Torres-Carrera et al., 2020).Even taking this into account, the use of morphological data and the combination of different molecular markers are convincing evidence sufficient to differentiate P. veracruzensis sp.nov.from the other known members of the genus.As well, the same integrative approach corroborates evidence of the presence of P. mirabilis in the localities sampled in this study and the previous report of the species from Tuxpan by (Caballero y Caballero & Bravo-Hollis, 1965) and Campeche (Caballero y Caballero & Bravo-Hollis, 1967).

Figure 8 .
Figure 8. Morphometric analyses of the characteristics of species of Protomicrocotyle. A. Principal components analyses.B. Discriminant analyses.

Figure 9 .
Figure 9. Cluster analysis showing similarities between specimens of Protomicrocotyle and other protomicrocotylids.A. Dendrogram of sequences of cytochrome c oxidase subunit I. B. Dendrogram of sequences of 28S rDNA.Note: Values in bold are the bootstrap values.

Table 1 .
List of species of fish sampled in the Coastal waters off Veracruz, Mexico.

Table 2 .
List of monogeneans included in the genetic distances analyses and Unweighted Pair Group Method with Arithmetic Mean analyses, and GenBank accession numbers of sequences from the partial CO1 and 28S genes.New sequences obtained for the present study are in bold.

Table 3 .
Morphometric comparison of different records of Protomicrocotyle mirabilis (taxonomic identity corroborated) and P. cf. mirabilis (taxonomic identity not corroborated).Measurements are presented in micrometers.

Table 4 .
Comparative morphological characteristics of the species of the genus Protomicrocotyle.Measurements are presented in micrometers.

Protomicrocotyle veracruzensis sp. nov. P. mirabilis (redescription) Hosts Caranx latus C. hippos and C. latus Location Casitas and Puerto de Veracruz, Veracruz, México Florida, EUA and Veracruz, Mexico
*Data obtained from the average length of the four clamps.Values in bold were taken of the line drawings.MCO = male copulatory organ.*Data obtained from the average length of the four clamps.Values in bold were taken of the line drawings.MCO = male copulatory organ.

Table 6 .
Cumulative variance of (A) the first two principal components and (B) factor values of discriminant analyses.

Table 7 .
Intraspecific and interspecific genetic distances estimated for (A) mitochondrial cytochrome c oxidase subunit I (CO1) and (B) 28S rDNA of Protomicrocotyle veracruzensis sp.nov.and other species of Protomicrocotylidae. Pairwise corrected p-distances are expressed as percentages (%).
The values in bold represents the intraspecific divergence in Protomicrocotyle mirabilis and P. veracruzensis sp.nov; The values in italics represents the interspecific divergence between P. mirabilis and P. veracruzensis sp.nov.*Sequences of Protomicrocotyle mirabilis; **Sequences of Protomicrocotyle veracruzensis sp.nov.The values in bold represents the intraspecific divergence in Protomicrocotyle mirabilis and P. veracruzensis sp.nov; The values in italics represents the interspecific divergence between P. mirabilis and P. veracruzensis sp.nov.*Sequences of Protomicrocotyle mirabilis; **Sequences of Protomicrocotyle veracruzensis sp.nov.