Two new species of Microcotyle (Monogenea: Microcotylidae) on intertidal fish from the south Pacific coast

Microcotyle is one of the most diverse and controversial genera within the family Microcotylidae. To date, 131 species have been described in Microcotyle; however, more than half have been transferred to other genera, and several others have poor descriptions. Therefore, less than half of all Microcotyle species may be considered valid. In Chile, two species have been recognized, and unidentified Microcotyle have been found on several littoral fish, but there has been no effort to properly identify them. In this study, two new species of Microcotyle are taxonomically described from intertidal fish of the central (33°S) and south-central (36°S) regions of Chile. In this study, Microcotyle sprostonae n. sp. (collected mainly from Scartichthys viridis in central Chile) and M. chilensis n. sp. (collected mainly from Calliclinus geniguttatus in south-central Chile) were identified based on morphological and molecular analyses (ITS2 and 18S genes). Both species of Microcotyle principally differed from one another and from other valid species in the number of testes and clamps. The two new species also differed from one another by one base pair in the ITS2 and 18S genes and differed from other species of Microcotyle by several base pairs of both genes. Intertidal fish are mostly endemic to the Pacific coast of South America, and they have a limited geographical distribution that does not overlap with the type hosts of other Microcotyle species. Therefore, the two new species described here are distinguished from other congeneric species by morphological, genetic, and biological characteristics.


Introduction
Microcotylidae Taschenberg, 1879 is one of the most controversial families within Monogenoidea Bychowsky, 1937, in which several genera and subgenera have been erected (e.g., Unnithan 1971, Caballero & Bravo-Hollis 1972. Of the 58 genera described within Microcotylidae (Bray 2001), Microcotyle van Beneden and Hesse, 1863 is the most diverse genus, comprising 131 species that have been described from 1863 (Van Beneden & Hesse 1863) to 2019(Bouguerche et al. 2019a. The genus Microcotyle is mainly characterized by a conspicuous genital atrium with well-developed radial muscles, armed with numerous small, conical spines, and by a female reproductive system composed of a long, convoluted ovary, with an unarmed, long, single vaginal duct (see Mamaev 1989 for the emended diagnosis of the genus). Possibly 64 species of all those described correspond to Microcotyle (Bray 2001); however, several still require exhaustive revisions.
In recent years, morphological descriptions have been complemented with genetic analysis, with both techniques allowing researchers to clarify species statuses (Verma et al. 2018) and verify the proposal of new species. Indeed, in the last decade molecular markers have been used in the identification of Microcotyle species (e.g., Hayward et al. 2007, Ayadi et al. 2017, Bouguerche et al. 2019a, although there are few species sequenced till now.

Collection of fish and monogeneans
Between 2014 and 2015, eight common fish species were collected from several rocky pools in different localities in Chile. There were 2,473 specimens obtained from central region (Valparaiso, between 33°26'S-71°41´W and 33°30'S-71°37´W) and 1,076 from the central-south region (Biobío, between 36°28´S-72°55´W and 36°41´S-73°08´W) of Chile (Table 1). Of these, it focused on the blenny Scartichthys viridis and the labrisomid Calliclinus geniguttatus, because they have the highest abundance and prevalence of microcotylid monogeneans. Fish were collected with hand nets during low tide. Of the fish obtained, some were dissected immediately to collect monogeneans for morphological analysis. Each fish was euthanized with an overdose of an anesthetic solution before dissection, according to the bioethics protocols of the Universidad de Valparaíso and Universidad Católica de la SSMA Concepción. The gills were removed from the fish and observed under a light microscope. The monogeneans were collected and fixed in 5% formalin for staining procedure or 96% ethanol for molecular analysis.
Monogeneans were stained with hematoxylin, dehydrated in a graded ethanol series (from 70% to 100%), cleared in methyl salicylate, and mounted in Entellan. The specimens stained and mounted which were sufficiently clear to observe internal morphology were selected for the taxonomical description. The monogeneans were measured with an eyepiece micrometer. For the descriptions, the mean ± the standard deviation, followed by the range of measurements in parentheses, were recorded and expressed in micrometers (µm). Drawings were made with a camera lucida attached to a light microscope (Leica ® DM LS2). The prevalence and mean intensity of the parasites was calculated according to Bush et al. (1997).

Molecular analyses
Total genomic DNA from single specimens of Microcotyle from five fish species of the intertidal zone of central and central-south of Chile (Table 2), was extracted using established salt extraction procedures (Aljanabi & Martinez 1997). A few microcotylid specimens of S. viridis from northern Chile (23°S) were also considered for molecular analyses. Amplification of the ITS2 nuclear ribosomal DNA region was performed with the 3S forward primer (5'-GGT ACC GGT GGA TCA CGT GGC TAG TG-3') (Bowles et al. 1993) and the ITS2.2 reverse primer (5'-CCT GGT TAG TTT CTT TTC CTCCG C-3') (Anderson & Barker 1993). The 18S rDNA fragments were amplified with the 18SF forward primer (5'-AAG GTG TGM CCT ATC AAC Table 2. Species of the Microcotylidae used in molecular analyses in the present study / Especies de Microcotylidae usadas en los análisis moleculares en el presente estudio T-3') and the 18SR reversed primer (5'-TTA CTT CCT CTA AAC GCT C-3'). The ITS2 and 18S regions in the sequences were determined using the Internal Transcribed Spacer 2 Ribosomal RNA Database website (from NCBI GenBank) 1 . PCR (Polymerase Chain Reaction) amplification for 18S rDNA was performed using 100-µl mixtures containing 200 ng of genomic DNA, 0.2 µM of each of the two primers, 50 µM of each of the dNTPs, 1× PCR buffer (with 2 mM MgCl2), and 2.5 U of ExTaq DNA polymerase (Takara). Thermocycling conditions were as follows: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min. A final extension was performed at 72 °C for 5 min.
PCR reactions for ITS2 rDNA were performed using the same reaction mixture as described above and the following thermocycling program: initial denaturation at 95 °C for 10 min, followed by a touchdown of 10 cycles of 95 °C for 15 s, 60-50 °C for 30 s, and 72 °C for 45 s. This was followed by the second stage of 35 cycles of 95 °C for 15 s, 50 °C for 30 s, and 72 °C for 45 s. A final extension of 72 °C for 30 min was performed (Peña et al. 2014). PCR products were visualized on 0.8% agarose gels using a 1× sodium borate (SB) buffer solution.
The final PCR products for the 18S and ITS2 rDNA genes were purified and sequenced using the service of Macrogen, South Korea. Sequencher™ version 4.5 (GeneCodes Corporation) was used to analyze the sequences. Analyses were conducted using the Kimura 2-parameter model (Kimura 1980). All positions with less than 95% site coverage were eliminated. More than 5% alignment gaps, missing data, or ambiguous bases were not allowed at any position. For the 18S rDNA gene, the analysis involved 22 nucleotide sequences and a total of 1,493 positions in the final dataset. For the ITS2 gene, the analysis involved 22 nucleotide sequences and a total of 371 positions in the final dataset.
Genetic sequences of Microcotyle species of this study were contrasted to other microcotylid species; M. sebastis and M. erythrinii were used for the 18S gene, and M. bassensis and M. pomatomi were used for the ITS2 gene, including other species of Microcotylidae listed in Table  2. Evolutionary analyses were conducted using MEGA6 (Tamura et al. 2013). The genetic distances were computed with the total number of mutations, and the divergences were calculated among individuals by applying three algorithms: NJ (neighbor-joining), ML (maximum likelihood), and MP (maximum parsimony) (Tateno et al. 1994).
Genital atrium cavity from oval to as a triangle shape with rounded corners, 101 ± 43 (60-250) long and 135 ± 23 (106-181) wide, with anterior wide and rounded border, covered by little spines. Spines also on the peripheral atrium cavity and inside the cavity, which are difficult to count, approximately 200 spines of similar size, 11 ± 2 (8-15) long. Posterior to the atrial cavity, a concave zone in which the cirrus evert, surround by muscular fibers. Two groups of spines located posterior-laterally to cirrus cavity, with 7 ± 1 (4-9) spines each group. Atrial cavity surrounded by radial muscles, making a conspicuous atrial zone, 179 ± 47 (125-262) long, including the cirrus cavity, and 185 ± 74 (215-475) wide, considering the radial muscles.
Ovary pre-testicular, intercaecal, glomerular and long. The portion with immature cells coiled. The portion of the ovary with mature cells as an inverted U, located at 1,519 ± 198 (1,200-2,050) from the anterior end of the body. Ovary maximum wide 109 ± 30 (75-175). Oviduct a long folded tube, connected to an oval seminal receptacle and posteriorly to the vitelline duct. Oviduct turns upwards, followed by the ootype. Ootype almost oval shape, 330 ± 28 (310-350) long. Mehlis' cells different sizes, short at the beginning and at the end of the ootype (11-18 µm), and large cells at the middle of the ootype (24-30 µm). Uterus Vol. 54, N°3, 2019 Revista de Biología Marina y Oceanografía tubular, intercaecal, extending up to the atrial cavity. Vitelline glands glomerulated, located laterally in both sides of the body, extending from gut bifurcation up to the haptor mid-level. Two vitelline ducts, intercaecal, at the middle of the body. Vitelline ducts replete with reserves in some specimens. Vitelline ducts united posteriorly and anteriorly; posterior union in short conduct which joined to oviduct; anterior union connect with the vitello-vaginal duct. Female genital pore dorsal, at 717 ± 45 (675-787) from the anterior edge. A short muscular vagina that gradually connects with a short vitelline duct. Female genital pore located at 490 ± 91 (425-595) from the vitelline duct bifurcation, and 308 ± 96 (240-475) from the posterior border of the atrium cavity.
Nine specimens with eggs. One mature egg per monogenean. In two specimens, another egg in development. Egg fusiform, 213 ± 22 (175-237) long and 76 ± 13 (56-95) wide, with polar filaments, one short 79 ± 29 (75-80) directed to posterior side and the other much longer than 10 times the egg length, directed to the genital opening.
The distribution of microcotylids and hosts also supports M. sprostonae n. sp. as a new species. This monogenean was recorded in northern and central Chile on six fish species, but with a notorious preference for S. viridis (Table 1). The fish hosts of M. sprostonae n. sp. are mostly endemic to the Pacific coast of South America, whereas M. ditrematis has been recorded on Ditrema temminki at the Japanese coast (Yamaguti 1940), and M. emmelichthyops has been found on Emmelicthys sp. in Hawaii (Yamaguti 1968).
Etymology: "sprostonae" is dedicated to Nora Georgina Sproston, who made important contributions to the taxonomy of monogeneans.
cavity. More than 200 spines of similar size in the atrium cavity. Posteriorly, a cirrus cavity as a concave depression, surround by muscular fibers, armed with two groups of spines located posterior-laterally to this cavity, with 11 ± 2 (7-14) spines each group. Atrial cavity surrounded by radial muscles, making a conspicuous atrial zone, 163 ± 44 (100-225) long and 187 ± 46 (113-250) wide including the cirrus cavity.
Ovary pretesticular, intercaecal glomerular and long twisted extremes; the portion of the ovary with mature cells as an inverted U, located at 1650 ± 440 (1050-2310) from the anterior end of the body. Ovary maximum wide 94 ± 35 (62-175). Ootype oval, 131 ± 33 (100-165) long, 41 ± 10 (31-60) wide, located posterior to mature part of the ovary. Oviduct a long folded tube, connected to an oval seminal receptacle and posteriorly to the vitelline duct. Oviduct turns upwards, followed by oval, long ootype. Uterus tubular, intercaecal, extending up to atrial cavity. Vitelline glands glomerulated, located laterally in both sides of the body, extending from some distance to gut bifurcation, at 600 ± 137 (350-800) from the anterior end of the body. Vitelline ducts united posteriorly. The anterior union of vitelline ducts was not observed in most specimens. Anterior union of vitelline conducts connect with the vitello-vaginal duct. Female genital pore dorsal, located at 638 ± 187 (475-900) from the anterior edge. A short muscular vagina that gradually connects with a short vitelline duct. Female genital pore located at 357 ± 201 (215-500) from the vitelline duct bifurcation, and 283 ± 80 (225-375) from the posterior border of the atrium cavity.

Remarks
Microcotyle chilensis n. sp. was compared to other species, which were selected according to two features: 10-20 testes and 40-60 clamps. Four species resembled M. chilensis n. sp.: M. hiatulae Goto, 1899, M. furcata Linton, 1940, M. pentapodi Sandars, 1944, and M. neozealanicus Dillon and Hargis, 1965 Microcotyle hiatulae has been poorly described, based only on body length, number of clamps and testes, and spine lengths on the genital atrium (Goto 1899). Thoney & Munroe (1987) redescribed M. hiatulae and considered it as a senior synonym of M. furcata. We used data from that redescription and found that, compared to the new species, M. chilensis n. sp. differs from M. hiatulae in a smaller oral sucker length (45-65 vs. 44-112) and in the ratio of haptor length to /body length (20-34% vs. 23.4-48.1%). M. chilensis n. sp. also has a genital atrium at a greater distance from the anterior edge compared to M. hiatulae . Moreover, M. pentapodi was described as having a particularly shaped genital atrium: a "sucker with a pair of saccular bags without spines" positioned to the right and to the left of the sucker (Sandars 1944). Sandars distinguished this species from others using this distinctive genital atrium shape. However, the general morphology of the genital atrium of M. pentapodi seems similar to most Microcotyle, although the way to represent this was confusing. Overall, the description of the morphology of this structure in M. pentapodi requires some revision.
It is worth noting that M. pentapodi has been transferred to the genus Manterella Unnithan, 1971, due to the shape of the genital atrium. However, Mamaev (1977) considered this genus to be a synonym of Cynoscionicola Yamaguti, 1963. However, the genital atrium of M. pentapodi was not well described and drawings conveyed a poor representation of this structure. Therefore, the status of this species is unclear.
Of the other Microcotyle species previously recorded in Chile, M. nemadactylus (clamps: 90-104; testes: 16-25) (Dillon & Hargis 1965) and M. moyanoi (clamps: 118-150; testes: 33-44) (Villalba & Fernández 1986) are morphologically distinct from M. chilensis n. sp. (clamps: 44-54; testes: 14-18). M. chilensis n. sp. shares several morphological and morphometrical traits with M. sprostonae n. sp., but they differ in the number of testes, and there is only a small overlap in the number of clamps (Table 4). The cirrus cavity length is 37-112 in M. chilensis n. sp., which is in contrast to 12-50 in M. sprostonae n. sp. There are also differences in the number of spines on the sides of the cirrus cavity (4-9 in M. sprostonae n. sp. vs. 7-14 in M. chilensis n. sp.). Other differences are in relative measurements, such as larger ratio between oral sucker length and pharynx length and larger distance from posterior edge to last vitellarium gland in M. sprostonae n. sp. than M. chilensis n. sp. (Table 3).
The distribution of parasites and hosts might support M. chilensis as a new species. Similar to the hosts of M. sprostonae n. sp., the fish hosts analyzed here are mostly endemic to the Pacific coast of South America, and they are farther away than the type localities of M. hiatulae, which are found in the fish Tautoga onitis from Newport, Rhode Island, in the United States (Thoney & Munroe 1987). M. pentapodi has been found on Pentapodus milii fish in Western Australia (Sandars 1944), and M. neozealanicus has been found in Helicolenus percoides in New Zealand (Dillon & Hargis 1965). M. chilensis n. sp. was recorded in south-central Chile on five intertidal fish species, but with a notorious preference for C. geniguttatus (Table  1), contrasting with the distribution of M. sprostonae n. sp. in northern and central Chile. However, with the information obtained in this study, it is not possible to confirm the existence of geographical overlapping between M. sprostonae n. sp. and M. chilensis n. sp.

Molecular analyses
Based on the 18S gene, there were two clades of Microcotyle in the samples used in this study. One included a specimen M. sprostonae n. sp. from S. viridis (Fig. 3) from central Chile (33°S), and the other was obtained from different fish species (C. geniguttatus, Auchenionchus variolosus, B. chilensis, and H. sordidus) from the southcentral region of Chile (36°S) (Fig. 3). Table 3. Pairwise sequence divergences for the 18S gene of Microcotyloid species obtained by averaging all sequence pairs between groups. The divergence distance was calculated using the maximum composite likelihood model, and it is shown as a percentage (below the diagonal). The mean number of mutations between pairwise comparisons is also shown for each clade (above the diagonal) / Divergencias de secuencia por pares para el gen 18S de las especies de microcotílidos promedio obtenidas de todos los pares de secuencias entre los grupos. La distancia de divergencia se calculó mediante el modelo de probabilidad máxima compuesta, indicada en porcentaje (debajo de la diagonal). El número medio de mutaciones entre comparaciones por pares también se muestra para cada clado (por encima de la diagonal) Based upon the ITS2 gene, there were also two clades of Microcotyle: one clade composed of monogeneans attached to the fish S. viridis and H. sordidus from central and from northern Chile (Antofagasta, 23°S) (Fig. 4). These specimens were described as M. sprostonae n. sp. Another clade was only composed Microcotyle from fish from the south-central region of Chile. These specimens were described as M. chilensis n. sp.
Specimens of M. sprostonae n. sp. did not differ in the genetic sequences for the ITS2 or 18S rDNA genes (Tables 4 and 5) even when the monogeneans were collected in localities far away from one another (Valparaíso 33°S and Antofagasta 23°S, Table 1). However, M. sprostonae n. sp. and M. chilensis n. sp. differed by one base pair in both genes (ITS2 and 18S), which was supported by the phylogenetic analyses (NJ, ML, and MP) (Tables 3 and 4). Hence, molecular analyses confirmed the presence of two Microcotyle species in intertidal fish on the coast of Chile.   Figure 3 / Árbol filogenético que muestra las relaciones entre las especies de microcotílidos, incluidas las dos nuevas especies descritas en este estudio, basado en los análisis de NJ de las secuencias del gen ITS2. Los números a lo largo de las ramas indican los porcentajes de los valores de respaldo resultantes de los diferentes análisis en el siguiente orden: NJ, ML y MP. Los valores bajos se indican con guiones (<0,5 para NJ y <50% para ML y MP). Abreviaciones como en la Figura 3

Discussion
In this study, two new species of Microcotyle were morphologically described, which was also supported by molecular data. Relative measurements, as suggested by Machkewskyi et al. (2013) were also useful for making comparisons among species, especially between M. sprostonae n. sp. and M. chilensis n. sp. The two species described here are parasites of sympatric fish, which are distributed along the Chilean coast. However, these Microcotyle species have different distributions. One is in south-central Chile (M. chilensis n. sp.), and the other is in northern and central Chile (M. sprostonae n. sp.).
Distinctions in morphology between these two species cannot be attributed to the geographic distance for the following four reasons. (1) Molecular data were based on a variable gene (ITS2) and a conserved gene (18S). Both genes differed between species, and that difference was consistent in all host species and geographical zones. (2) Almost 200 specimens of S. viridis were collected in southcentral Chile (Table 1), but none of them were parasitized by M. sprostonae n. sp., despite C. geniguttatus being collected in the same habitat at the same time with a high level of parasitization by M. chilensis n. sp. In contrast, in central Chile, most S. viridis collected were parasitized by microcotylids (M. sprostonae n. sp.). In 109 specimens of C. geniguttatus, only two were parasitized by microcotylids (one parasite per fish). One of these monogeneans was fixed in ethanol, but, unfortunately, DNA amplification was not successful. However, its morphology is consistent with that of M. sprostonae n. sp. In addition, three specimens of S. viridis were collected in northern Chile. All of them were M. sprostonae n. sp. (Figs. 3-4). (3) Central (33°S) and south-central (36°) zones of Chile are 600 km away. In both locations, different Microcotyle species were found. Table 4. Pairwise sequence divergence for the ITS2 gene of Microcotyloid species obtained by averaging all sequence pairs between groups. The divergence distance was calculated using the maximum composite likelihood model, and it is shown as a percentage (below the diagonal). The mean number of mutations between pairwise comparisons is also shown for each clade (above the diagonal) / Divergencia de secuencias por pares para el gen ITS2 de las especies de microcotiloides promedio obtenidas de todos los pares de secuencias entre los grupos. La distancia de divergencia se calculó mediante el modelo de probabilidad máxima compuesta, indicada en porcentaje (debajo de la diagonal). El número medio de mutaciones entre comparaciones por pares también se muestra para cada clado (por encima de la diagonal) While the central (33°S) and northern (23°S) zones of Chile are 1300 km apart, M. sprostonae n. sp. was in both localities. Therefore, the geographic distance alone does not explain the presence of different Microcotyle species. It is probable that environmental conditions, which change across latitudes, may affect the distribution and host specificity of Microcotyle. (4) The presence of Microcotyle species in one zone versus another was not due to fish body sizes, because S. viridis and C. geniguttatus were similar in body length between sampling zones (Table 1). Altogether, there is sufficient morphological, molecular, and ecological evidence to confirm the validity of the two Microcotyle species described here.
Microcotyle sprostonae n. sp. and M. chilensis n. sp. were present on hosts of different families, although each species showed a preference for a certain host species (based on the high abundance and prevalence of the parasite). Also, a host species, Hypsoblennius sordidus, can be parasitized by both monogeneans separately, depending on the geographical distribution of the parasite. This result indicates that it is not possible to assume fish of the same species from different localities have the same Microcotyle species or that Microcotyle from different host species are different (Martínez & Barrantes 1977).
The genus Microcotyle requires the reassessment of many species, which is a very difficult task to carry out because several of the species considered valid need revision. Moreover, over time, more morphological details have been incorporated, and thus descriptions have become more complex. Consequently, the characteristics and distinctions of the recent new species are well understood, but the simplicity of the original descriptions for many of the species described long ago generates more doubts about the validity of those species. Also, it is worth noting that some characteristics may change with age, such as body width, number of clamps, and egg size (Sproston 1946). Thus, future studies need to considerate several specimens in order to establish the variability of any morphological trait. The descriptions of a species should consider as many morphological features as possible, which should be documented in measurements and figures. Molecular techniques are good tools for species descriptions; however, the molecular approach has been applied only recently, considering so far just a few species of Microcotyle, and therefore the advantages of genetic analyses for identification purposes are still limited for this genus. Therefore, future studies may include molecular methods to complement the species descriptions (new and already known), in order to clarify the status of numerous species in Microcotyle and in any other genera within Microcotylidae.