Record of Caromiobenella (Copepoda, Monstrilloida) in Brazil and Discovery of the Male of C. brasiliensis : Morphological and Molecular Evidence

: Monstrilloid copepods are protelean parasites with a complex life cycle that includes an endoparasitic juvenile phase and free-living early naupliar and adult phases. The monstrilloid copepod genus Caromiobenella Jeon, Lee and Soh, 2018 is known to contain nine species, each one with a limited distribution; except for two species, members of this widespread genus are known exclusively from males. Hitherto, members of Caromiobenella have not been recorded from tropical waters of the South Western Atlantic (SWA). The nominal species Monstrilla brasiliensis Dias and Su á rez-Morales, 2000 was originally described from female specimens collected in coastal waters of Esp í rito Santo and Rio de Janeiro (Brazil), but the male remained unknown. The failure to reliably link both sexes of monstrilloid species is one of the main problems in the current taxonomy of the group, thus leading to a separate treatment for each sex. New zooplankton collections in coastal waters and intertidal rocky pools of the SWA yielded several male and female monstrilloid copepods tentatively identiﬁed as Monstrilla brasiliensis . Our results of both morphologic and molecular (mtCOI) analyses allowed us to conﬁrm that these males and females were conspeciﬁc. We also found evidence suggesting that Caromiobenella is not a monophyletic taxon. Our male specimens are morphologically assignable to Caromiobenella , therefore, females of the nominal species Monstrilla brasiliensis , are matched here with the aforementioned males and, thus, the species should be known as C. brasiliensis comb. nov. (Dias and Su á rez-Morales, 2000). This ﬁnding represents the third documented discovery of a female of Caromiobenella , the ﬁrst record of the genus in the Southwestern Atlantic, and the ﬁrst documented record of monstrilloids from coastal tidepools. With the addition of C. brasiliensis , Caromiobenella now includes 10 valid species worldwide. This work represents the second successful use of molecular methods to link both sexes of a monstrilloid copepod. The male of C. brasiliensis is herein described, and a key to the known species of Caromiobenella and data on the habitat and local abundance of C. brasiliensis are also provided.


Introduction
Monstrilloid copepods are protelean parasites of benthic invertebrates, including polychaetes, molluscs, and sponges [1][2][3]; most juvenile stages are endoparasitic and free-living adult individuals lacking mouthparts are non-feeding reproductive forms that Zooplankton samples were collected monthly between August 2017 and December 2018 in marine coastal waters and rocky tidepools at five localities in the State of Rio de Janeiro, Southeastern Brazilian coast (Figure 1). Some of these samples contained male and female monstrilloid copepods that were taxonomically examined. Monstrilloids were firstly found in three rocky tidepools of the municipality of Rio das Ostras (Areias Negras beach: 22 • Table 1).
The water in the rocky tidepools was drained with an electric bilge pump (12 V) and the zooplankton was retained in a 100 µm mesh filter attached to the end of the water pipe. The drained water volume was recorded to calculate the zooplankton densities (individuals/m 3 ). The water temperature and salinity were recorded with a thermosalinometer (YSI Yellow Spring Pro 2030). The samples used in taxonomic examination and descriptions were fixed in 4% formaldehyde, analyzed in a stereomicroscope, and quantified in a Dollfus chamber. To complete the genetic analysis, five additional collections were carried out between September and December 2018 (Table 1) in Cemiterio beach, coastal region beyond the surf zone, in an area without rocky shores. These collections were made with a 100 µm mesh plankton net towed by a kayak without using a flowmeter, thus without density estimates. These samples were preserved in 92.8 • GL ethanol and monstrilloids were sorted and taxonomically examined in the laboratory.

Morphologic Analysis
A few male specimens, including those presented here, were tentatively assigned to the genus Monstrilla Dana, 1849. A reexamination of these male individuals allowed us to recognize them as a species of Caromiobenella, largely known from males and with distinctive morphological characters [3]. One of these specimens (MNRJ30136) was selected to be described. Our description of this male followed the current upgraded descriptive standards in monstrilloid taxonomy [1, 13,14] and included the distinctive characters of Caromiobenella [3,15]. The morphologic terminology follows Huys and Boxshall (1991) [16]. The Brazilian male specimens were deposited in the collections of the Museu Nacional-Federal University of Rio de Janeiro (MNRJ) and Invertebrate Collection of the Instituto de Biodiversidade e Sustentabilidade (NUPEM/UFRJ) (CIN-NPM), where they are available for inspection.

Molecular Analysis
Total DNA was extracted from four females and two male copepods by the Chelex resin protocol [17,18] as follows. Ethanol-preserved copepods were picked up with plastic pipettes under a stereomicroscope and then transferred to 0.6 mL Eppendorf vials. The excess of ethanol was then removed and left to dry at room temperature for at least 1 h. Afterwards, 10 µL proteinase-k (10 mg/µL) was added directly over the copepod body, followed by 75 µL lysis buffer 2× (0.1 mM Tris, 0.01 mM EDTA, pH 8.0), 75 µL 12% chelating resin (Chelex 100), SIGMA, shaken in vortex, and incubated overnight at 55 • C. The anterior region of the mitochondrial cytochrome c-oxidase, subunit I (COI) was amplified by the polymerase chain reaction (PCR) from 5 and 10 µL of extracted DNA, using Folmer et al. (1994) [19] primers, HCO2198 (TAAACTTCAGGGT GACCAAAAAATCA) and LCO1490 (GGTCAACAAATCATAAAGATATTGG) in a 25 µL final reaction volume containing 1× reaction buffer, 3 mM MgCl2, 0.24 µM of each dNTP, 0.12% Triton-X-100, 0.4 µM of each primer, 2 U of DNA polymerase. PCR was performed in a thermocycler (Gradient Mastercycler, Eppendorf) as follows: 1 cycle at 94 • C for 4 min; 35 cycles at 94 • C, 48 • C and 72 • C for 1 min each; and a final cycle at 72 • C for 7 min. DNA fragments were visualized after electrophoresis over ultraviolet (UV) light using the fluorescent stain UniSafe dye (Uniscience) and PUC19 ladder for fragment size determination. Amplicons were sequenced by the automation system of capillary electrophoresis sequencing (ABI 3730xl System; Sanger method). Sequences were edited using the Chromas Pro, v. 2.1.8 software and then aligned with Clustal W online tool. Edited and revised sequences were analyzed by the Basic Local Alignment Search Tool (BLAST) to find similar sequences deposited in GenBank and verify their proximity to the expected taxonomic group. Multiple alignment and gap insertions were performed retaining conserved coding regions at the same positions.
Pair-wise p-distances and Tamura-Nei distances were obtained for the Brazilian group. For comparisons with species from different genera, Tamura-Nei and Kimura-2 parameters distances were used. Maximum likelihood (ML) trees were obtained using Tamura-Nei distance and using 1000 bootstrap iterations for the branch confidence. A parsimony tree was also obtained. Phylogenetic and molecular evolutionary were conducted using MEGA version 6 [20,21]. Sequences of copepod species of other genera obtained from GenBank were used for comparisons and to verify the position of sequences of local copepods. Some of the sequences of other monstrilloid genera available in GenBank were shorter than those found in the present study for the Brazilian copepods. Therefore, to include a greater number of species in the comparison, it was necessary to work with a smaller region, but present in all of them. These comparisons were only possible at over 582 bp DNA fragment of the Brazilian copepods. It was not possible to include Caromiobenella ohtsukai (MH638358) and Monstrillopsis longilobata (MF447160; MF447163) because they were shorter at the 5 end.

Diagnosis (Female and Male)
Female Monstrilla with medium-sized, robust body, cephalothorax with dorsal and ventral scattered fields of striae. Urosome 4-segmented, ovigerous spines short: fifth legs elongate, bilobed, divergent; outer lobe long, slender, with three setae, inner lobe short, cylindrical, with single seta. Antennule 4-segmented, first segment partially fused to cephalothorax, segments 3-4 fused; combined third segment with outer surface produced, forming proximal rounded process furnished with field of coarse cuticular ridges. Caudal rami with 6 setae. Male: Monstrilla-like, medium-sized, body segmentation as in Caromiobenella (body length~0.98 mm), cephalothorax representing~49% of total body length. Pedigerous somites 2-4 representing 39% of total body length. Oral papilla at 34% of way back along ventral surface of cephalothorax. Cephalothorax with dorsal and ventral scattered pores and fields of cuticular striae. Antennules 5-segmented, representing 35.6% of total body length, geniculated between segments 4-5; first segment partially fused to cephalothorax; second segment with outer margin produced into rounded process ornamented with field of deep transverse wrinkles. Distal antennulary segment with usual armature of genus. Fifth pedigerous somite with reduced fifth legs represented by pair of knob-like processes. Preanal somite with medial pair of small keel-like acute processes on postero-ventral surface. Genital complex of type I, represented by short robust shaft with short, thick distal lappets, branches separated by deep longitudinal slit. Caudal rami armed with 6 setae including short, slender biserially plumose innermost seta. Body robust, total body length of examined individual = 0.98 mm, measured from anterior end of cephalothorax to posterior margin of anal somite. Additional measurements in Body short, robust, cephalothorax incorporating first pedigerous somite representing~49% of total body length. Succeeding pedigerous somites 2-4 each bearing pair of biramous swimming legs; pedigerous somites 2-4 combined accounting for 39% of total body length in dorsal view. Dorsal surface of pedigerous somites 2 and 3 each with pair of "craterlike" cuticular processes; those on third somite being smaller (Figure 2A,C). Cephalic region of cephalothorax wide, smooth, bilaterally protuberant in dorsal view, slightly narrower than cephalothorax; outer margin of cephalic protuberances weakly corrugate. Pair of small dorsal pit setae between antennulary bases. Forehead moderately produced, weakly rounded, with transverse striation fields on dorsal anterior and lateral surfaces; no other cephalic ornamentation was observed on dorsal anterior surface (Figure 2A). Cephalothorax robust, 0.36 mm long, representing almost 37% of total body length; dorsal surface with scattered dorsal pores (Figure 2A). Anterior ventral surface with rounded preoral projection ( Figure 3C) between antennule bases and oral papilla, visible in lateral view. Oral papilla at 34% of way back along ventral surface of cephalothorax, with adjacent field of transverse cuticular striae ( Figure 3C). Cephalothorax with eyes consisting of relatively small, unpigmented paired lateral cups separated medially by length of less than one eye diameter plus medial cup slightly larger than lateral cups. One pair of relatively small nipple-like cuticular processes present on anterior ventral surface between antennule bases and oral papilla (arrowed in Figure 2B); nipple-like cuticular processes surrounded by field of wrinkles (Figure 2A).  Antennule length = 0.35 mm, representing~35.6% of total body length. Antennule relatively short, 5-segmented, type III [16] representing 36% of total body length, and 73% of cephalothorax length; antennules indistinctly 5-segmented, segments 1-4 separated by incomplete sutures. First antennulary segment subrectangular, partially fused to cephalothorax proximally and to second segment distally. Second segment with bulging lateral process on outer proximal half; process furnished with deep transverse cuticular wrinkles (arrow in Figure 3A). Fourth segment being longest, representing 37% of total antennulary length. Geniculation between segments 4 and 5 ( Figure 3A). In terms of the pattern described by Grygier and Ohtsuka (1995) [14] for antennular armature of segments 1-4 and complemented with Huys et al.'s (2007) [1] nomenclature for elements on the male fifth antennule segment, element 1 present on first segment; element spiniform, lightly pinnate, relatively short, barely reaching midlength of succeeding second segment. Second segment armed with long, lightly plumose elements 2d 1,2 , 2v 1-3 , and slender seta IId. Third segment partially fused with second, subquadrate, armed with setiform elements 3, IIId, and IIIv. Setal element 3 lightly pinnate, reaching proximal 1/3 of succeeding fourth segment. Fourth segment subrectangular, elongate, about 3.5 times as long as wide, bearing normally developed elements 4d 1,2 and 4v 1,2 as well as long setae IVd and IVv; long, slender aesthetasc 4aes on mid-ventral position. Elements of group 4v short, setiform, lightly pinnate; element 4v 3 not discernible in the specimen examined. Distal segment with conspicuous proximal geniculation, armed with 11 setal elements (sensu Huys et al., 2007) including elements 1-5 and A-E plus apical aesthetasc 5aes.

Molecular Analysis
Six monstrilloid copepods including four females (identified as M. brasiliensis) and two males (tentatively assigned to M. brasiliensis), underwent molecular analysis to verify that they belong to the same species. We amplified all specimens' 5 end of the COI gene successfully, corresponding to a fragment of 681 bp (Table S1). The region showed 21 variable sites including 12 that were phylogenetically informative (shared). These copepods are conspecific, as shown by the distances among them (Table 4) with mean distances of 0.014 ± 0.003, characteristic of intraspecific individuals. The male MZ223434 clustered with MZ223430 female and the male MZ223435 clustered with the other females ( Figure 6), confirming that the males belong to the same species of the studied females. The branch in the ML tree ( Figure 6) that contains exclusively the six Brazilian copepods (Rio das Ostras, RJ, Brazil) was 100% supported by bootstrap. This Brazilian branch appears as a sister group of Monstrilla ilhoii (see Table 5), followed by Caromiobenella branch, which includes also in its branch the species Caromiobenella hamatapex (Monstrilla hamatapex in Genbank and in Figure 6). More distantly is grouped Caromiobenella helgolandica (as Monstrilla helgolandica in Genbank and in Figure 6) and the branch of Maemonstrilla. The most distant group from the examined Brazilian copepods was the genus Cymbasoma and Monstrillopsis longilobata (KR049000 and KY553229). Table 5. Mean pair-wise genetic distances over 582 pb COI fragment among different monstrilloid genera or branches as shown in Figure 6 for the comparisons with Brazilian copepods from Rio de Janeiro coast. See Table S1 for sequences obtained from GenBank. Distances: (below the diagonal) Kimura-2 Parameter; (above the diagonal) Tamura-Nei distance.    Table S1 for specific names and GenBank accession numbers for the partial sequences of C. brasiliensis used.

Discussion
The males examined herein can be morphologically identified as members of Caromiobenella Jeon, Lee and Soh, 2018 by their possession of distinctive genus characters [3], as follows: (1) the modified distal half of the male fifth antennulary segment, with a series of brush-like processes is the main distinguishing character of Caromiobenella [3] and our specimens clearly have this character ( Figure 3A,B); (2) the body shape and tagmosis and the urosome segmentation are also typical of Caromiobenella [3] (see Figure 1A-C); (3) the presence of 6 caudal setae with innermost apical caudal seta VI clearly shorter and slenderer than the others is another synapomorphic character shared with Caromiobenella ( [3], Figure 5A,B); the male genital complex is also of the same type (type I) as that described for this genus ( [3] Figure 5B-E). The results of our mtCOI analysis and comparison support the designation of our specimens as a species of Caromiobenella.
A reliable morphologic method to link the sexes of a monstrilloid species consists of finding particular autapomorphies shared by both sexes [2]. Thus, having a complete description of both sexes of the nominal M. brasiliensis ([8], present data), it was possible to use this morphologic criterion. It was previously applied to designate a male preliminarily identified as a subspecies of M. wandelli Suárez-Morales and Islas-Landeros, 1993 as the true male of M. mariaeugeniae Suárez-Morales, 1994 [23]. During our examination of the males and females from our samples, we were able to find a distinctive autapomorphy shared by both sexes of Caromiobenella brasiliensis comb. nov. This key character is the peculiar wrinkled protuberance on the male and female antennules; it is sexually dimorphic, present on segment 2 in the male and on segment 3 in the female ( [8], Figure 4A,B); this character has not been observed in any other monstrilloid species. In addition, both sexes of C. brasiliensis share a short, spiniform element 1 ( [8,14], Figure 3A) and a first antennulary segment partially fused with the cephalothorax ( [8], Figures 3B and 5A). Another option to link both sexes is by using molecular and genetic markers, a method that was successfully tested to match males and females of Korean monstrilloid copepods [24]. Overall, Caromiobenella brasiliensis can be morphologically distinguished from its known congeners by the distinctive process on the second antennulary segment and by the paired keel-like processes on the ventral surface of the preanal somite. Currently

Ecology
As far as we are aware of, monstrilloids have not been hitherto reported from tidal pools of rocky reefs. Rocky reefs are coastal shores made from solid rock and considered reef-like ecosystems. Monstrilloids parasitize of marine benthic invertebrates including benthic polychaetes, but also pyramidellid and vermetid gastropods [1,2] and mussel [4]. Interestingly, the largest known aggregations of monstrilloids have been recorded from reef-related areas [25][26][27].
In addition, the occurrence of monstrilloids in this coastal region, and particularly in the rocky tide pools, may be related to the abundance of host species. So far, the hosts of C. brasiliensis remain unknown, but a potential, locally abundant host species is the brown mussel Perna perna (Linnaeus, 1758), previously found, either directly [4] or indirectly [28], in association with Monstrilla sp. in southern Brazil.
Most (78.3%) individuals of C. brasiliensis from both coastal waters and rocky tidepools were collected during spring. In the rocky tidepools, the densities of C. brasiliensis ranged between 2.5 and 15 ind./m 3 and contributed up to 6% of the total copepod community in August (Table 1). Caromiobenella brasiliensis was found in water with temperatures ranging between 20.4 and 28.8 • C and salinities between 28.1 and 32.0.

Molecular Remarks
The genetic analysis based on the 681 bp COI fragment confirmed that the six copepods from Rio das Ostras (State of Rio de Janeiro, Brazil) investigated here form a single intraspecific group, and confirm that males showing the described morphology are conspecific with the females found in the surveyed area, designated here as Caromiobenella brasiliensis comb. nov. The phylogenetic tree (Tamura-Nei), based on 582 pb COI fragment, showed that the Brazilian species clustered in a major branch (69% bootstrap confidence) containing Monstrilla ilhoii, the most closely related species (D = 0.299, Kimura-2-Parameter), followed by a distance of 0.302 (D, Kimura-2-Parameter) by the branch containing three species of Caromiobenella, C. polluxea, C. castorea (the type species), and C. hamatapex. All other groups showed distances over 0.450 (D, Kimura-2-Parameter) with respect to the Brazilian copepods. The obtained phylogenetic tree (ML) suggests that the genus Caromiobenella is not monophyletic.
in Caromiobenella, thus representing the first discovery of this genus in the Southwestern Atlantic Ocean and the first record of monstrilloids from a restricted habitat (i.e., coastal rocky tidepools). This work represents the second successful use of integrative taxonomy (molecular + morphologic) methods to link both sexes of a monstrilloid copepod. Caromiobenella now includes 10 species worldwide.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/d13060241/s1, Table S1: GenBank sequences used for comparisons with Brazilian copepods sequences (present study) and for the construction of Maximum Likelihood Tree.

Data Availability Statement:
The data presented in this study are available on request.