A new species of Macrocypraea (Gastropoda, Cypraeidae) from Trindade Island, Brazil, including phenotypic differentiation from remaining congeneric species Author Simone, Luiz Ricardo L. Author Cavallari, Daniel C. Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto da Universidade de São Paulo, Ribeirão Preto, SP, Brazil text PLoS ONE 2020 e 0225963 2020-01-08 15 1 1 26 http://dx.doi.org/10.1371/journal.pone.0225963 journal article 301512 10.1371/journal.pone.0225963 42907c40-ea0a-41c1-aff4-6e16e16eb615 1932-6203 PMC6948752 31914132 12630957 C8E6E515-508F-47DE-9753-4EA619C1DDFD Macrocypraea zebra (Linnaeus, 1771) Fig 12G–12L Synonymy see [ 3 ]. Complement: Macrocypraea zebra : Meyer, 2003[ 16 ]: 411; Simone, 2004[ 3 ]: 35, Figs 1–4 , 70, 116–143; Soriano, 2006[ 17 ]: 49, Table 3; Rosenberg et al, 2009[ 18 ]: 636; Rios, 2009[ 14 ]: 132, text figure. Lorenz, 2017[ 2 ]: 288, text figure. Macrocypraea zebra zebra : Lorenz, 2017[ 2 ], 288, text figure. Macrocypraea zebra dissimilis : Lorenz, 2017[ 2 ]: 289, text figure. Distribution. North Carolina to southern Brazil . Habitat. Rocks and corals, subtidal, 0–37 m depth [ 3 ]. Material examined. Specimens of Macrocypraea zebra listed in Simone [ 3 ], with the addition of the following: DOMINICAN REPUBLIC : Puerto Plata Province ; Puerto Plata , 2–3 m depth MZSP 115363 , 1 shell (local people coll., XI/2011 ). HAITI : Nippes Department ; Anse-àVeau Commune , MZSP 115364 , 1 shell (local people coll., 1986). BRAZIL : Bahia State ; reefs off Alcobaça , 20–25 m depth , MZSP 72653 , 20 shells ( A. Bordart coll., VII/2004 ). Rio de Janeiro State ; Arraial do Cabo , 1–2 m , MZSP 73553 , 8 shells ( P. Gonçalves coll., XII/2003 ). São Paulo State ; Ilhabela , 23˚46’S, 45˚21’W, MZSP 121109 , 20 shells ( J. Colella coll., VII/1968 ). BRAZIL : Santa Catarina State ; precise locality unknown, MZSP 38946 , 1 shell ( De Fiore coll., date unknown) . Measurements (mean values ± standard deviation, in mm; n = 50). H = 26 ± 7, L = 64.5 ± 16.9, W = 33.7 ± 8.7. Remarks. Based on the specimens analyzed by Simone [ 3 ] and in the present samples, there are no significant anatomical or conchological differences between individuals of the Caribbean (i.e., M . zebra zebra ) and Brazilian ( M . zebra dissimilis ) populations. As such, there are not enough characters to distinguish these subspecies other than the supposed range gap. Lorenz [ 2 ] mentioned that the division is corroborated by molecular data, bud did not mention any reference or study in progress and provided no additional evidence to endorse the separation. In our understanding, these subspecies must remain invalid until further evidence is presented. The anterior pair of projections franking canal is slightly longer than that of M . cervinetta , but shorter than that of M . mammoth , about 1/10 of shell length ( Fig 12G and 12I ). The inner lip slope has fewer and shorter teeth than M . mammoth ( Fig 12K ), disposed perpendicularly to the longitudinal shell axis; those teeth from anterior concave region are interrupted at middle, with weak ventral nodes ( Fig 12K ). The left edge of canal is more uniform and arched, oblique positioned ( Fig 12K and 12L ). Discussion Individuals of Macrocypraea mammoth have been reported from Trindade and referred to as M . zebra or its older combination Cypraea zebra in the literature and museum collections for quite a long time. Even so, most of the specimens deposited in museum collections consist of fragmentary and empty shells. The oldest lots, which are deposited in the Museu Nacional do Rio de Janeiro ( MNRJ ), date back to 1916. Leal [ 13 ] had already pointed out the large size of this species as distinctive from the continental specimens, regarding it as conspicuous and abundant among beach drift in Trindade. By the time Leal reported his findings, however, no other specimens had been collected in the island since 1976, raising the possibility of extinction. The lack of living specimens and the fact that most of the recovered shells were badly damaged did nothing but reinforce this suspicion. Still, the live individuals collected in the latest samplings nearly a hundred years since the first shells were found at Trindade dismissed this suspicion. They also revealed crucial anatomical information that further distinguished the newly introduced M . mammoth from its ally species, M . zebra . Some of the samples studied herein with precise collection data were obtained during the daytime, since nocturnal diving in the region is forbidden by the Brazilian maritime authority. It is widely known that related species such as M . zebra are mostly active during the night [ 27 ], and we believe the same could be assumed for M . mammoth . The lack of nocturnal samplings may thus help explain why live specimens were never collected in the past. Fig 12. Macrocypraea , 3D reconstructions of shells (CT-Scan). (A-F) M . cervinetta MZSP 131503 (L 58 mm). (A) Frontal view. (B) Same, more ventral portions removed. (C) Anterior view; (D) Posterior view. (E) Right view, right half removed. (F) Same, longitudinal section along columellar axis. (G-L) M . zebra MZSP 38946 (L 100 mm). (G) Frontal view. (H) Same, more ventral portions removed. (I) Anterior view. (J) Posterior view. (K), Right view, right half removed. (L) Same, longitudinal section along columellar axis. https://doi.org/10.1371/journal.pone.0225963.g012 Conchologically, Macrocypraea mammoth is immediately distinguished from the remaining species by its proportionally much heavier shell ( Fig 4G, 4J and 4K ), with thick walls. From M . zebra it differs by its wider and more rounded outline ( Fig 4F and 4J ), and color pattern with proportionally larger beige spots. It differs from M . cervus by having a slightly more elongated outline, with a more tapered posterior end (both laterally and dorso-ventrally, Fig 4H and 4K), fewer teeth on the outer lip, and by a color pattern with larger, less coalescent beige spots, the most basal of which can present a dark brown (ocellated) central area. Moreover, M . mammoth differs from M . cervinetta in having a much larger and heavier shell, more inflated outline, more pointed anterior projections flanking the siphon area, and more spaced apertural teeth. M . mammoth can be further told apart from both other Western Atlantic cypraeid species by having a longer tapered posterior end and a more inflated base ( Figs 2A , 3F and 4F– 4K). Additionally, the shell of M . mammoth has a longer anterior and more flattened anterior end than those of the remaining congeners; the anterior, siphonal region actually looks like a pair of small spatulas laterally flanking the siphon, projected anteriorly. The closest looking species regarding this character is M . zebra , but this pair of anterior projections are shorter and the right one is slightly swollen. Differences in the average number of labial teeth between of the Western Atlantic Macrocypraea had already been reported, with M . cervus showing 15% more teeth on the outer lip than M . zebra [ 27 ]. M . mammoth , is similar to M . zebra in this regard, having an equivalent number of columellar and outer lip teeth. Macrocypraea zebra was so far the single species of the genus that occurred in Brazilian coast. It has 11 nominal synonyms usually found in literature (e.g., [ 3 , 28 , 29 ]), all them described for the North Atlantic and Caribbean. Some synonyms were described without stated locality ( Cypraea dubia Gmelin, 1791 ; C . clauca Röding, 1789 ; C . dissimilis Schilder, 1924 ), with no indication of they are based on South Atlantic specimens. Those that were figured show more elongated shells that are comparable to M . zebra , rather than M . mammoth . Therefore, no synonym of M . zebra can be revalidated in order to designate the new species from Trindade. The CT-Scan examination of the four Macrocypraea species ( Figs 4 , 11 and 12 ) revealed further shell differences. M . mammoth has the thicker shell walls ( Fig 4G, 4J and 4K ), which is common in gastropods living in oceanic islands (personal observation), which is possibly an adaptation against the local parrotfishes ( Scaridae ). The inner slope of the inner lip is particularly informative, as M . mammoth has radially, more densely arranged, and longer teeth ( Fig 4J ), while the remaining species have the shell region has shorter and more spaced teeth, arranged perpendicularly to the longitudinal shell axis ( Fig 11E , Fig 12E and 12K ). The density of teeth of M . mammoth is only comparable to M . cervus ( Fig 11E ). The anterior concavity preceding the canal is larger in M . mammoth ( Fig 4J ) than the remaining species ( Figs 11E, 11F , 12E and 12K ), and has ventral nodes at the end of the teeth, which are absent in the other species. The highly developed inner lip slope is visible in apertural view ( Figs 2B, 2H, 2J , 3B and 4F ), a region that remains hidden in the other species ( Figs 11A , 12A and 12G ). The inner shape of the spire is more spherical in M . cervus ( Fig 11B and 11F ), while it is elongated in the remaining species ( Figs 4G, 4K , 12B, 12F, 12H and 12L ). The left edge of the anal canal usually is thick in M . cervinetta ( Fig 12D ) and in M . mammoth ( Fig 4I ), while it is a narrow fold in M . zebra ( Fig 12J ) and in M . cervus ( Fig 11D ). Regarding the fossil species of Macrocypraea [ 19 , 30 – 32 ], M . mammoth can be distinguished from M . veintensis Perilliat, Avendano & Vega, 2003 (Eocene, Mexico ), in having a much larger shell (that species is about 40 mm ), wider aperture, not so rounded outline, slightly more projected anterior region, and fewer apertural teeth. It differs from M . spengleri Petuch, 1990 ( Figs 5 and 6 in [ 31 ]) and from M . joanneae Petuch, 2004 ( Fig 8.1 in [ 31 ]) (both Pleistocene, Florida) in lacking an inflated shell, with widely rounded posterior region, narrower anterior region of the aperture, in having fewer apertural teeth, and by the less truncated anterior region; from M . joanneae , in particular, M . mammoth still differs in having a much more bulged posterior region of the shell, mainly on its left side; and from M . spengleri , in particular, in having a narrower aperture, mainly anteriorly. It differs from M . anguillana (Cooke, 1919) (Miocene, Anguilla ) in being larger (that species is about 30 mm ) and having a more visible spire. From M . trinidadensis (L Miocene, Trinidad ) by the larger size and by the slightly wider outline. Anatomically, M . mammoth can be easily distinguished from the remaining species through the set of characters listed in its diagnosis. Notably, the presence of pallial papillae bearing 3–5 small aligned terminal projections ( Fig 5B : mp), as opposed to a single terminal projection in the remaining species. Remarkably, the osphradium in M . mammoth looks somewhat deformed, with a shortened posterior branch as if it were severed, and an elongated and slightly narrower anterior branch ( Fig 5B : os). This bizarre osphradium topology was found in all M . mammoth specimens examined herein. The pair of odontophore dorsal protractor muscles ( Fig 6B : m1d) also looks exclusive since the remaining species have different arrangements. In M . cervinetta , the arrangement is somewhat similar (Fig 149: m1d in [ 3 ]), but much smaller, while in M . zebra (Figs 123, 125: m1b in [ 3 ]) and M . cervus ( Fig 9A ), they comprise several small pairs branched far from the median line. The pair of odontophore ventral protractor muscles of M . mammoth , on the other hand, is different in being rather lateralized and bearing 3–4 separated branches ( Fig 6B : m10). The folded portion of inner surface of the esophageal origin ( Fig 7B : ff) is a single set of folds, running along the anterior esophagus, while this folded region is normally separated into a pair of triangular region on posterior half of the dorsal folds of buccal cavity (Fig 124: ff in [ 3 ]) ( Fig 9B : ff). The middle esophagus lacking longitudinal folds flanking the esophageal gland (ec) is also exclusive ( Fig 7B : es), which is normally found in the other species (Fig 124: es in [ 3 ]) ( Fig 9B ). The seminal vesicle of M . mammoth is also very irregularly coiled, as usual for Macrocypraea , however, the structure looks with the same width along its length ( Fig 10A : sv), while those of the other congeneric species have two clearly uniform regions, a left narrow one, and a wide right one (e.g., Fig 10A : sv). The pallial sperm duct ( Fig 7C : ps) is flanked by similar-sized folds, while in the other species an asymmetry is present. The penis of M . mammoth is somewhat as usual for Macrocypraea – an elongated, simple structure curved at the tip. However, it has an exclusive well-developed glandular region, bulging in a side of middle penial region ( Fig 7C : pl). The bursa copulatrix of M . mammoth is also different in having a long duct and a single wide, thin-walled region at tip ( Fig 7D : bc), while in the remaining species the bursa is strongly bent, with a short or absent duct, and bearing strong inner folds (Figs 143, 153: bc in [ 3 ]) ( Fig 8C : bc). With the descriptions of Macrocypraea mammoth and M . cervus , the anatomy of every extant species of Macrocypraea has now been described in detail. Even so, the anatomical diagnosis of the taxon remained unchanged. Nevertheless, adding the present data to the morphology-based phylogeny by Simone [ 3 ], which already contained M . zebra and M . cervinetta , reinforces the monophyly of Macrocypraea . The main characters are: 1) osphradium separated from posterior region of the gill; 2) pair of odontophore muscles mc comprising two bundles; 3) pair of m11 with insertion surrounding ventral region of radular sac base; 4) bursa copulatrix located at middle level of pallial oviduct. Another set of character are present in remaining tree species excepting M . cervus , as follows: 1) pallial papillae stubby, broad, with narrow base; 2) odontophore pair m7 originating at anterior border of ventral m4 branch; 3) bursa copulatrix U-shaped, bearing inner folds. A close relationship of Macrocypraea with Cypraea s.s. was obtained by morphological phylogenetic approach [ 3 ], and it was somewhat corroborated by molecular approach ( Fig 4 in [ 16 ]; Fig 2 in [ 20 ]), although other two genera not included in the morphological study resulted still closer– Leporicypraea Iredale, 1930 , and Mauritia Troschel, 1863 . The discovery of a new cypraeid species is an uncommon event in recent times and serves two immediate purposes. First, it is as a reminder of how poorly known the Brazilian mollusk fauna is, particularly in remote areas such as oceanic islands. Second and most importantly, it draws attention to the necessity of protecting these animals, which, because of the endemicity and rarity, may become endangered in the near future.