Holothuriophilus trapeziformis Nauck, 1880 (Decapoda: Pinnotheridae) from the Pacific coast of Mexico: taxonomic revision based on integrative taxonomy

Morphology

Fifty-five crabs belonging to Holothuriophilus trapeziformis were extracted from the coelom and intestine of the sea cucumber Holothuria (Halodeima) inornata. These were collected in 11 locations from the Pacific coast of Mexico (Fig. 1). These localities were grouped into two arbitrary regions designated as the northern region (Sinaloa state; which include the type locality Mazatlan) and the southern region (Guerrero and Oaxaca states). Hosts were manually collected through skin and SCUBA diving at a maximum depth of 10 m. The collected material was labeled and fixed according to Elías-Gutiérrez et al. (2018) protocol for tissue preservation and DNA analyses. Due to the size of the specimens and the thickness of the cuticle, we injected ethanol into the body through the joints of the appendices with insulin syringes.

All biological material (Table S1) was classified and deposited in the Scientific Collection of Marine Invertebrates of the Laboratorio de Sistemática de Invertebrados Marinos (LABSIM) at Universidad del Mar (UMAR), Oaxaca, Mexico (OAX-CC-249-11). Hosts were identified with specialized literature (Solís-Marín et al., 2009; Honey-Escandón & Solís-Marín, 2018).

For the analysis of the taxonomic status of Holothuriophilus trapeziformis specialized literature from Nauck (1880), Manning (1993), Ng & Manning (2003), Ahyong & Ng (2007), and Campos, Peláez-Zárate & Solís-Marín (2012) was reviewed. Likewise, for H. pacificus, Poeppig (1836), Nobili (1901), Rathbun (1918), and Garth (1957) were reviewed.

The species description follows the terminology of Manning (1993). We consider the gonopod terminology as first gonopod and second gonopod of the male, according to Becker, Türkay & Brandis (2012). The setae terminology is based on Garm & Watling (2013). Drawings were made with the help of a camera lucida and then digitalized in a vector format. Pictures were taken with a Nikon D5100 digital camera. Measurements are given in millimeters and latitude and longitude were obtained from Google Earth^TM.

Because we were only able to obtain eight specimens (three males and five females) from the northern region (Pinitos Beach in the type locality Mazatlan, Sinaloa, Mexico), in contrast to 47 (six males and 41 females) from localities in the southern region, and due to morphological variability observed between these two regions, it was necessary to standardize the observations by using specimens in the same stage of development. Eight millimeters carapace width was the common size in both, the northern and southern regions, in males and adult ovigerous females. This size-group was used to illustrate and discuss the observed morphological variability. The specimen and the dissected appendages were mounted on a plastic clay base for standardizing the observations, and to make the drawings. For the carapace contour, the samples were mounted, so the dorsal view of the posterior margin line of the carapace was observed. For the Mxp3, we extracted it from its base to obtain both endopod and exopod and to mount it with the articles in the same perspective. The cutting edge of the fingers’ chelae was cleaned of dirt to see all the teeth. The first gonopod was extracted from its base, and the setae cleaned of dirt.

Abbreviations used in the text: CL, carapace length (taken as the middle line from the frontal margin to the posterior margin of the carapace); CW, carapace width (measured in its medium-anterior portion); Mxp2, second maxilliped; Mxp3, third maxilliped; WL, walking legs 1 to 4 (thoracopods 2–5).

DNA extraction and PCR amplification

Genomic DNA of individuals of Holothuriophilus trapeziformis was extracted from biological material collected in the field and some individuals from the OAX-CC-249-11 regional collection of the UMAR, using tissue from the walking legs, the chelae, or eggs from the ovigerous females. Tissues were placed into 96-well microplates with a drop of 96% ethanol, and DNA extraction was carried out following the standard glass fiber method consisting of a mix of Proteinase K with an invertebrate lysis buffer according to Ivanova, De Waard & Hebert (2006). Following the DNA extraction, the PCR mixture with a final volume of 12.5 μl contained 2 μl of Hyclone ultrapure water (Thermo Fisher Scientific, Waltham, MA, USA), 6.25 μl of 10% trehalose (previously prepared: 5 g D-(+)- trehalose dihydrate (Fluka Analytical, St. Louis, MO, USA) in a total of 50 ml of molecular grade ddH₂O), 1.25 μl of 10X PCR Platinum Taq buffer (Invitrogen, Waltham, MA, USA), 0.625 μl of 50 μmol/L MgCl2 (Invitrogen, Waltham, MA, USA), 0.0625 μl of 10 μmol/L dNTP (KAPA Biosystems, Wilmington, MA, USA), 0.125 μl of each 10 μmol/L primer, 0.06 μl of PlatinumTaq DNA polymerase (Invitrogen, Waltham, MA, USA), and 2 μl of DNA template. All specimens were amplified with the Zooplankton primers (ZplankF1_t1 and ZplankR1_t1, see Prosser, Martínez-Arce & Elías-Gutiérrez, 2013 for details). The reactions were cycled at 94 °C for 1 min, followed by five cycles of 94 °C for 40 s, 45 °C for 40 s and 72 °C for 1 min, followed by 35 cycles of 94 °C for 40 s, 51 °C for 40 s and 72 °C for 1 min, with a final extension of 72 °C for 5 min. PCR products were visualized on a pre-cast 2% agarose gels (E-Gel^© 96, Invitrogen, Waltham, MA, USA), and the most intense positive products were selected for sequencing.

Sequencing and DNA barcode

Selected PCR products were sequenced using a modified (Hajibabaei et al., 2005) BigDye^© Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystem, Inc., Waltham, MA, USA), and then sequenced bidirectionally on an ABI 3730XL automated capillary sequencer using M13F and M13R sequence primers at the Biology Institute at the National Autonomous University of Mexico and at the Eurofins Genomics Louisville Laboratory. Sequences were edited using CodonCode^© v 3.0.1 (CodonCode Corporation, Dedham, MA, USA) and uploaded to BOLD. In some cases, the original forward and reverse trace files uploaded to BOLD were checked again, consensus assembly was generated, and edited manually with Sequencher^© 4.1.4. (Gene Codes Corporation, Ann Arbor, MI, USA), and then they were aligned using BioEdit^© (Hall, 1999).

Likelihood tree and distance analysis

COI sequences generated for Holothuriophilus trapeziformis in this study were compared with COI sequences from other pinnotherids collected in the Eastern Pacific coast of America, available in BOLD and/or GeneBank (Table S2). Sequence data, trace files, and primer details for all H. trapeziformis specimens and for the outgroup species are available under the dataset name “Holothuriophilus trapeziformis from Mexico” (DOI: dx.doi.org/10.5883/DS-PINMX1HT) in the Barcode of Life Data System (barcodinglife.org). Additionally, H. trapeziformis sequences were uploaded to GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Accession numbers are noted in Table S2.

We calculated the best-fitting evolution model of nucleotide substitution for distance based on COI alignments for the Maximum Likelihood (ML) tree accordingly to the Akaike (AIC) and Bayesian (BIC) criterions (Darriba et al., 2011) using jModelTest^© 2.1.10 (Posada & Buckley, 2004). The final tree was estimated with 1,000 bootstrap replicates in MEGA^© 7.0 (Tamura et al., 2013). With the compress/expand feature of MEGA we prepared the final tree. Also, we estimated the interspecific COI genetic distances for the dataset using the Kimura-2 parameters distance method in MEGA. Values greater than 3% were considered the threshold for the delimitation of the species (Hebert et al., 2003).

Acronyms used in the text: BOLD, barcode of life database (boldsystems.org); BIN, barcode index number (sensu Ratnasingham & Hebert, 2013); BOLD-ID, Specimen ID in the Barcode of Life Data System; CNE-ICML-UNAM, National Collection of Echinoderms of the Institute of Marine Sciences and Limnology of the National Autonomous University of Mexico; DC-NHM, Division of Crustacea, Natural History Museum, Smithsonian Institution; SMF-ZMG, Senckenberg Museum für Naturkunde, Zoologisches Museum Göttingen University, Humboldt Universität, Berlin; UABC, Autonomous University of Baja California, Mexico; UMAR, Universidad del Mar campus Puerto Angel, Oaxaca, Mexico.

Collectors: AEV, Aidé Egremy Valdés; AGF, Andrea Glockner Fagetti; CCA, Carlos Cruz Antonio; AHM, Adanely Hernández Muñoz; FBV, Francisco Benítez Villalobos; FCC, Fernando Cortés Carrasco; HMC, Humberto Mesa Castillo; KFL, Karen Lizbeth Flores López; KMB, Karen Mesa Buendía; RGF, Rebeca Granja Fernández; VCH, Valeria Chavez García.

For this study, we obtained a field collecting permit for non-commercial scientific research purposes from Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (SAGARPA) and Comisión Nacional de Acuacultura y Pesca (CONAPESCA) (Collecting permit: PPF/DGOPA-301/17).

Holothuriophilus trapeziformis Nauck, 1880

(Figs. 2A–2G, 3A–3D, 4A–4K, 5A–5K, 6A–6D)

Holothuriophilus trapeziformis Nauck, 1880: 24, 66 [ovigerous female type]. —De Man, 1887: 721–722 [female (CW = 13.8 mm, CL = 10.5 mm)]. —Manning, 1993: 524–528, Fig. 3c [resurrected to Holothuriophilus]. —Ng & Manning, 2003: 903, 916–918, Figs. 7C–7F [designated female lectotype (CW = 7.7 mm, LC = 4.8 mm): SMF-ZMG 67/565a]. —Ahyong & Ng, 2007: 213–214, Fig. 20 [redescribed and refigured]. —Campos, Peláez-Zárate & Solís-Marín, 2012: 57–62, Figs. 1A, 1B, 2A–2D [female (CW = 9.1 mm, CL = 5.5 mm)].

Pinnotheres trapeziformis.—Bürger, 1895: 380–381, plate 9, Fig. 26, plate 10, Fig. 25 [female type (CW = 14 mm, CL = 10 mm), male (CW = 5 mm, CL = 8.5 mm)]. —Adensamer, 1897: 107. —Schmitt, McCain & Davidson, 1973: 5, 13, 89 [list].

Pinnoteres trapeziformis Balss, 1957: 1,417 [not 1956 fide Schmitt, McCain & Davidson, 1973].

Pinnixa barnharti (no Rathbun, 1918) Caso, 1958: 329; 1965: 254–26.

Holothuriophilus sp. Campos, Díaz & Gamboa-Contreras, 1998: 377, Fig. 1E.

Material examined: 56 specimens: 25 ovigerous females, 22 females, nine males (Table S1).

General distribution: Tropical Mexican Pacific.

Previous records: Mazatlan, Punta Tiburón (Sinaloa); Ixtapa (Guerrero).

New records: Pinitos Beach (Sinaloa); Nudista Beach, Zacatoso Beach, Caleta de Chón Beach (Guerrero); Agua Blanca Beach, Coral Beach, Camarón Beach, Panteón Beach, Estacahuite Beach, La Tijera Beach, San Agustín Beach, El Tejón Beach (Oaxaca).

BIN: BOLD:ADE9974

Carapace size (mm): See Table S1.

Diagnosis. Carapace transversely subrectangular, suboval or subtrapezoidal. Crestated anterolateral margin, a hepatic notch with a blunt tooth inside. Inner surface of merus and carpus of chelipeds densely setose; ventral inner margin of the propodus with a row of short setae, without a gap when the cutting edge of propodus and dactylus meet; cutting edge of the dactylus with proximal denticles, a conspicuous medial tooth, and a distal convex or acute projection. Dorsal surface of the merus of WL1, WL 3 and WL 4 with setae, WL2 without seta. Abdomen with six somites plus free telson; on male, margin of somite 4 to 6 concave, telson subrounded. First gonopod notably curved outward from its mid-distal portion.

Description: Male (Figs. 2A–2C; UMAR-DECA-308; CW = 11 mm, CL = 7 mm): Carapace, transversely subtrapezoidal, wider than long, CW/CL ratio ca. 1.6, mid-anterior portion widest; anterolateral margins slightly projected, crested, a hepatic notch with a blunt middle tooth (Figs. 3A, 3B; bold arrow); dorsal surface convex, smooth, undefined regions; mid-posterior and posterolateral surface with microscopic pits with variable pilosity and size (Figs. 3A); inferior lateral margin of the carapace with abundant plumose setae (Fig. 3A).

Front bilobed, scarcely visible in dorsal view, margin granulated, surface slightly pubescent (Fig. 3B).

Orbits small; eyes pigmented, same size as the orbits; ocular peduncle scarcely pubescent.

Antennules robust; peduncle with two segments, biflagellate, transversely folded inside fossae; upper flagellum with two articles, second article the longest and sharpened distally with apical setae (Fig. 4Ba); lower flagellum conic with four articles decreasing in size, the first three articles with a transverse line of simple setae, fourth article with two transverse lines of simple seta (Fig. 4Bb).

Antennae long, slender, with 12 articles, last article with short apical setae (Fig. 4A).

Pterygostomian region pubescence (Fig. 3B). Buccal frame trapezoidal, completely covered by Mxp3. Mxp2 endopod with five articles, with setae (Fig. 4Ca), dactylus subrounded and shorter than propodus (Fig. 4C); exopod with an article, wider distally, external surface with an elevated ridge (Fig. 4Cb), flagellum with long apical setae (Fig. 4Cc), epipodite long, distal margin rounded (Fig. 4Cd). Mxp3 ischiomerus fused without suture line, width/length ratio 0.7, mesial margin convex with setae, medial margin with a conspicuous projection (Fig. 4Da); carpus subconical, with short setae; propodus subconical (Fig. 4Dc); subspatuliform dactyl, widens distally (Fig. 4D), exceeds the length of the propodus, external surface with short plumose setae, external margin with long plumose setae; exopod with one article and a flagellum, external margin and external surface with short simple setae, slender flagellum, with plumose long setae (Fig. 4E).

Third sternal plate with anterior margin sinuous, anterolateral angles with crenulated margin (Fig. 3C), surface scarcely pilose (Fig. 3C); fourth plate slightly globose, surface with microscopic pits (Fig. 3C), anteroexternal angle curved outward, with margin crenulated (Fig. 3C).

Chelipeds subequal (Fig. 2A); external and lower surface of merus and lower margin of carpus with plumose setae (Figs. 2B); chelae wide with ventral margin microscopically granulated (Figs. 4F, 7C), dorsal margin slightly crested and bent inwards; fingers wider than longer, spooned with acute tip subdistally (Fig. 4F), without a gap where the cutting edge of propodus and dactyl meet (Fig. 7C). Movable finger shorter than fixed finger, crossed inward when closed. Movable fingers of both chelae with cutting edge sinuous, three medial teeth (Fig. 4F) and a subdistal convex projection (Fig. 4F). Fixed finger cutting edge of both chelae with nine teeth and subdistal surface flatted (Fig. 4F), ventral margin with short setae.

Walking legs similar in both sides of the body, relative length WL3 > WL2 > WL1 > WL4, segments short, robust, compressed and dorsal margin crested, ventral surface with plumose setae; dorsal margin of merus of the WL1, WL3, WL4 with plumose setae, and WL2 naked; dactylus with acute tips, curved, and stout; length of the dactylus subequal to propodus of the WL1–WL3, and WL4 shorter than propodus (Fig. 3A).

Abdomen symmetrical, subtriangular, six free somites and telson margin with short setae, lateral margin from somites 4–6 slightly concave and narrowing, telson subrounded (Fig. 3D).

First gonopod slender, margins sinuous, mid-distal portion notably curved outwards, surface with abundant plumose setae (Fig. 3E). Second gonopod small, slightly bent inwards, tip pointing upwards, distal margins convex with shallow notch (Fig. 3F).

Female (Figs. 2D–2F; UMAR-DECA-307; CW = 10.50, CL = 7): Same as the male but with less abundant setae in the pterygostomian region and ventral margin of the chelae; dorsal surface of the merus of the WL1–4 and inner surface of the merus and carpus of the cheliped with long and abundant setae. Carapace slightly more convex. Abdomen subovate. See variation section for more details.

Color in life: Body beige or creamy white, dorsal surface of carapace and chelipeds carpus, and on the external surface of the chelae with red patches. In fixed and preserved specimens this pattern of color remains, or it could change from red to light or dark brown (Figs. 2A–2F).

Habitat: Marine. Associated with the sea cucumber Holothuria (Halodeima) inornata, living in its coelom and inside its intestine (Fig. 2G). This holothurian inhabits rocky-sand sea floor in shallow waters (0–18 m).

Variation: Morphological variation observed between the 8 mm individuals from the two regions (northern and southern) showed three general carapace shapes: subrectangular, suboval and subtrapezoidal.

The subrectangular carapace (Figs. 5A and 6A) was observed in 33% of the males and in 11% of females (three and five specimens, from the northern region). It has projected and straight margin frontal lobes, and straight anterolateral margin with a deep hepatic notch, eroded, and extended over the carapace (Fig. 5A) in males but in females it is less conspicuous (Fig. 6A). Males have truncated and scarcely projected anterolateral margins in which the anterior portion is concave (Fig. 5A) compared to straight in the females (Fig. 4A).

The subovate carapace (Figs. 5E, 5I, 6D, 6G) was observed in 56% of the males and in 85% of females (five and 40 specimens from the southern region). It has oblique and scarcely projected frontal lobes; anterolateral margin continues smoothly to the lateral margin forming a convex lobe (Figs. 5E, 5I), the hepatic notch in the males is deep, eroded and extended (Figs. 5E, 5I), while in the females is shallow, slightly eroded, and less extended over the carapace (Figs. 6D, 6G).

The subtrapezoidal carapace (Figs. 2D, 7A) is present in 11% of the males and in 2% of females (one and two specimens from the southern region). It has scarcely projected frontal lobes, oblique and straight anterolateral margin ending lateral lobes.

Regardless of the carapace shape, the females have a more convex shell and margin of the frontal lobes and the eyes are not visible in dorsal view, and only a slight hepatic notch can be seen (Figs. 6A, 6D, 6G). This shape was more frequent in ovigerous females (10 specimens, 67%) than in non-ovigerous (five specimens, 33%).

Despite the variation in the carapace shape of both sexes, all the ratios (v.g. CW/CL ratio, length between the notch of the frontal lobes to the external orbital angle, length between the external orbital angle to the anterolateral angle, ishiomerus width/length ratio of the Mxp3) were constant.

Regardless of the shape of the carapace, the Mxp3 features show very subtle variations resulting from mounting and drawing. However, the observed variations are: The ischiomerus inner margin has a blunt or slightly acute projection (Figs. 5Ca, 5Ga, 5Ka, 6Ca, 6Fa, 6Ia). The dorsal margin of the carpus from convex (Figs. 5Kb, 6Cb, 6Fb, 6Ib) to straight (Figs. 5Cb, 5Gb). Distally expanded subspatulated dactylus (Figs. 5Kd, 6Cd) or narrower suboblong one (Figs. 5Cd, 5Gd, 6Fd, 6Id). However, in all cases there is a projected ridge on the internal surface of the carpus with setae, and the distal margin of dactylus slightly overreaches the propodus (Figs. 5Cd, 5Gd, 5Kd, 6Cd, 6Fd, 6Id).

The chelae fingers ornamentation is most variable and does not show a relationship with carapace shape or size. In both sexes the cutting edge of the movable finger has two or three proximal teeth (Figs. 5B, 5F, 5J, 6B, 6E, 6H). These teeth could be blunt or acute, but in the females, they always are acute. The medial tooth in males could be simple (Figs. 5B, 5J) or bicuspid (Fig. 5F), but in females it is always simple (Figs. 6B, 6E, 6H). The subdistal projection in both sexes can be blunt (Figs. 5B, 5J, 6B, 6H) or acute (Figs. 5F, 6E). The cutting edge of the fixed finger is more variable between sexes. In males it has six to nine proximal teeth (Figs. 5B, 5F, 5J), but in females it has 4 to 13 (Figs. 6B, 6E, 6H). In males it has one middle tooth which is always bicuspid (Figs. 5B, 5F, 5J) but in females could be bicuspid (Figs. 6B, 6E) or simple (Fig. 6H). Only one specimen (DECA-1172) had different sized chelae and a different teething pattern on the cutting edge of the fixed finger (Figs. 6J, 6K).

The first gonopod is related to the carapace shape. In caudal view it is similar between males of the northern region with a subrectangular carapace and that of the southern region of Oaxaca with suboavate and subtrapezoidal carapace. In these, the external and internal margins are sinuous (Figs. 5D, 5L), the curvature degree is approximately 90° (Fig. 5D) and 75° (Fig. 5L). The tip of the external margin is truncated (Figs. 5De–5Df, 5Le–5Lf), and the ventral margin of the tip has a blunt projection (Figs. 5De, 5Le).

Instead, other males from Guerrero, in the southern regions, with a subovate carapace, have the external and internal margins of the first gonopod less sinuous and the curvature degree is approximately 65° (Fig. 5H). The tip of the external margin is convex (Figs. 5He–5Hf), and the ventral margin of the tip has an acute projection (Fig. 5He).

Remarks: The taxonomic history of Holothuriophilus trapeziformis was synthetized by Campos, Peláez-Zárate & Solís-Marín (2012), and they highlighted the fact that the specimen identified by Bürger (1895) as a male based on the shape of the abdomen is actually a female. We observed the same situation in several young individuals. The presence of pleopods in all of the abdominal somites confirms that they are also females. In juvenile males the lateral margins of the abdomen are straight instead of concave and the first and second gonopods are present. This finding allows us to present the complete male morphology of H. trapeziformis.

All the biological material examined shows phenotypic variation, particularly between the individuals from the type locality in Mazatlan (northern region) with respect to those from of Guerrero and Oaxaca (southern region), but COI gene shows no differences.

With our detailed description of the male morphology, it is now possible to differentiate Holothuriophilus trapeziformis from H. pacificus with certainty by the carapace shape and CW/CL ratio. In H. trapeziformis, it can be subrectangular (Figs. 5A, 6A; CW/CL = ∼1.6), suboval (Figs. 5E, 5I, 6D, 6G; CW/CL = ∼1.6) or subtrapezoidal (Figs. 1A, 1D, 2A, 7A; CW/CL = ∼1.6), while in H. pacificus it is subcuadrangular (Fig. 7E; CW/CL = ∼1.2).

Nevertheless, the mxp3 does not present differences between both species, except for the flagellum of the exopod that according to the illustrations is robust (Figs. 7B, 7J, 7K, 8A) in H. trapeziformis, and slender in H. pacificus (Figs. 7F, 8D).

The abdomen of Holothuriophilus trapeziformis is subtriangular in males, with lateral margins narrowing from the fourth to the sixth somite. The third somite has convex lateral margins. The sixth somite has concave lateral margins, and the telson is subrounded and wider than long (Fig. 8B). In H. pacificus, it is triangular, the lateral margins are almost straight. The third and sixth somite lateral margins are concave, and the telson is subtriangular and more extended than wide (Fig. 8E).

In the case of adult ovigerous and non-ovigerous females of Holothuriophilus trapeziformis, the abdomen is suboval and wider than long. The first somite has convex lateral margins. The second somite has sinuous distal margins. The third somite has oblique and downward lateral margins. The sixth somite has oblique and outward lateral margins, and the telson has a length to width ratio ca. 0.2 (Fig. 7D). In contrast, in H. pacificus it is suboval and longer than wide. The first somite has concave lateral margins. The second somite has almost straight distal margins. The third somite has oblique and upwards lateral margins. The sixth somite has convex lateral margins, and the telson has a length to width ratio ca. 0.3 (Fig. 7H).

The first gonopod of Holothuriophilus trapeziformis has a sinuous lateral margin with a larger distal portion curved outwards, with abundant setae (Fig. 8C). In H. pacificus it is straight, with the distal portion slightly curved outwards, and less setae (Fig. 8F).

Distribution and ecological comments: With this study we increased the previous known distribution range that went from Punta Tiburón (Sinaloa state) to Playa Tejón (Oaxaca state). We found crabs in the coelom cavity and near the cloaca of the host, as mentioned by Manning (1993), Campos, Peláez-Zárate & Solís-Marín (2012), and Honey-Escandón & Solís-Marín (2018). For the first time, we found the crab within the intestine (Fig. 1G).

Holothuria (Halodeima) inornata is distributed throughout the Tropical Eastern Pacific from the Gulf of California, Mexico to Ecuador, and in the temperate island Lobos de Afuera, Peru (Prieto-Rios et al., 2014; Honey-Escandón & Solís-Marín, 2018). It also represents an important fishery resource throughout its distribution range (Santos-Beltrán & Salazar-Silva, 2011). There are no records for Holothuriophilus trapeziformis outside the Pacific coast of Mexico.

DNA Barcodes

From the 56 crabs examined (Table S1), 51 were processed. The number of base pairs was between 549 bp and 648 bp for 37 specimens with a sole Barcode Index Number (BIN; Ratnasingham & Hebert, 2013) in the BOLD database: ADE9974. Of those, 35 produced high quality barcodes. The 14 crabs that could not be amplified correspond to old museum material and recent collections that were not fixed according to the Elías-Gutiérrez et al. (2018) protocol. A BLAST query in GenBank confirmed our sequences to belong to a brachyuran lineage.

Maximum likelihood tree and genetic distance analysis

The best nucleotide substitution model according to the AIC and BIC criterion was General Time Reversible under a gamma distribution (GTR+G) model (Nei & Kumar, 2000). The Maximum-Likelihood (ML) distance method under the selected model delimited the 37 sequences of Holothuriophilus trapeziformis from the dataset (DS-PINMX1HT) in a single cluster. The cluster of H. trapeziformis is well separated from H. pacificus as shown in Fig. 9, with a 12% to 14% divergence among all specimens. Holothuriophilus is also close to the Calyptraeotheres clade, but far from other species (Fig. 9) with an interspecific divergence ranging from 12% to 19%. The intraspecific divergences in H. trapeziformis ranged from 0 to 2.2%. This result is congruent with the BOLD distance summary analyses, which shows an average distance of 0.73% and a maximum of 2.27% for sequences with more than 500 bp.