Alheli Rivera

Ilse E. Segura-Zarzosa ^{a, b}, Hortencia Obregón-Barboza ^c, Gopal Murugan ^c,
Christopher B. Boyko ^{d, e}, Gabino A. Rodriguez-Almaraz ^a, Humberto García-Velazco ^f, Alejandro M. Maeda-Martínez ^c, *

^a Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Avenida Universidad s/n, Ciudad Universitaria, 66455 San Nicolás de los Garza, Nuevo León, Mexico

^b Universidad Juárez del Estado de Durango, Facultad de Ciencias Biológicas, Centro de Estudios Ecológicos, Avenida Universidad s/n, Fraccionamiento Filadelfia, 35010 Gómez Palacio, Durango, Mexico 

^c Centro de Investigaciones Biológicas del Noroeste, S.C., Calle IPN 195, 23096 La Paz, Baja California Sur, Mexico 

^d American Museum of Natural History, Division of Invertebrate Zoology, Central Park West @79th St., New York, NY 10024, USA 

^e Hofstra University, Department of Biology, 114 Hofstra University, Hempstead, NY 11549, USA

^f Unidad de Educación Media Superior Tecnológica Agropecuaria y Ciencias del Mar, CBTA-198, kilómetro 25.5 carretera Ensenada – La Paz, 22790 Ensenada, Baja California, Mexico

*Corresponding author: almaeda04@cibnor.mx (A.M. Maeda-Martínez)

Received: 18 April 2024; accepted: 22 October 2024

Abstract

As part of a research project on the taxonomy of the woodlice (Oniscidea) of northern Mexico, isopod collections of Mexican academic institutions were examined. For the first time, the main diagnostic morphological traits of 4 native species are documented using SEM micrographs: Alloniscus mirabilis (Stuxberg, 1875) (Alloniscidae) and Littorophiloscia richardsonae (Holmes & Gay, 1909) (Halophilosciidae) from the northern Pacific coast of Baja California, Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) (Armadillidae) from northern central Durango, which is also the first record of this species in Mexico, and Porcellio virgatus (Budde-Lund, 1885) (Porcellionidae) from the northeastern states of Nuevo León and Tamaulipas. The first 2 COI haplotypes (mtDNA) for P. virgatus are reported. Bayesian phylogenetic analysis of COI haplotypes placed P. virgatus in a clade with a Porcellio dilatatus haplotype with high support, forming a group with Porcellio scaber.

Keywords: Alloniscidae; Armadillidae; Halophilosciidae; Porcellionidae; Systematics; Taxonomy

© 2025 Universidad Nacional Autónoma de México, Instituto de Biología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Revisión taxonómica de cuatro isópodos terrestres (Crustacea: Oniscidea) de México

Resumen

Como parte de un proyecto de investigación sobre taxonomía de las cochinillas (Oniscidea) del norte de México se revisaron colecciones de isópodos en instituciones académicas mexicanas. En este trabajo presentamos una relación taxonómica de 4 especies nativas del norte de México y se documentan por primera vez sus características diagnósticas morfológicas principales con micrografías de MEB: Alloniscus mirabilis (Stuxberg, 1875) (Alloniscidae) y Littorophiloscia richardsonae (Holmes y Gay, 1909) (Halophilosciidae) de la costa norte del Pacífico de Baja California, Venezillo apacheus (Mulaik en Mulaik y Mulaik, 1942) (Armadillidae) de Durango, lo cual representa el primer registro de la especie en México, y Porcellio virgatus (Budde-Lund, 1885) (Porcellionidae) de los estados norteños de Nuevo León y Tamaulipas. Reportamos los primeros haplotipos de COI (ADNmt) para P. virgatus. El análisis filogenético Bayesiano de haplotipos COI colocó a P. virgatus en un clado con Porcellio dilatatus con alto soporte formando un grupo con Porcellio scaber. 

Palabras clave: Alloniscidae; Armadillidae; Halophilosciidae; Porcellionidae; Sistemática; Taxonomía

Introduction

The Oniscidea (Crustacea: Isopoda) from Mexico have received little attention (Souza-Kury, 2000). The last taxonomic revision of terrestrial isopods from this country was published 64 years ago by Mulaik (1960). Jass and Klausmeier (2004) concluded that 86 oniscidean species belonging to 33 genera and 16 families have been recorded in Mexico; 10 of them are considered as exotic species (Garthwaite & Sassaman, 1985; Garthwaite et al., 1995; Mulaik, 1960; Rodríguez-Almaraz et al., 2014; Segura-Zarzosa et al., 2020; Souza-Kury, 2000; Treviño-Flores & Rodríguez-Almaraz, 2012). For the northern part of Mexico (including the states of Aguascalientes, Baja California, Baja California Sur, Chihuahua, Coahuila, Durango, Guanajuato, Jalisco, Nayarit, Nuevo León, San Luis Potosí, Sinaloa, Sonora, Tamaulipas, and Zacatecas) 7 exotic species and only 28 native species have been recorded in this region (Jass & Klausmeier, 2004; Rodríguez-Almaraz et al., 2014; Segura-Zarzosa et al., 2020, 2022). Surprisingly, states with large geographic areas like Chihuahua, Durango, Guanajuato and Zacatecas do not have any native species registered (Jass & Klausmeier, 2004). As part of a research project on the taxonomy of the oniscideans of northern Mexico, we examined 258 isopod collection lots deposited in 4 academic institutions, from which 11 species, belonging to 8 genera and 5 families, were identified. Out of 1,820 specimens, 1,417 belong to 6 exotic species (see Segura-Zarzosa et al., 2020), and 160 specimens belong to 5 native species. The remaining 243 specimens are still under study. The first native species reported from this study, Venezillo stuckchensis (Mulaik, 1960), is endemic to the Baja California Peninsula, and was already redescribed (Segura-Zarzosa et al., 2022). In the present work we give a taxonomic account of 4 native species: Alloniscus mirabilis (Stuxberg, 1875) (Alloniscidae) and Littorophiloscia richardsonae (Holmes & Gay, 1909) (Halophilosciidae) from the northern Pacific coast of Baja California, and Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) (Armadillidae) from Durango, which represents the first record of the species in Mexico. After examination and analysis of molecular data and morphological characteristics, we agree with Schultz’s (1975, 1977, 1982) placement of Porcellionides virgatus (Budde-Lund, 1885) in the genus Porcellio Latreille, 1804. Thus, we report the fourth species, Porcellio virgatus (Budde-Lund, 1885) (Porcellionidae) from the northeastern states of Nuevo León and Tamaulipas with its first 2 identified COI haplotypes (mtDNA). 

Materials and methods

The material examined is deposited in 2 scientific collections housed in the Facultad de Ciencias Biológicas, Universidad Juárez del Estado de Durango (UJED), Gómez Palacio, Durango, and Centro de Investigaciones Biológicas del Noroeste, S.C. (CIB), La Paz, Baja California Sur. Only adult specimens were examined and sorted by morphotypes according to the general shape of the cephalothorax, second antennae, pereon, pleon, uropods and pleotelson. Specimens of each morphotype were differentiated as males and females based on sexual dimorphism (Schmidt, 2002). Specimens were examined with a stereomicroscope and a digital caliper (Mitutoyo 700-113, Kawasaki, Japan) was used to record their total lengths (TL). The identity of the material examined was established following morphological descriptions published by Stuxberg (1875), Budde-Lund (1885), Holmes and Gay (1909), Van Name (1936, 1942), Mulaik and Mulaik (1942), Mulaik (1960), Schultz (1975, 1984), and Taiti and Ferrara (1986). The type material of Venezillo apacheus deposited in the Division of Invertebrates, American Museum of Natural History (AMNH) was also examined. Representative specimens of each species were prepared for SEM examination. They were dissected and dehydrated individually in 100% ethanol baths for 24 h and critical-point dried (Samdri-PVT-3B, Tousimis, Rockville, MD, USA), sputter coated with gold (desk II, Denton Vacuum, Moorestown, NJ, USA), and analyzed with a scanning electron microscope (Hitachi S-300N), at CIB.

Table 1

GenBank accession number and origin of COI sequences of Porcellionidae species used in the genetic distance and phylogenetic analyses.

The classification and order of the families follow Ahyong et al. (2011). The assignation of the species to their respective genera and families follows Schmalfuss and Wolf-Schwenninger (2002), Schmalfuss (2003) and Schmidt and Leistikow (2004). As we mentioned above, we agree with Schultz’s (1975) placement of Porcellionides virgatus in the genus Porcellio. Citations of authorship for species described in Mulaik and Mulaik (1942) are given as “Mulaik in Mulaik and Mulaik, 1942”, rather than “Mulaik and Mulaik, 1942” as is usually seen in publications (e.g., Schmalfuss, 2003; Schultz, 1965). This is because all species in the 1942 paper are listed as being described by “Mulaik” alone, although with no indication of which Mulaik (Stanley or Dorothea) should be so credited.

The taxonomic account includes: name, author, and year of description of the family, genus, species, species synonymy (restricted to Mexican material, except for V. apacheus and P. virgatus where a complete synonymy is provided), diagnosis, and a taxonomic summary including the type locality, distribution in Mexico, general distribution, material examined, and remarks on the material examined. The diagnoses of the species are mainly based on the publications of several authors, which are indicated in each corresponding species account, and are updated according to morphological variations found in the material examined. Types of habitus are based on the eco-morphological categories proposed by Schmalfuss (1984). For each species we present their main diagnostic morphological traits using SEM micrographs. The anatomical terminology used in this work is according to Schmidt (2002, 2003). The section of examined material is organized by state, site, geographical coordinates, collection date, collector’s name, number of males and females examined (with TL range, mean ± 1 sd, between brackets), and catalog code. 

Attempts to obtain DNA from specimens of A. mirabilis, L. richardsonae, and P. virgatus were carried out, but useful genetic material was obtained only from specimens of the latter species, from 2 different localities. Genomic DNA was isolated from pereopods using the Gentra Puregene kit (Qiagen). Fragments of COI were amplified with primers 22F (modified primer of LCO1490) (CAA CAA ATC ATA AAG ATA TTG GAA C) and HCO2198 by adopting the cycling conditions of Tizol-Correa et al. (2009). The sequences were edited in DNA Baser 4.5 (www.dnabaser.com) and aligned in Clustal X (Thompson et al., 1997). Genetic distances (uncorrected p-distance) between the 2 P. virgatus sequences and also among the sequences of the porcellionids used in this study (Table 1) were determined in MEGA X (Kumar et al., 2018). The best nucleotide substitution model determined in jModeltest 2.1.10 (Darriba et al., 2012; Guindon & Gascuel, 2003) was used in the Bayesian phylogenetic analysis with MrBayes v3.2 (Ronquist et al., 2012). COI sequences of other species of Porcellionidae were obtained from GenBank (Table 1). The phylogenetic analysis was run for 10 million generations and a consensus tree was generated after eliminating 0.25 of the trees generated during the analysis. The consensus tree was viewed using the program FigTree v1.4.3.

Results

Descriptions

Order Isopoda Latreille, 1817

Suborder Oniscidea Latreille, 1802

Alloniscidae Schmidt, 2003

Alloniscus Dana, 1854

Alloniscus mirabilis (Stuxberg, 1875)

(Fig. 1A-N)

Rhinoryctes mirabilis Stuxberg, 1875, 51-55 (original description).

Alloniscus mirabilis (Stuxberg, 1875).- Schultz, 1984, 155-160; Schmalfuss, 2003, 10; Jass and Klausmeier, 2004, 4, 6, 18.

For a more comprehensive synonymy see Schultz (1984).

Diagnosis. Habitus type runner. Pleon not narrower than pereon. Dorsal surface covered by small tubercles with triangular setae or scales (Fig. 1A, B, M, N). Cephalothorax approximately 1.3 times as wide as long (Fig. 1A, B); frontal shield (lamina frontalis) with medial prominent quadrangular process (Fig. 1B, D); lateral lobes large and conical (Fig. 1B, D). Compound eyes on sides of cephalothorax, each with 17-20 ommatidia (Fig. 1B, C). First antennae 3-jointed, distal article with subapical and apical aesthetascs, basal article longest and broadest (Fig. 1E). Second antennae not reaching pereon-tergite 3 (Fig. 1A), first article short, second approximately 2 times as long as first (Fig. 1A, D), third and fourth subequal, each a little longer than second, fifth approximately 1.5 times longer than fourth (Fig. 1A, F); flagellum 3-jointed, first article little longer than second, third article lanceolate with apical cone, approximately 1.5 times longer than second; complete flagellum approximately 1.2 times longer than fifth article (Fig. 1F, G). Pereon with pereon-tergite 1 approximately 1.5 longer than rest, which are subequal in size (Fig. 1A), proximal-lateral angles produced forward to surround cephalothorax (Fig. 1A, D) up to anterior margin of compound eyes (Fig. 1A, C), pereon-tergites 1-3 with rounded posterolateral margins, pereon-tergites 4-7 produced posteriorly (Fig. 1A), pereopod 1 with transversal antennal brush composed of groove covered by fringed scales on anterior (frontal) side of carpus (Fig. 1H, I), pereopods 1-3 of male with brush of long setae on ventral side of carpus and merus (Fig. 1H), pereopod 7 with pattern of setae similar in males and females (Fig. 1J). Pleon with pleon-tergites 1 and 2 with lateral parts undeveloped, covered at sides by pereon-tergite 7, pleon-tergites 3-5 broadly expanded laterally, lateral margins forming continuous line with lateral margins of pereon-tergites (Fig. 1K, M), pleopod 1 endopodite (copulatory appendage) elongated with tip slightly divergent (Fig. 1K). Pleotelson (pleon-tergite 6) triangular, of same length or shorter than posterior margins of pleon-tergite 5 (Fig. 1M, N), uropod sympodites with wide lobed posterolateral margins, endopodites inserted in internal margin of sympodites bearing group of long setae on apex, exopodites lanceolate (Fig. 1L, N). Sources: Stuxberg (1875) and Schultz (1984).

Taxonomic summary

Type locality. San Pedro, Los Angeles, California, USA (Schultz, 1984; Stuxberg, 1875).

Distribution in Mexico. Only in northwestern Mexico on the Pacific coast of Baja California Sur in Bahía Magdalena, and in Baja California in Isla Cedros (Schultz, 1984), and Bahía San Quintín (this study).

General distribution. From Pacific Grove, Monterey, California, USA in the north, to Bahía Magdalena, Baja California Sur, Mexico in the south (Jass & Klausmeier, 2004; Schultz, 1984).

Material examined. Bahía Falsa, San Quintín, Baja California, Mexico (30°27’14.34” N, 116°00’06.50” W), on a sand beach, 02-12-2006, coll. A. Maeda, 1 male (8.8 mm), 5 females (8.1-9.7, x 8.8 ± 0.63 s.d. mm TL) (CIB-41B). 

Remarks

As reported by Stuxberg (1875) for the A. mirabilis type specimens from California, the color of the dorsal side of alcohol-fixed specimens examined exhibited a light brown color with a dorsal medial dark line and lateral light and dark spots. Schultz (1984) reported A. mirabilis from Oxnard, California with 12-13 ommatidia, while our specimens have 17-20 ommatidia. 

Armadillidae Brandt in Brandt and Ratzeburg, 1831

Venezillo Verhoeff, 1928

Venezillo apacheus (Mulaik, 1942 in Mulaik & Mulaik, 1942)

(Figs. 2-8)

Cubaris apacheus Mulaik, 1942 in Mulaik and Mulaik, 1942, 8-9 (original description). 

Cubaris apachea Mulaik, 1942.- Van Name, 1942, 313.

Cubaris apacheus Mulaik, 1941 [sic].- Mulaik and Mulaik, 1943, 9.

Venezillo (Venezillo) apacheus (Mul.).- Arcangeli, 1957, 119.

Cubaris apacheus Mulaik and Mulaik, 1942.- Boyko, 1997, 7-8.

Venezillo apacheus (Mulaik, 1942).- Leistikow and Wägele, 1999, 46.

Venezillo apacheus (Mulaik & Mulaik, 1942).- Schmalfuss, 2003, 285.

Figure 1. SEM micrographs of adult male Alloniscus mirabilis (Stuxberg, 1875) from Bahía Falsa, San Quintín, Baja California, Mexico. A, Cephalothorax, second antenna and pereon-tergites in dorsal view; B, cephalothorax in dorsal view; C, left compound eye with ommatidia in lateral view; D, cephalothorax in anteroventral view; E, left first antenna in lateral view; F, left second antenna in ventral view; G, flagellum of left second antenna in ventral view; H, left pereopod 1 in medial view; I, carpal brush of left pereopod 1 in medial view; J, left pereopod 7 in medial view (central appendage in the micrograph); K, pleopods 1-5, endopodites (copulatory appendages) of pleopods 1, uropod sympodites with endopodites and exopodites; L, uropod sympodites with endopodites and exopodites in ventral view; M, pleon-tergites 1-5, triangular pleotelson and uropod sympodites with exopodites in dorsal view; N, pleon-tergite 5, and triangular pleotelson with uropod sympodites and exopodites in dorsal view. Scale bars: A = 3 mm; B = 500 μm; C, G = 300 μm; D, F, H, J, K, L, N = 1 mm; E = 100 μm; I = 200 μm; M = 2 mm.

Figure 2. Adults of Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) from Gómez Palacio, Durango, Mexico. A, C-F are SEM micrographs; B is a stereo light microscope micrograph. A, Habitus of male in right lateral view; B, habitus of female in left lateral view; C, habitus of male in ventral view; D, cephalothorax and pereon-tergite 1 of male in right anterolateral view; E, external surface of frontal shield of male covered by semi-curved scales with scattered tricorn scales; F, external surface of vertex of cephalothorax of male covered by semi-curved scales with scattered tricorn scales. Scale bars: A-C = 2 mm; D = 1 mm; E = 100 μm; F = 200 μm.

Diagnosis. Habitus type endoantennal conglobator (Fig. 2A, B). Color pale yellowish (alcohol preserved specimens). Dorsal surface with scales and tricorn process inserted in diverse positions (Figs. 2E, F; 3E; 7E, F). Cephalothorax approximately 1/3 longer than wide (Fig. 2A-D); frontal shield (lamina frontalis) with proximal lateral antennal lobes for holding proximal portion of second antenna during conglobation (Fig. 2D), posterior margin expanded, remaining a groove behind frontal shield (Figs. 2B, D; 3A). Compound eyes on sides of cephalothorax, each with 8-9 ommatidia in 2 rows (Fig. 3A-D).

Figure 3. SEM micrographs of adult Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) from Gómez Palacio, Durango, Mexico. A, Cephalothorax and pereon-tergite 1 of male in lateral view showing ommatidia of the complex eye; B, left complex eye of male in anterolateral view showing ommatidia; C, right eye of female in lateral view showing ommatidia; D, left eye of female in lateral view; E, frontal shield of male in dorsal view covered with semicircular scales and tricorn scales; F, third article of right first antenna of male with aesthestascs. Scale bars: A, B = 500 µm; C, D, F = 300 µm; E = 100 µm.

First antenna 3-jointed with 13 aesthetascs on distal article (Figs. 2F; 4A), basal article wide and subequal in length as terminal one (Fig. 3A). Second antennae with first article short, second approximately 2 times as long as first, third subequal to second, fourth 1.5 times longer than third, fifth approximately 1.3-1.5 times longer than fourth (Fig. 4B); flagellum 2-jointed, distal article lanceolate, 3 times longer than first, complete flagellum approximately 2/3 of fifth antennal article (Fig. 4B, C). Right and left mandibles with similar pars incisive, lacinia mobilis, and pars molaris (Fig. 5A-D); lateral side of mandibles covered by semi-curved scales with some acute scales (Fig. 5B), pars incisive with 3 round projections, central one largest (Fig. 5C), lacinia mobilis with basal setose lobe (Fig. 5D); second maxilla membranous, laminate, inner lobe smaller, covered by abundant short setae, outer lobe wide, rounded, covered by pubescence; maxilliped palp with 3 articles (Fig. 5E, F). Pereon with pereon-tergite 1 with notch (schisma) along epimeron (Fig. 6A) into which anterior corner of pereon-tergite 2 epimeron fits when animal conglobates; pereon-tergite 2 with large digitiform lobe on anterior ventral side (Fig. 6B); pereon-tergites 3-5 with rounded epimeron (Fig. 2A, B); pereopod 1 with antennal brush composed of groove covered by transversal scales on anterior (frontal) side of carpus, inner side of merus, carpus and propodus with spines; sternite 7 with medial process apically bilobate, covering proximal portion of genital papilla (Fig. 6E, F). Pleon with pleon-tergites (pleon-segments) 1-2 with lateral parts undeveloped and covered at sides by pereon-tergite 7 (Fig. 2A, B); pleon-tergites 3-5 broadly expanded laterally, lateral margins forming continuous line with lateral margins of pereon-tergites (Fig. 2A, B); male genital papilla with lanceolate ventral shield (Fig. 6F); pleopod 1 endopodite (copulatory appendage) elongated with acute apex (Fig. 7A). Pleotelson wide at base, becoming constricted approximately at 3/4 of length, then expanding to truncate caudal margin, dorsomedial surface slightly raised, some specimens with line (somewhat keeled) (Fig. 7A-C); uropod sympodites (uropod protopodites) enlarged and flattened (Fig. 7C), filling space between caudal side of pleon-tergites 5 and lateral side of pleotelson (Fig. 7C, D), exopodites conical and small, inserted on notch in medial margin of sympodites (Fig. 7C, D), endopodites oblong, flat, covered by scales and setae, base covered by basal plate of uropodal sympodite (Fig. 8A-D). Sources: Mulaik in Mulaik and Mulaik (1942) and Van Name (1942).

Taxonomic summary

Type locality. Alice, Brooks County, Texas, USA (Mulaik in Mulaik & Mulaik, 1942).

Type material examined. Holotype male (AMNH 17515) and allotype female (AMNH 17516) with the original label: “Cubaris apacheus M&M, Alice, Brooks Co., Texas, Dec 1939 S-D Mulaik.” Holotype (damaged), ca. 7 mm TL, allotype (partially conglobated), ca. 5 mm TL. Paratypes (AMNH 17517) with the original label: “Cubaris apacheus M&M, Alice, Brooks Co., Texas, Dec 1939 S-D Mulaik.” 12 specimens intact, partly conglobated, mixture of males and females (ca. 4.5-6.5 mm TL), and fragments of 5 specimens.

Figure 4. SEM micrographs of adult Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) from Gómez Palacio, Durango, Mexico. A, Left first antenna of male in lateral view; B, right second antenna of female in dorsal view; C, flagellum of right second antenna of female in dorsal view; D, apex of distal article of flagellum of right second antenna of female in dorsal view; E, labrum and clypeus of male in anteroventral view; F, clypeus and frontal shield of female in anterior view. Scale bars: A = 100 µm; B = 500 µm; C = 200 µm; D = 50 µm; E, F = 300 µm.

Mexican material examined. Gómez Palacio, Durango, Mexico, 23-06-1994, coll. A. Bedolla, 1 male (7.0 mm TL), 2 females (7.6-9.7 mm TL) from the garden of a house (UJED-09).

Distribution in Mexico. Known only from Gómez Palacio, Durango. This is the first record of the species in Mexico, and the first record of a native oniscidean species in the state of Durango.

General distribution. Known from Alice, Kerville, Laredo (Mulaik in Mulaik & Mulaik, 1942), and Jim Wells County (Mulaik & Mulaik, 1943), Texas, USA, and Durango, Mexico.

Remarks

The type material examined fits well the description published by Mulaik in Mulaik and Mulaik (1942). The only difference noted between the type material and the Mexican specimens is that the fifth article of the second antenna is approximately 1.3 times longer than the fourth article in the type specimens, while in the Mexican specimens it is approximately 1.5 times longer than the fourth article (Fig. 4B). According to Mulaik in Mulaik and Mulaik (1942) the most similar species to V. apacheus is V. chamberlini (Mulaik in Mulaik & Mulaik, 1942), a oniscidean from Edinburg, Hidalgo County, Texas, USA, that can be differentiated by having 6 ommatidia, pleon-tergite 5 posterior margins visible in dorsal view, and the inner border of the epimeral schisma not visible in lateral view.

Figure 5. SEM micrographs of female Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) from Gómez Palacio, Durango, Mexico. A, Left mandible in posterior view; B, left mandible in anterior view; C, pars incisus and lacinia mobilis of left mandible, from A; D, pars incisus, lacinia mobilis, and pars molaris of left mandible, from B; E, left maxilliped in anteroventral view; F, left maxilliped palp with 3 articles in anteroventral view. Scale bars: A, B, E = 500 µm; C, F = 100 µm; D = 200 µm.

Halophilosciidae Verhoeff, 1908 (cf. Schmidt, 2003; Schmidt & Leistikow, 2004)

Littorophiloscia Hatch, 1947

Littorophiloscia richardsonae (Holmes & Gay, 1909)

(Fig. 9A-N)

Philoscia richardsonae Holmes and Gay, 1909, 378-379 (original description).

Philoscia richardsonae Holmes and Gay, 1909.- Mulaik, 1960, 158.

Littorophiloscia richardsonae (Holmes & Gay, 1909).- Leistikow and Wägele, 1999, 18; Souza-Kury, 2000, 245; Schmalfuss, 2003, 133; Jass and Klausmeier, 2004, 3, 10; Taiti and Ferrara, 1986, 1350-1354.

For a more comprehensive synonymy see Taiti and Ferrara (1986).

Diagnosis. Habitus type runner (Fig. 9A). Pleon abruptly narrower than pereon. Color brownish with spots on dorsal and ventral sides of body, pereon-tergites with 3 longitudinal dark lines, 1 medial and 2 lateral. Dorsal surface with short minute scale-spines, margins with line of small spines (Fig. 9B). Cephalothorax 2 times as wide as long (Fig. 9B), vertex convex (Fig. 9D), frontal shield prominent, rounded with posteromedial border concave (supra-antennal line) (Fig. 9D), lateral lobes small, subtriangular (Fig. 9B). Compound eyes at anterolateral sides of cephalothorax, each with 12-15 ommatidia (Fig. 9C). First antennae 3-joined with aesthetascs on distal article, basal article longest, broadest (Fig. 9E). Second antennae long, approximately 1/2 of total length, first article short, second and third 2 times as long as first, fourth 2 times longer than third, fifth 1.5 times longer than fourth (Fig. 9F); flagellum 3-joined, first and third articles subequal, slightly longer than second (Fig. 9G). Pereon with pereon-tergite 1 broadest (Fig. 9A), pereon-tergites 3-7 with rectangular lateral borders, pereopod 1 antennal brush with transversal scales on anterior (frontal) side of carpus (Fig. 9H, I). Pleon with pleon-tergites 3-5 posterolateral margins strongly produced and curved backwards, margins with line of small scale-spines (Fig. 9J, M); male pleopod 1 endopodite (copulatory appendage) elongated with acute apex (Fig. 9J); pleopods 1-5 with triangular exopodites (Fig. 9J). Pleotelson triangular with rounded tip and concave lateral borders, margins with line of small scale-spines (Fig. 9M, N); uropod sympodites as long as wide, complete structure of endopodites approximately 2/3 as long as exopodites, in dorsal view endopodites approximately 1/3 as long as exopodites, with long apical setae, inserted in sympodites before exopodites, exopodites long, subconical, lateral margin nearly straight, medial margin somewhat convex (Fig. 9J, L). Sources: Holmes and Gay (1909), Van Name (1936) and Taiti and Ferrara (1986).

Figure 6. SEM micrographs of male Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) from Gómez Palacio, Durango, Mexico. A, Cephalothorax and pereon-tergite 1 with notch (schisma) along epimeron, pereon-tergite 2 with large digitiform lobe on anterior ventral side; B, lateral margins of pereon-tergites 1-4, and left pereopod 2 in ventral view; C, left pereopod 1 in anterior view; D, propodus and dactylus of left pereopod 1 in anterior view; E, sternite 7 with medial bilobate process covering proximal portion of genital papilla in posteroventral view; F, genital papilla with lanceolate ventral shield in posteroventral view. Scale bars: A = 1 mm; B, C = 500 µm; D-F = 200 µm.

Taxonomic summary

Type locality. San Diego, California, USA (Holmes & Gay, 1909).

Material examined. Bahía Falsa, San Quintín, Baja California (30°27’14.34” N, 116°00’06.50” W), on a sand beach, 02-12-2006, coll. A. Maeda, 8 males (4.8-8.3 mm, x 6.9 ± 1.02 s.d. mm TL), 10 females (4.7-8.0 mm, x 6.6 ± 0.94 s.d. mm TL) (CIB-42B).

Distribution in Mexico. Isla Cedros, Baja California (Mulaik, 1960; Taiti & Ferrara, 1986), and Bahía Falsa, San Quintín, Baja California, a new locality record for the species. 

General distribution. From northwestern Mexico to Vancouver Island, Canada (Garthwaite et al., 1985; Schmalfuss, 2003; Taiti & Ferrara, 1986).

Figure 7. Adults of Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) from Gómez Palacio, Durango, Mexico. A, C, D-F are SEM micrographs; B, is a stereo light microscope micrograph. A, Pleon-tergites 3-5, pleotelson and uropod sympodites of male in posterior view; B, pleon-tergites 1-5, pleotelson and uropod sympodites of male in posterodorsal view; C, pleotelson and uropod sympodites with exopodites of female in posterior view; D, left uropod exopodite in sympodite from C; E, dorsal surface of female pleotelson; F, dorsal surface of pleotelson with semicircular scales and tricorn scales from E. Scale bars: A, C = 500 µm; D = 300 µm; E = 100 µm; F = 50 µm.

Remarks

Taiti and Ferrara (1986) reviewed Littorophiloscia and recognized 15 species, 2 of them recorded in Mexico: L. richardsonae from Baja California and L. tropicalis Taiti and Ferrara, 1986 from Yucatán. Taiti and Ferrara (1986) reported specimens of L. richardsonae up to 7 mm of length with 12 or 13 ommatidia and pereopod 1 with an antennal brush with transversal scales on the anterior (frontal) side of the carpus and propodus; our examined specimens have a mean TL of 6.9 mm for males, 6.6 mm for females, the number of ommatidia varied from 13 to 15, and the antennal brush is only on the carpus (Fig. 9H, I).

Figure 8. SEM micrographs of adults of Venezillo apacheus (Mulaik in Mulaik & Mulaik, 1942) from Gómez Palacio, Durango, Mexico. A, Pleotelson and uropod sympodites with endopodites of male in ventral view; B, pleotelson and uropod sympodites with endopodites of female in ventral view; C, uropod endopodites of male in ventral view; D, uropod endopodites of female in ventral view. Scale bars: A, B = 500 µm; C, D = 100 µm.

Porcellionidae Brandt in Brandt and Ratzeburg, 1831

Porcellio Latreille, 1804 

Porcellio virgatus (Budde-Lund, 1885)

(Fig. 10A-L)

Metoponorthus virgatus Budde-Lund, 1879, 4 (nomen nudum).

Metoponorthus virgatus Budde-Lund, 1885, 182 (original description).

Metoponorthus virgatus Budde-Lund, 1885.- Richardson, 1905, 630; Mulaik, 1960, 174.

Porcellionides virgatus (Budde-Lund, 1885).- Van Name, 1936, 241; 1940, 137; 1942, 327; Mulaik and Mulaik, 1942, 7; 1943, 7; Leistikow and Wägele, 1999, 36; Souza-Kury, 2000, 245; Jass and Klausmeier, 2004, 5. 

Porcellionides mulaiki Van Name, 1936, 522.- Van Name, 1940, 137.

Porcellio virgatus (Budde-Lund, 1885).- Schultz, 1975, 185-193; 1977, 152; 1982, 2, 5, 6, 20-21.

“Porcellionides” virgatus (Budde-Lund, 1885).- Schmalfuss, 2003: 214.

Diagnosis. Habitus type runner. Color brown, mottled with light spots, 2 light lateral and 1 medial lines. Body surface with small tubercles and rounded tricorn scales (Fig. 10A, B, D, L). Cephalothorax approximately 2 times as wide as long (Fig. 10B), vertex with convex medial part (Fig. 10B), frontal shield with small rounded lateral lobes (Fig. 10B), posterior frontal line forming slightly medial convex margin (Fig. 10B, D), supra antennal line V-shaped absent (Fig. 10D). Compound eyes each with 19-30 ommatidia (Fig. 10C). First antennae 3-joined; basal article longest and broadest, second short, third with subapical and apical aesthetascs (Fig. 10F). Second antennae with first article short, second 2 times as long as first, third subequal to second, fourth 1.5 times longer than third, fifth approximately 1.5 times longer than fourth (Fig. 10G); flagellum 2-joined, proximal article shorter or subequal to distal article, distal article lanceolate with apical cone (Fig. 10H). Maxilliped palp with 3 articles (Fig. 10E). Pereon-tergites 1-7 with a well marked dorsal transverse furrow (Fig. 10A); pereon tergite 1 with projected anterolateral sides not reaching cephalothorax anterior margin, 1.5 times longer than pereon-tergites 2-7 (Fig. 10A); sternite 7 without medial process (Fig. 10I). Pleon not abruptly narrower than pereon (Fig. 10K); pleon-tergites with acute lateral margins posteriorly directed (Fig. 10K); male pleopod 1 endopodites (copulatory appendages) lanceolate (Fig. 10I); pleopods 2-5 with exopodites semi-quadrangular (Fig. 10I). Pleotelson triangular with rounded tip (Fig. 10K, L); uropod sympodites twice as long as wide, as long as exopodites, endopodites thin, lightly flattened, reaching tip of exopodites, exopodites subconical (Fig. 10J). Sources: Budde-Lund (1885), Richardson (1905), Van Name (1936), Mulaik (1960), and Schultz (1982).

Figure 9. SEM micrographs of male Littorophiloscia richardsonae (Holmes & Gay, 1909) from Bahía Falsa, San Quintín, Baja California, Mexico. A, Cephalothorax with right antenna and pereon-tergites 1-6 in dorsal view; B, cephalothorax in dorsal view; C, left compound eye with ommatidia in anterolateral view; D, cephalothorax in anterior view (left second antenna removed); E, left first antenna in anterior view; F, right second antenna in dorsal view; G, flagellum of right second antenna in dorsal view; H, right pereopod 1 in medial view; I, carpus with brush, protopod and dactyl of pereopod 1 in medial view (from H); J, pleon-tergites 1-5 with pleopods, copulative appendages of pleopod endopodites, uropods sympodites with endopodites and exopodites in ventral view; K, endopodites of uropod sympodites in ventral view; L, right uropod sympodite with exopodite in posteroventral view; M, pleon-tergites 2-5, triangular pleotelson and uropod sympodites with endopodites and exopodites in posterodorsal view; N, triangular pleotelson in dorsal view. Scale bars: A = 2 mm; B, G, I = 300 μm; C, E = 100 μm; D, F, H, L, M = 500 μm; J = 1 mm; K, N = 200 μm. 

Figure 10. SEM micrographs of male Porcellio virgatus (Budde-Lund, 1885) from San José, Gómez Farías, Tamaulipas, Mexico. A, Cephalothorax and pereon-tergites 1-7 in dorsal view; B, cephalothorax in dorsal view; C, right compound eye in dorsal view; D, cephalothorax, first antennae, clypeus, and labrum in ventral view; E, maxillipeds in ventral view; F, left first antenna with aesthetascs in ventral view; G, second antenna in medial view; H, flagellum of second antenna in medial view; I, posterior part of body showing pereon-tergite 7, pleon-tergites 3-5, and pleopods 1-5 in ventral view; J, left uropod with sympodite, endopodite, and exopodite in ventral view; K, posterior part of body showing pereon-tergite 7, pleon-tergites 1-5, and pleotelson in dorsal view; L, pleon-tergite 5, and triangular pleotelson in dorsal view. Scale bars: A, I, K = 2 mm; B, E, H, J = 500 μm; C = 200 μm; D, L = 300 μm; F = 100 μm; G = 1 mm.

Taxonomic summary

Type locality. New Orleans and Florida, USA (Budde-Lund, 1885).

Material examined. Nuevo León: Cola de Caballo, Santiago (25°26’12.18” N, 100°09’12.68” W), under stones, 06-05-2006, coll. A. Maeda, 2 females (5.4-6.8 mm) (CIB-49B); Parque Chipinque, Km 2, San Pedro Garza García (25°36’38.1” N, 100°21’20.8” W), under stones, 06-05-2006, coll. A. Maeda, 1 male (11.0 mm) (CIB-30B1). Tamaulipas: San José, Gómez Farías (23°05’23.1” N, 99°06’40.1” W), natural zone below stones, 30-07-2006, coll. A. Maeda, 3 males (6.8-9.6 mm, mean 7.9 ± 1.21 s.d. mm TL), 2 females (7.5-7.6 mm) (CIB-09B). 

Haplotypic identity. COI haplotypes were obtained from a specimen from Chipinque, Nuevo León (GenBank accession number OK376218), and a specimen from San José, Tamaulipas (GenBank accession number OK376217). The genetic distance between these haplotypes is 2.5% (Table 2). Prior to this study there was no record of DNA sequences for Porcellionides virgatus/Porcellio virgatus in GenBank. The Bayesian analysis placed these haplotypes in a well supported clade within the genus Porcellio with high support rather than with species in the genus Porcellionides (Fig. 11).

Distribution in Mexico. Santiago and San Pedro Garza García, Nuevo León, and San José, Tamaulipas. Mulaik (1960) reported Porcellio virgatus (as Porcellionides virgatus) from Villagrán, Tamaulipas, and Tepic, Nayarit. 

Table 2

Genetic p-distance (%) among COI sequences (623 bp) of Porcellionidae species used in the phylogenetic analysis.

Figure 11. Bayesian consensus tree of phylogenetic relationships of Porcellio virgatus (Budde-Lund, 1885) COI haplotypes from Nuevo León and Tamaulipas, Mexico, with selected GenBank haplotypes of Porcellionides and Porcellio species. GenBank sequences of palaemonids Periclimenes imperator Bruce, 1967 (= Zenopontonia rex [Kemp, 1922]) (DecaNet eds., 2024), and Periclimenes rathbunae Schmitt, 1924 were used as outgroup.

General distribution. Budde-Lund (1885), Van Name (1936), Mulaik and Mulaik (1942, 1943), and Mulaik (1960) reported the species (as Porcellionides virgatus)from the USA in Florida, Louisiana, Mississippi and Texas. Schultz (1982) reported Porcellio virgatus from North Carolina and Georgia, USA.

Remarks

The species has been cited as belonging to Metoponorthus Budde-Lund, 1879(Mulaik, 1960; Richardson, 1905), and to Porcellionides (the senior synonym, see Schmalfuss & Ferrara, 1978) (Jass & Klausmeier, 2004; Leistikow & Wägele, 1999; Souza-Kury, 2000; Van Name, 1936, 1940, 1942). Schmalfuss (2003) listed it as “Porcellionides” virgatus, indicating that the species does not belong to this genus. Schultz (1975, 1977, 1982) was the first carcinologist to recognize the species as Porcellio virgatus from Georgia and North Carolina, USA. The morphological similarity between Porcellio and Porcellionides has been, to date, an issue in recognizing them as separate genera (Dimitriou et al., 2018; Mattern, 2003; Mulaik, 1960; Schmalfuss & Ferrara, 1978; Van Name, 1936). Dimitriou et al. (2018) analysed 2 mitochondrial and 3 nuclear genes and found evidence against the monophyly of both Porcellionidae and Porcellio. The genetic distances they found were considerably high between Porcellionidae genera; e.g. for COI the variation was between 16.9-50.3% (Dimitriou et al., 2018). Mattern (2003) applied small subunit rRNA analyses to study the molecular phylogeny of Oniscidea and found that the genera Porcellio and Porcellionides are not monophyletic groups. He reported that Porcellio scaber Latreille, 1804 is more closely related to Porcellionides sexfaciatus Budde-Lund, 1885 than to Porcellio spinicornis Say, 1818, the latter branching as the sister group of Porcellionides pruinosus (Brandt, 1833). However, considering Mattern’s (2003: Fig. 2) molecular phylogenies, it can be concluded that Porcellio scaber and Porcellionides pruinosus represent 2 different lineages. Herein, we report the first COI haplotypes of the species obtained from specimens from Tamaulipas and Nuevo León. Our molecular analysis is not intended as a revision of the phylogenetic systematics of the group but rather to provide the first information on the molecular identity and the generic placement of these species. The results showed that the genetic distance of our haplotypes is larger when compared with haplotypes of Porcellionides (19.40 to 21.19%) than with haplotypes of Porcellio (15.57 to 19.58%) (Table 2). Besides, the Bayesian analysis placed our haplotypes in a clade with a specimen of Porcellio dilatatus Brandt in Brandt and Ratzeburg, 1831 haplotype with high nodal support, forming a group with Porcellio scaber (Fig. 11). Our specimens fit the diagnostic morphological features of the genus Porcellio proposed by Schmalfuss and Ferrara (1978): 2 pairs of pseudotracheae, unable to conglobate, pereon tergite 1 with caudally concave epimera, eyes with more than 20 ommatidea. The typical supra antennal line V-shape showed by most species of Porcellionides (Schmalfuss & Ferrara, 1978) is absent in our specimens (Fig. 10D) and in specimens examined by Van Name (1936, Fig. 135). Our specimens have the pereon-tergites 1-7 with a well marked dorsal transverse furrow (Fig. 10A), as figured for Porcellionides virgatus by Van Name (1936, Fig. 135). Mulaik (1960) recorded specimens of P. virgatus from Nayarit and Tamaulipas with 21 ommatidia and 10 mm TL. The specimens examined herein have 6.8-11.00 mm TL for males and 5.4-7.6 mm TL for females, with 19-30 ommatidia. The molecular data and morphological characteristics mentioned support Schultz’s assignation of the species as Porcellio virgatus.

Discussion

According to Schultz (1984), 3 Alloniscus species are recognized in North America, A. perconvexus Dana, 1854, known from Laguna Beach, California, USA to Vancouver Island, British Columbia, Canada; A. mirabilis recorded from Magdalena Bay, Mexico to the Pacific Grove, California; and A. thalassophilus Rioja, 1963 known from Isla de Ixtapan, Zihuatanejo, Guerrero, Mexico. The presence of Alloniscus in Mexico was first recorded by Mulaik (1960) who reported A. perconvexus based on specimens from Bahía Magdalena, Baja California Sur, and Isla Cedros and Bahía San Quintín, Baja California. Schultz (1984) mentioned that A. mirabilis and A. perconvexus are similar species, but can be separated by the uropod sympodite which is larger in A. mirabilis resulting in a broadened, extended posterolateral margin (Schultz, 1984, Fig. 3B, H) (Fig. 1L-N), and by the cephalothorax anterolateral lobes, which are well developed and extending far beyond the eyes in A. mirabilis (Schultz, 1984, Fig. 3D, E) (Fig. 1B, D), but small and well defined in A. perconvexus (Schultz, 1984, Fig. 1D). Schultz (1984) argued that Mulaik’s (1960) A. perconvexus illustrations actually represented A. mirabilis. We disagree with Schultz’s (1984) statement because the uropod sympodite illustrated by Mulaik (1960, Fig. 175) shows a straight posterolateral margin typical of A. perconvexus as also figured by Schultz (1984, Fig. 2B), while A. mirabilis has a wide-lobed posterolateral margin (Schultz, 1984, Fig. 3B, H) (Fig. 1L-N). Thus, we agree with Jass and Klausmeier (2004) that these 2 species are native to the northwest Mexican Pacific coast. As with all Mexican oniscideans, the populations of these similar species deserve more detailed morphological and genetic studies, including examination of material from their type localities.

The record of V. apacheus, formerly known only from Texas, USA, increases the number of species of Venezillo in Mexico from 18 (Jass & Klausmeier, 2004; Schmalfuss, 2003) to 19. Venezillo tanneri (Mulaik in Mulaik & Mulaik, 1942) also has been recorded in both USA (Texas) and Mexico (Nuevo León) (Mulaik & Mulaik, 1942; Schultz, 1965). In the southern USA Venezillo arizonicus (Mulaik in Mulaik & Mulaik, 1942) has also been recorded from Robles, Nogales, Olberg and Rock Springs, Arizona, and V. chamberlini (Mulaik in Mulaik & Mulaik, 1942) from Edinburg, Texas; both species are likely also inhabiting northern Mexico (Mulaik, 1960).

The genus Littorophiloscia is not well known in Mexico. Taiti and Ferrara (1986) recognized the presence of L. richardsonae from Baja California and L. tropicalis Taiti and Ferrara, 1986 from the Yucatán. In this study, L. richardsonae was recorded as co-occurring with A. mirabilis on a sand beach of Bahía Falsa in San Quintín, Baja California, sharing a habitat under marine macroalgae.

In Mexico, the genus Porcellio is now represented by the exotic P. laevis and P. scaber, and the putative natives P. scabriusculus Mulaik, 1960 (Jass & Klausmeier, 2004; Schmalfuss, 2003; Segura-Zarzosa et al., 2020), and P. virgatus (this study). Mulaik (1960) described and figured P. scabriusculus as very different from P. virgatus, with 9 ommatidea and spinous tubercles on the dorsal side of the cephalotorax, pereion terguites, and pleon terguites (except the pleotelson). Schmalfuss (2003) suggested that P. scabriusculus is probably not a member of the genus Porcellio. Mulaik (1960) mentioned his decision to keep Porcellionides saussurei (Dollfus, 1896) (a species known from Córdova, and San Luis Potosí, Mexico) in this genus until the Porcellio–Porcellionides complex is clarified. As mentioned above, these morphologically  similar genera are not monophyletic groups (Dimitriou et al., 2018; Mattern, 2003) and delimiting them is an important unsolved problem that limits the understanding of the systematics of the species involved. 

Acknowledgments

We thank an anonymous reviewer and Fernando Álvarez for helpful corrections to improve this article. Ariel A. Cruz Villacorta, Laboratorio de Microscopía Electrónica, CIB, provided expert assistance in the production of SEM images, and Gerardo Rafael Hernández García, Departamento de Extensión y Divulgación Científica, CIB edited the SEM plates and cladogram. Ilse E. Segura-Zarzosa received a doctoral fellowship from Consejo Nacional de Ciencia y Tecnología (No. 551538).

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Alejandro Valdez-Mondragón *, Samuel Nolasco-Garduño

Centro de Investigaciones Biológicas del Noroeste, S.C., Programa Académico de Planeación Ambiental y Conservación, Laboratorio de Aracnología, Km. 1 Carretera a San Juan de La Costa “El Comitán”, 23205 La Paz, Baja California Sur, Mexico

*Corresponding author: lat_mactans@yahoo.com.mx (A. Valdez-Mondragón)

Received: 11 March 2024; accepted: 29 October 2024

Abstract

We describe the morphological and genetic variation in Ixchela azteca Valdez-Mondragón & Francke (Pholcidae) from central Mexico, based on morphological and DNA barcoding evidence. Species delimitation employing the barcoding locus included 3 methods under the corrected genetic p-distances neighbor-joining (NJ) criteria: 1) Assemble Species by Automatic Partitioning (ASAP), 2) General Mixed Yule Coalescent model (GMYC), and 3) Bayesian Poisson Tree Processes (bPTP). The genetic analyses found a genetic p-distance of 3.5% between 2 populations of I. azteca. The molecular methods and morphology were not congruent in delimiting and recognizing 2 possible different species. Instead, 2 allopatric populations of I. azteca are recognized. Also, an updated taxonomic revision of I. azteca is included. In conclusion, the incongruence between the molecular delimitation methods for species delimitation and the morphological, genetic, ecological, and biogeographic evidence maintain I. azteca as a widespread species across central Mexico, with allopatric populations in temperate and semiarid regions.

Keywords: Species delimitation; Integrative taxonomy; CO1; Ecology; Biogeographic provinces

© 2025 Universidad Nacional Autónoma de México, Instituto de Biología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Ixchela azteca (Araneae: Pholcidae), una especie de araña ampliamente distribuida en el centro de México: ¿diversidad subestimada o variación morfológica y genética?

Resumen

Describimos la variación morfológica y genética en Ixchela azteca Valdez-Mondragón y Francke (Pholcidae) del centro de México, basado en evidencia morfológica y código de barras de DNA. La delimitación de especies con el locus de código de barras incluyó 3 métodos bajo el criterio de distancias genéticas p corregidas “neighbor-joining” (NJ): 1) “Assemble Species by Automatic Partitioning” (ASAP), 2) “General Mixed Yule Coalescent model” (GMYC), and 3) “Bayesian Poisson Tree Processes” (bPTP). Los análisis genéticos entre 2 poblaciones de I. azteca encontraron una distancia genética p de 3.5%. Los métodos moleculares y morfología no fueron congruentes para delimitar y reconocer 2 posibles especies, pero sí se reconocen 2 poblaciones alopátricas de I. azteca. Adicionalmente, se incluye una revisión taxonómica actualizada de I. azteca.En conclusión, la incongruencia de los métodos de delimitación molecular para la delimitación de especies en combinación con la evidencia morfológica, genética, ecológica y biogeográfica, corrobora a I. azteca como una especie ampliamente extendida en la región central de México, con poblaciones alopátricas en zonas templadas, pero también en regiones semiáridas.

Palabras clave: Delimitación de especies; Taxonomía integrativa; CO1; Ecología; Provincias biogeográficas

Introduction

The spider family Pholcidae is the ninth-largest spider family in the world and the most diverse within the Synspermiata clade (WSC, 2025). Commonly known as cellar spiders or daddy long-legs spiders, Pholcidae is currently composed of 97 genera and 1,901 species (WSC, 2025). This family is among the most diverse and abundant web-building spiders in the world, mainly occurring in tropical and subtropical forests and with a high number of synanthropic and troglobite species (Huber, 2000, 2011, 2018; Valdez-Mondragón, 2010). 

The genus Ixchela Huber, 2000 (subfamily Modisiminae) currently contains 22 described species (Valdez-Mondragón, 2013, 2020; Valdez-Mondragón & Francke, 2015; WSC, 2025). The genus is composed of relatively large pholcid spiders mainly distributed in Mexico and Central America. These spiders inhabit temperate climate zones in pine, oak, or pine-oak forests at moderately high elevations (1,000-2,950 m) (Valdez-Mondragón, 2013, 2020; Valdez-Mondragón & Francke, 2015). The monophyly of the genus is supported based on morphological and molecular evidence (CO1+16S), with the genus’ divergence time estimated to have occurred in the late Miocene (Valdez-Mondragón & Francke, 2015).

Although pholcid spiders generally have a relatively uniform and conservative somatic morphology, their genitalia (palps and epigyna) and secondary sexual characters (male chelicerae) are highly informative for morphological identification at species level. These morphological features are a source of important and robust evidence and are commonly used for species delimitation in most groups of spiders (Huber, 2003). Spider genitalia usually show little intraspecific, but conspicuous interspecific variation (Eberhard, 1985; Eberhard et al., 1998). However, in certain groups with highly conservative morphology, or species with a wide geographic distribution, species delimitation and identification require additional diagnostic features to differentiate underestimated species diversity. In contrast, other species of spiders have wide geographic variation and even show intrasexual dimorphism within males or females. In the last 4 decades, research has focused on understanding the variation among individuals, leading sexual variation to be understood as the differences exhibited not only between males and females but also among individuals of the same sex within a species (Andersson, 1994; Barraclough, 1995; Gross, 1996; Huber, 2021).

The mitochondrial cytochrome c oxidase subunit 1 (CO1) DNA barcode gene has emerged as the standard animal barcoding marker for many taxa, including spiders (Astrin et al., 2006; Correa-Ramírez et al., 2010; Graham et al., 2015; Hamilton et al., 2011, 2014, 2016; Hebert, Cywinska et al., 2003; Hebert, Ratnasingham et al., 2003; Hebert et al., 2004; Ortiz & Francke, 2016; Planas & Ribera, 2015; Nolasco & Valdez-Mondragón, 2022a; Tahami et al., 2017). The integrative taxonomy approach is generally accepted as being more effective in the diagnosis, delimitation, and description of species or even to understand the variation in different populations within species.

Ixchela azteca Valdez-Mondragón & Francke, 2015from central Mexico and Ixchela abernathyi (Gertsch, 1971) from northeast Mexico represent the species with the widest distributions in comparison with other species of the genus, mainly in temperate regions. However, Valdez-Mondragón and Francke (2015) reported body size variation for populations of I. azteca from temperate regions, bringing into question if this is merely geographic variation in the same species or is evidence of the presence of different species not yet described. Because of that, morphological and molecular evidence are needed to test if I. azteca has a widespread distribution in temperate and semiarid habitats or represents different species.

In this study, we described based on morphological evidence and DNA barcoding (CO1) the variation of different populations of I. azteca including 3 populations from temperate regions of central Mexico and a population from a semiarid habitat in Guanajuato state, Mexico. In addition, an updated taxonomic revision of I. azteca is included herein, including new records for the species.

Materials and methods

The specimens used in this study are deposited in the Colección Nacional de Arácnidos (CNAN), Instituto de Biología, Universidad Nacional Autónoma de México (IBUNAM), Mexico City; and the Colección Aracnológica (CARCIB), Centro de Investigaciones Biológicas del Noroeste, CIB, La Paz, Baja California Sur, Mexico. The specimens from Guanajuato, Mexico are deposited at the CARCIB. Morphological structures, such as female epigyna and male palps, were dissected in ethanol (80%) and cleared in potassium hydroxide (KOH-10%). Habitus, chelicerae, palps, and epigyna were placed on 96% gel alcohol to facilitate positioning and covered with a thin layer of liquid ethanol (80%) to minimize diffraction during photography. The analyzed morphological structures were female epigyna and male palps, dorsal and ventral habitus, and male chelicerae.

A Zeiss Axiocam 506 color camera attached to a Zeiss AXIO Zoom.V16 stereoscope was used to photograph specimen structures. For measurements, the specimens were observed using a Zeiss Discovery V8 stereoscope. All measurements are in millimeters (mm). The distribution map was generated using QGIS v. 2.18.17 “Las Palmas”. Photographs and a map were edited using Adobe Photoshop CS6. Morphological abbreviations follow Valdez-Mondragón (2013, 2020) and Valdez-Mondragón and Francke (2015). Abbreviations: ALE, anterior lateral eyes; AME, anterior median eyes; E, embolus; FAC, frontal apophysis of chelicerae; MSE, median septum of epigynum; PAB, prolateroventral apophysis of bulb; PLE, posterior lateral eyes; PME, posterior median eyes; PP, pore plates; PR, procursus; SAC, sclerotized apophysis of chelicerae; VPP, ventrobasal protuberance of procursus.

The DNA barcoding locus was obtained from a total of 59 specimens belonging to 19 species of Ixchela previously described by Valdez-Mondragón (2013, 2020) and Valdez-Mondragón and Francke (2015), and combined with available CO1 sequences, including I. azteca and a single outgroup (Physocyclus dugesi Simon) for the genetic p-distances analysis. Species used in the molecular analyses are listed in Supplementary material (S1). Physocyclus dugesi (subfamily Arteminae) was used only to root the tree. The comparison among sequences of I. azteca and the rest of the species’ sequences was to analyze the intra and interspecific genetic variation. For samples of I. azteca,1 sequence (CNAN Ara-0160) from Km 46 highway Toluca-Valle de Bravo, Estado de México (type locality of the species); 2 sequences (CNAN Ara-0158 and Ara-0159) from Cueva del Fraile, Gustavo A. Madero, Mexico City; and 2 sequences (CNAN Ara-0169 and Ara-0170) from Cueva del Diablo, Tepoztlán, Morelos were used for the analysis. Specimens used in this study, including GenBank accession numbers and locality information are listed in S1.

DNA was isolated from legs (1 complete leg per sample), and extractions were performed with a Qiagen DNeasy Tissue Kit following a modified protocol described in Valdez-Mondragón and Francke (2015) and Valdez-Mondragón (2020). DNA fragments corresponding to 658 bp of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene were amplified using the same primers (LCO1490 and HCO2198) as in Valdez-Mondragón and Francke (2015) and Valdez-Mondragón (2020). Amplifications were carried out using a Veriti 96 Well Thermal Cycler (Applied Biosystems) following the modified protocol of Valdez-Mondragón and Francke (2015) and Valdez-Mondragón (2020). PCR products were checked to analyze length and purity on 1% agarose gels with a marker of 100 bp and purified directly from the QIAquick PCR Purification kit of Qiagen. DNA extraction and PCR amplification were performed at Laboratorio de Biología Molecular, Laboratorio Regional de Biodiversidad y Cultivo de Tejidos Vegetales (LBCTV), Instituto de Biología, Universidad Nacional Autónoma de México (IB-UNAM), Tlaxcala. Bi-directional Sanger sequencing was performed at the Laboratorio de Secuenciación Genómica de la Biodiversidad y la Salud (LANABIO), IB-UNAM, Mexico City. The generated sequences were saved in FASTA format. All newly generated sequences were deposited in GenBank with the accession numbers OR106002-OR106006 (see S1).

Sequences were aligned using the default gap opening penalty of 1.53 in the online MAFFT platform (multiple alignment using fast fourier transform), version 7 (Katoh & Toh, 2008) using the following alignment strategy: auto (FFT-NS-2, FFTNS-I, or L-INS-i; depending on data size). Inspection and editing of sequences and alignments was done using GENEIOUS version 10.2.6 (Kearse et al., 2012) and BioEdit version 7.0.5.3 (Hall, 1999). The matrix obtained from the multiple sequence alignments was then used for subsequent analyses.

To assess species delimitation with the barcoding locus, we first conducted a phenetic analysis with the neighbor joining (NJ) method to visualize the uncorrected p-distances using the program MEGA v. 7.0 (Tamura et al.,2007). We subsequently used the reconstructed NJ tree to carry out analyses with the following 3 methods: 1) Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al., 2021): we ran this analysis on the online platform (https://bioinfo.mnhn.fr/abi/public/asap/) with the parameters suggested by Nolasco and Valdez-Mondragón (2022a); 2) General Mixed Yule Coalescent model (GMYC) (Pons et al., 2006): this analysis was performed using the GMYC web server (https://species.h-its.org/gmyc/); and 3) Bayesian Poisson Tree Processes (bPTP) (Kapli et al., 2017; Zhang et al., 2013): this method was conducted using the web server (https://species.h-its.org/ptp/).

p-distances under neighbor joining (NJ) 

The genetic distances tree was reconstructed with MEGA v. 7.0 (Tamura et al.,2007), using the following commands: No. replicates = 1,000, bootstrap support values = 1,000 (significant values ≥ 50%), substitution type = nucleotide, model = p-distance, substitution to include = d: transitions + transversions, rates among sites = gamma distributed with invariant sites (G+I), missing data treatment = pairwise deletion.

Automatic Barcode Gap Discovery (ABGD): this method is implemented to find gaps in genetic divergence, considering that the intraspecific genetic variation is smaller than the interspecific divergences. First, the method generates a prior data partition of the data into putative species (initial partitions, IP). Then, these initial partitions are recursively partitioned until there is no further partitioning of the data (recursive partitions, RP). ABGD analyses were carried out in the online platform (https://bioinfo.mnhn.fr/abi/public/abgd/) using the following options: K2P distances non-corrected, pmin = 0.001, pmax = 0.1, steps = 10, relative gap width (X) = 1, nb bins = 20. Assemble Species by Automatic Partitioning (ASAP): this is an ascending hierarchical clustering method, merging sequences into groups that are successively further merged until all sequences form a single group. A partition is the equivalent to each sequence merge step, then, the software analyzes all partitions and scores the most probable groups on the tree (Puillandre et al., 2021). ASAP analyses were run in the online platform (https://bioinfo.mnhn.fr/abi/public/asap/) using Kimura (K80) distance matrices and configurated with following parameters: substitution model = p-distances, probability = 0.01, best scores = 10, fixed seed value = -. General Mixed Yule Coalescent (GMYC): this species delimitation method applies single (Pons et al., 2006) or multiple (Monaghan et al., 2009) time thresholds to delimit species in a maximum likelihood context, using ultrametric trees (Ortiz & Francke 2016). To generate the ultrametric tree, phylogenetic analyses were carried out in BEAUti and BEAST v.1.10.4 software (Drummond et al., 2012) using a coalescent (constant population) tree prior. Independent log normal clock uncorrelated was applied with its respective evolutionary model and substitution rates. The models of sequence evolution were selected using jModelTest version 2.1.10 using the Akaike information criterion (AIC) (Posada & Buckley, 2004). The models selected for CO1for each partition block were GTR+G+I (1^st and 2^nd codon positions) and GTR+G (3^rd codon position). For the analyses, 40 million iterations were run 5 times, independently. The program Tracer 1.6 (Rambaut & Drummond, 2003) was used to evaluate the convergence values. Tree annotator v. 2.6.0 (BEAST package) was used to construct maximum clade credibility trees, after discarding the first 25% of generations (“burnin”) of the 5 independent runs. Finally, the GMYC method was implemented in the web service (https://species.h-its.org/gmyc/), which uses the original R implementation of the GMYC model (Fujiwasa & Barraclough, 2013). Bayesian Poisson Tree Processes (bPTP): this species delimitation method is similar to GMYC, but it does not use an ultrametric tree as input. This is because the models of speciation rate are implemented directly using the numbers of substitutions calculated from the branch lengths. The Bayesian and maximum likelihood variants were carried out on the online version (https://species.h-its.org/ptp/), with the following options: rooted tree, MCMC = 1,000,000, thinning = 100, burn-in = 0.1, seed = 123. The trees obtained in all analyses were edited with iTOL online version (https://itol.embl.de/) (Letunic & Bork 2021) and Photoshop CS6. We used the congruence integration criteria to delimit species, which is based on the correspondence among the different molecular methods to generate high support for a species hypothesis (DeSalle et al., 2005, Hamilton et al. 2011, Navarro-Rodríguez & Valdez-Mondragón, 2020, 2024; Nolasco & Valdez-Mondragón, 2022a; Valdez-Mondragón, 2020).

The approaches for DNA barcoding tree-based delimitation explicitly use the phylogenetic species concept, where the analysis recognizes a monophyletic cluster by searching differential intra and inter-specific branching patterns (Ortiz & Francke, 2016). 

Results

According to the morphology of primary (male palps and female epigyna) and secondary (male chelicerae) sexual structures, the populations of Ixchela azteca showed geographical variation in morphological traits (Fig. 1). The males of the 3 populations have the same shape of the palp (Figs. 1, 8, 9; Valdez-Mondragón & Francke, 2015: Figs. 38, 39); however, there are differences in the shape of the male chelicerae (Figs 1, 4, 6, 7; Valdez-Mondragón & Francke, 2015: Figs. 36, 37, 40) and female epigyna (Figs. 10-13; Valdez-Mondragón & Francke, 2015: Fig. 36). The populations from the temperate region of Cueva del Fraile, Mexico City (CNAN Ara-0158 and Ara-0159), the population from the type locality (Estado de México, CNAN Ara-160), and the population from Cueva de Diablo, Morelos (CNAN Ara-0169 and Ara-0170) have the frontal apophysis of chelicerae (FAC) shorter (Valdez-Mondragón & Francke, 2015: Fig. 40) than the males from the population of the dry region of Guanajuato, which have a longer and conical FAC (Fig. 7). The female epigynum from Guanajuato is square-shaped in ventral view (Fig. 10), whereas in the other 3 populations from temperate region of central Mexico (Mexico City, Estado de México and Morelos) it is more trapezoidal (Valdez-Mondragón & Francke, 2015: Fig. 42). In frontal view, the conical projection in the population from Guanajuato is smaller (Fig. 12) than the one observed in females from central Mexico (Valdez-Mondragón & Francke, 2015: Fig. 44). Finally, females from the 3 populations of the temperate region of central Mexico have a longer and straighter epigynum in lateral view (Valdez-Mondragón & Francke, 2015: Fig. 41) than the population from the dry region of Guanajuato, which have a shorter and curved epigynum (Fig. 13). The dorsal pattern of the carapace is similar between populations of I. azteca,with lateral spots on the carapace and a dark region along the fovea (Fig. 5; Valdez-Mondragón & Francke, 2015: Fig. 35). In comparison with the rest of the species of the genus, I. azteca so far is the only species with morphological variation in the primary (male palps and female epigyna) and secondary (male chelicerae) sexual structures.

The CO1 matrix included 59 terminals (including 1 outgroup) and 601 aligned positions. The uncorrected CO1 average distances between the populations of I. azteca from temperate regions and the dry region of Guanajuato was 3.5% (Table 1), whereas the average genetic distances among the remaining species of Ixchela was 12.1% (Table 1). Bootstrap support for the populations from Guanajuato was 100% (Fig. 1, red branch), whereas the group with the populations from temperate regions in central Mexico had a bootstrap support of 59% (Fig. 1, green branch). 

Molecular methods for species delimitation

In the maximum likelihood (ML) phylogram obtained with CO1 (Fig. 2), most of the species assignments based on the current taxonomy were congruent with all species delimitation methods, except for the specimens assigned to I. azteca (Fig. 2). The GMYC model recovered a total of 21 species, and the 2 clades of I. azteca (Fig. 2, green and red branches) were recovered as a single species exclusive of I. azteca CNAN-Ara0169 and Ara0170, which were recognized as a different species (Fig. 2). The ASAP method recognized a total of 18 species, recovering both clades of I. azteca as a single species (Fig. 2). Finally, the bPTP method recovered a total of 25 species of Ixchela under both variants (BI and ML); however, similar to the GMYC analysis, the specimens CNAN-Ara0169 and Ara0170 were recognized as a different species from I. azteca (Fig. 2). The first clade of I. azteca (Fig. 2, green branch)did not receive significant bootstrap support (58%), a result also observed in the NJ analysis, whereas the clade from Guanajuato (Fig. 2, red branch) was supported by a high bootstrap value (100%). The I. azteca samples CNAN-Ara0169 and Ara0170 had a high bootstrap support of 99% for their sister relationship (Fig. 2).

Family Pholcidae C. L. Koch, 1850

Genus Ixchela Huber, 2000

Diagnosis and description. For an updated diagnosis and description of the genus, see Valdez-Mondragón (2013, 2020) and Valdez-Mondragón and Francke (2015).

Taxonomic summary

Type species. Coryssocnemis furcula F. O. Pickard-Cambridge, 1902, by original designation of Huber (2000). Type locality: female holotype from Tecpam, región de los Altos (cerro Tecpam, Departmento Chimaltenango), Guatemala, around 2,300 m, Otto Stoll. Godman & Salvin Coll., in BMNH (F. O. Pickard-Cambridge, 1902; Huber, 1998).

Composition. Twenty-two species. See the World Spider Catalog (2025) for the specific list of species. 

Distribution. Ixchela is widely distributed from northeastern Mexico to Nicaragua. Although specimens from Nicaragua were not examined in this work, Huber (2000) examined 1 male of an undescribed species from this country deposited in the Museo Entomológico Nicaraguense (León, Nicaragua). 

Ixchela azteca Valdez-Mondragón & Francke, 2015

I. azteca Valdez-Mondragón & Francke, 2015: 29, f. 1-6, 18-44 (Description ♂, ♀).

Figs. 3-13, 16-19 

Diagnosis and description. See Valdez-Mondragón and Francke (2015).

Taxonomic summary

Type material. Mexico: Estado de México: ♂ holotype (CNAN T0763) [26 August 2011; A. Valdez, J. Mendoza, D. Barrales, R. Monjaraz, E. Miranda Cols.] from Km 46 highway Toluca-Valle de Bravo (19°15’21.6” N, 100°04’00.8” W; 2,315 m asl) (examined). Paratypes: 1 f# (CNAN T0764); 1 ♀, 3 juv. (CNAN T0765), same data as holotype (examined). See Valdez-Mondragón and Francke (2015) for the complete list of material examined from Estado de México, Guerrero, Mexico City, Michoacán, and Morelos.

Table 1

Average genetic p-distance under NJ among the different species of Ixchela analyzed with CO1. The value in bold indicates the average p-distance between the populations from temperate regions (A) and the population from the dry region in Guanajuato (B) of I. azteca (3.5%) (bold). The average genetic p-distance under NJ between the species of Ixchela was 12.1%.

Figure 1. Neighbor-joining tree constructed with p-distances of the CO1 barcode sequences from different specimens and species of Ixchela. Branch colors indicate species, red and green branches and asterisks represent populations of Ixchela azteca. Male chelicera, male palp, and female epigyna are shown to illustrate the variation at species level and within I. azteca. Numbers on branches indicate bootstrap support values (> 50%).

Figure 2. Maximum likelihood tree of Ixchela constructed with CO1. Bar colors represent putative species in the tree and in the columns, which represent the different species delimitation methods. Red and green branches and asterisks represent populations of Ixchela azteca. Numbers below the columns represent the species recovered in each species delimitation method (not considering Physocyclus dugesi). Numbers above branches represent bootstrap support values for ML (> 50%). Column abbreviations: morphology (M); GMYC with single (SN) and multi (MT) thresholds; ASAP; bPTP with maximum likelihood (ML) and Bayesian inference (BI) variants.

Figures 3-9. Ixchela azteca Valdez-Mondragón & Francke, 2015 from Cortazar, Guanajuato, Mexico. Male: 3-4, habitus, lateral and dorsal views, respectively; 5, carapace and chelicerae, frontal view; 6-7, chelicerae, frontal and left lateral views, respectively; 8-9, left palp, prolateral and retrolateral views, respectively. E: Embolus, PAB: prolateroventral apophysis of bulb, PR: procursus, SAC: sclerotized apophysis of chelicerae, VPP: ventrobasal protuberance of procursus. Scale bars: Figs. 6, 7 = 0.5 mm, Figs. 5, 8, 9 = 1 mm, Figs 3, 4 = 2 mm.

New material examined. Mexico: Guanajuato: 1 ♂, 2 ♀, 2 juv. (CARCIB) [14 October 2021; A. Valdez, A. Juárez, L. Cabrera, S. Nolasco Cols.] from 7.5 km southeast of Cortazar (20°25’31.1” N, 100°54’51.8” W; 2,160 m asl), Municipality Cortazar (night collecting). 

Morphological variation. Male specimens from Cueva del Diablo (Morelos, Mexico) and from Gruta de Tziranda were notably smaller than specimens from the type locality and the other localities, including the new specimens from Guanajuato. Female specimens from Grutas de Tziranda (Michoacán, Mexico) and road to Cueva del Fraile (Mexico City, Mexico) were notably smaller than specimens from the other localities, including the new specimens from Guanajuato.

Figures 10-13. Ixchela azteca Valdez-Mondragón & Francke, 2015 from Cortazar, Guanajuato, Mexico. Female epigynum: 10, ventral view; 11, dorsal view; 12, frontal view; 13, left lateral view. MSE: Median septum of epigynum, PP: pore plates. Scale bars: 0.5 mm.

Specimens from Cueva del Diablo have paler coloration on carapace and legs than specimens from other localities. The opisthosomal coloration can vary from grey, pale grey, blue, or pale blue, potentially related to the prey consumed. Males:Cueva del Diablo (Morelos, Mexico) (n= 4), tibia I: 10.87-12.50 (x= 11.74). Cueva del Diablo, La Peña (Estado de México, Mexico) (n= 1): tibia I: 18.12. Cueva de Peña Blanca (Estado de México, Mexico) (n= 2), tibia I: 13.75, 14.00. Gruta de Tziranda (Michoacán, Mexico) (n= 1): tibia I: 11.00. Road to Cueva del Fraile and Cueva del Fraile, respectively (Mexico City, Mexico) (n= 2): tibia I: 9.50, 12.25. 7.5 km southeast of Cortazar (Guanajuato, Mexico) (n= 1): tibia I: 13.63. Females:Cueva del Diablo (Morelos, Mexico) (n= 7): tibia I: 7.50-16.00 (x= 12.77). Km 34 highway Toluca-Zitácuaro (Estado de México, Mexico) (n= 3): tibia I: 6.60-12.5 (x = 9.50). 5 km W of Casahuates (Guerrero, Mexico) (n= 3): tibia I: 9.37-15.25 (x= 11.95). El Naranjo (Michoacán, Mexico) (n= 2): tibia I: 9.50, 11.75. 7 km SE of Ciudad Hidalgo (Michoacán, Mexico) (n= 2): tibia I: 9.7, 11.37. Gruta de Tziranda (Michoacán, Mexico) (n= 4): tibia I: 7.10-10.2 (x= 8.70). Road to Cueva del Fraile (Mexico City, Mexico) (n= 2): tibia I: 10.12, 11.87. 7.5 km southeast of Cortazar (Guanajuato, Mexico) (n = 2): tibia I: 9.87, 11.87 (x = 10.87).

Figures 14-19. Habitat and live specimens of I. azteca. 14-15, Thorny scrub forest at 2,160 m asl. from 7.5 km southeast of Cortazar, Municipality Cortazar, Guanajuato, Mexico (red arrow indicates the microhabitat where the specimens were collected); 16-17, live male specimens; 18-19, live female specimens.

Natural history. Valdez-Mondragón and Francke (2015) reported that the specimens from Estado de México and Guerrero were collected on their sheet webs in oak-pine and pine forests, inside cavities on walls along road-cuts in wet and shaded areas covered with roots. The male collected in the Instituto de Biología, UNAM was walking on a wall inside a classroom. The specimens from Cueva del Fraile, Mexico City, and Gruta de Tziranda, Michoacán were collected on their sheet webs inside the caves, close to the walls. Specimens collected outside the Cueva del Fraile were among boulders in shady moist areas, whereas specimens from Cueva del Diablo, Morelos were collected in the cave entrance and inside the cave, where humidity was c.70% and temperatures cold. These specimens were collected on their sheet webs, and it was very common to find prey remains in their webs, mainly large leafcutter ants of the genus Atta (subfamily Myrmicinae). The specimens from Cortazar, Guanajuato, were collected in a disturbed thorny scrub forest (Figs. 14, 15) with columnar cacti and nopales (Opuntia sp.). These specimens were observed on their sheet webs built among walls of big rocks (Figs. 14, 15 red arrow, 16-19). 

Figure 20. Distribution records of I. azteca in the Chihuahuan Desert biogeographic province (Southern Semiarid Elevations ecoregion) and in the Transmexican Volcanic Belt biogeographic province (Temperate Mountains ecoregion). Map by Mayra Cortez Roldán.

Distribution. Mexico: Estado de México, Guanajuato, Guerrero, Mexico City, Michoacán, Morelos (Fig. 20).

Discussion

The evolutionary aspects of morphological variation, including dimorphism and polymorphism within and among sexes, has been widely studied in several animal groups. Also, assessing species boundaries is a central debate in modern systematics, particularly in cases where morphology presents wide variations or lacks information at deep levels. The use of DNA barcoding approaches has been applied in modern systematics in 2 different ways: first, to distinguish among described species (equivalent to species identification or species diagnosis), and second, to discover new species (incorporating species delimitation methods and species descriptions) (DeSalle et al., 2005). In this way, another taxonomic task is to establish the limits between species or identify those species with wide morphological and genetic variation among populations.

Regarding the genus Ixchela,molecular methods for species delimitation have been used in parallel with morphological features to obtain the most robust evidence for species delimitation and diagnoses. Valdez-Mondragón (2020) described I. panchovillai and I. zapatai using 4 molecular species delimitation methods and morphology. All molecular methods were congruent with the morphology of primary and secondary sexual features (male palps, male chelicerae, and female epigyna). However, in the case of I. azteca,despite the population from Guanajuato (Fig. 2, green branch) being genetically divergent from the populations from central Mexico (Fig. 2, red branch) by a p-distance value of 3.5% (Table 1), the molecular species delimitation methods were not congruent with morphology, recovering the 2 clades as a single species. Previous studies on species delimitation in pholcid spiders have usually found congruence between species limits hypothesized a prioribased on morphology and those recovered using mitochondrial markers (Nolasco & Valdez-Mondragón, 2022a; Valdez-Mondragón, 2020). However, similar to I. azteca, morphological variation of sexual structures within the same species has been previously reported in other pholcid spiders. Huber and Pérez-González (2001) reported a case of morphological variation in the female genitalia of 3 species in the genus Ciboneya Huber & Pérez-González, 2001, which even have different female morphs in the same population. Valdez-Mondragón (2010) described discontinuous interspecific variation in the epigyne shape in females of Physocyclus enaulus Crosby, 1926 with 3 distinct morphotypes. Such variation has been observed in insular pholcids as Huber and Acurio (2021) reported for Aymaria conica (Banks, 1902), with wide morphological variation in the female´s genitalia. In the case of I. azteca, the epigyna of the population from Guanajuato is wider and more curved than in the populations from central Mexico, which are longer and less curved. Males of the different I. azteca populations also showmorphological variation, mainly in the chelicerae, a robust and very important feature at species level in the family Pholcidae. Males from Guanajuato have longer apophyses than males from central Mexico. 

Pholcid spiders show a great variety of morphological features that allow for delimitation and diagnosis to the species level in most cases. Primary sexual structures such as male palps and female genitalia, as well as secondary sexual structures such as male chelicerae, are important and useful characters for identifying and diagnosing pholcids species (Huber et al., 2018; Nolasco & Valdez-Mondragón, 2022b; Valdez-Mondragón, 2020). However, as Huber and Pérez-González (2001) suggest, it is possible that taxonomists are strongly biased against discovering genitalic variation such as dimorphisms or even polymorphisms, simply because the genitalia have traditionally been used with an overwhelming priority for species delimitation and identification. This is why taxonomic descriptions should (ideally) be based on a large series of specimens to analyze both intra- and inter-specific morphological and genetic variation. This practice also allows us to identify cases of morpho-clines, dimorphisms, or even polymorphisms.

Although the genetic p-distance under CO1 between both populations of I. azteca was 3.5% (Table 1), this mitochondrial marker is susceptible to over- and underestimating diversity in some cases; therefore, the limits interpretation must be considered with caution (Astrin et al., 2006; Ortiz & Francke, 2016). Additional informative mitochondrial (e.g., 16S) or even nuclear (e.g., ITS1, ITS2, or 28S) markers, as well as further morphological evidence, would help to obtain better resolution of species hypotheses (Astrin et al., 2006; Agnarsson, 2010; Navarro-Rodríguez & Valdez-Mondragón, 2020; Nolasco & Valdez-Mondragón, 2022a; Ortiz & Francke, 2016; Planas & Ribera, 2015; Valdez-Mondragón et al., 2019).

For the most part, the natural distribution of the genus Ixchela is in temperate climate zones, principally in pine, oak, or pine-oak forests, between 1,000-2,950 m of elevation (Valdez-Mondragón, 2013). However, species such as Ixchela juarezi Valdez-Mondragón, 2013have been recorded in thorny scrub forest. The population of I. azteca from Cortazar, Guanajuato, was similarly collected in a disturbed thorny scrub forest in the Chihuahuan Desert biogeographic province. Although intensive collecting efforts have targeted low elevations, Ixchela seems to occur only above 1,000 m asl mainly in temperate climate regions (Valdez-Mondragón, 2013, 2020; Valdez-Mondragón & Francke, 2015). Species such as I. juarez and I. azteca that are found in arid and semiarid elevations might represent relict species or populations resulting from vicariant events, and the temperate mountains likely served as a conduit for dispersal. Allopatric populations of I. azteca may be a product of the repeated appearance and disappearance of orographic barriers and habitat fragmentation across a complex landscape formed in response to past orogenic events and climate oscillations, mainly in the Transmexican Volcanic Belt (TVB) biogeographic province (transition zone between the Nearctic and Neotropical regions), as was demonstrated by Valdez-Mondragón and Francke (2015) in the genus Ixchela.

Based on the available evidence, we propose that the allopatric populations of I. azteca assessed herein belong to the same species considering the following: 1) although morphological variation is evident, this variation could be influenced by environmental effects, genetic mutations, developmental perturbations, or even geographic variation as was suggested in other groups by Debat and David (2001); 2) although 2 clades of I. azteca were recovered under ML with a genetic p-distance of 3.5%, the populations from central Mexico present a low bootstrap support value (59%) and 2 subclades were found; 3) incongruence among molecular species delimitation methods was observed with regards to I. azteca, with some methods recovering both clades as a single species; and 4) the population from Guanajuato was collected in a thorny scrub forest (Chihuahuan Desert province-Southern Semiarid Elevations ecoregion), while the rest of the specimens from central Mexico where collected in temperate pine-oak forests (TVB province-Temperate Mountains ecoregion) (Fig. 20). We suggest that the observed morphological and genetic variation might be due to the geographic variation of the species.

In conclusion and considering the suggestions of Carstens et al. (2013), while probing different methods or lines of evidence is necessary for properly implementing species delimitation, it is better to be conservative about species delimitation when the information and results are incongruent to avoid over-splitting species.

Acknowledgments

The first author thanks SEP-Conahcyt for financial support of the project of Basic Science (Ciencia Básica) 2016, No. 282834. We also thank Edmundo González Santillán (curator) and Oscar F. Francke (ex-curator) of the Colección Nacional de Arácnidos (CNAN), Instituto de Biología, UNAM, for providing specimen loans. We are grateful to Laura Márquez Valdelamar for help in the molecular sequencing of the samples, to Brett O. Butler for the English language review of the manuscript, and to the reviewers for their comments and suggestions that improved the manuscript. We thank Carlos Palacios-Cardiel (Technician of the Centro de Investigaciones Biológicas del Noroeste) for his support with storage of the material at the CARCIB. Specimens were collected under the Scientific Collector Permit FAUT-0309 from the Secretaría de Medio Ambiente y Recursos Naturales provided to Alejandro Valdez Mondragón.

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Maria Alejandra Villegas-Ojeda ^{a, b}, Guido Fernández-Marinone ^b, Mariana Jofré ^b, Cynthya González ^c, *

^a Universidad Nacional de San Luis – Consejo Nacional de Investigaciones Científicas y Técnicas, Almirante Brown 907, 5700 San Luis, Argentina

^b Universidad Nacional de San Luis, Facultad de Química, Bioquímica y Farmacia, Área de Biología, Departamento de Biología, Ejército de los Andes 950, segundo piso, 5700 San Luis, Argentina 

^c Universidad Nacional del Nordeste – Consejo Nacional de Investigaciones Científicas y Técnicas, Centro de Ecología Aplicada del Litoral, Ruta Provincial Nº 5 Km 2,5, 3400 Corrientes, Argentina 

*Corresponding author: cynthyaelizabethgonzalez@gmail.com (C. González)

Received: 16 October 2023; accepted: 2 October 2024

Abstract

The studies of helminth parasites in Argentine amphibians have focused on the Dry and Humid Chaco ecoregions; in the Dry Chaco the reports are from the Chaco and Formosa provinces. The aim of this work was to report the first record of Aplectana hylambatis (Baylis, 1927) Travassos, 1931 (Nematoda: Cosmocercidae) from San Luis province and the southernmost record for the Dry Chaco ecoregion. Specimens of Rhinella arenarum (Hensel, 1867) (n = 6), Leptodactylus mystacinus (Burmeister, 1861) (n = 13), and Odontophrynus cf. asper (n = 6) were analyzed. Specimens of A. hylambatis were collected from the large intestine of these hosts and were studied using light and scanning electron microscopy (SEM). In addition, relevant features are described, including the number and arrangement of caudal papillae, and of mamelon-like cuticular protuberances associated to the vulva. The morphological characteristics are compared and discussed in relation to those of specimens collected from other hosts in other localities of the country. The presence of A. hylambatis in localities of the Dry Chaco represents an expansion of the geographic range of this cosmocercid and the first record in L. mystacinus and O. cf. asper from Argentina.

Keywords: Aplectana hylambatis; Bufonidae; Cosmocercidae;Dry Chaco; Leptodactylidae; Odontophrynidae; Parasitic helminths

© 2025 Universidad Nacional Autónoma de México, Instituto de Biología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Primer reporte de Aplectana hylambatis (Nematoda: Cosmocercidae) en anfibios
de la provincia de San Luis, Argentina 

Resumen

Los estudios de helmintos parásitos de anfibios argentinos se han centrado en las ecorregiones del Chaco Seco y Húmedo; en el Chaco Seco los reportes se realizaron en las provincias de Chaco y Formosa. El objetivo de este trabajo fue reportar el primer registro de Aplectana hylambatis (Baylis, 1927) Travassos, 1931 (Nematoda: Cosmocercidae) para la provincia de San Luis y el registro más austral para la ecorregión del Chaco Seco. Se analizaron ejemplares de Rhinella arenarum (Hensel, 1867) (n = 6), Leptodactylus mystacinus (Burmeister, 1861) (n = 13) y Odontophrynus cf. asper (n = 6). Los ejemplares de A. hylambatis fueron colectados del intestino grueso de estos hospedadores y se estudiaron mediante microscopía óptica y electrónica de barrido (MEB). Se describen características relevantes, como el número y disposición de las papilas caudales, y el número y disposición de las protuberancias cuticulares tipo mamelón asociadas a la vulva. Los caracteres morfológicos encontrados se comparan y discuten con los especímenes colectados en otros hospedadores de diferentes localidades del país. La presencia de A. hylambatis en localidades del Chaco Seco representa una ampliación del rango geográfico de este cosmocércido y el primer registro para L. mystacinus y O. cf. asper de Argentina.

Palabras clave: Aplectana hylambatis; Bufonidae; Cosmocercidae; Chaco Seco; Leptodactylidae; Odontophrynidae; Helmintos parásitos

Introduction 

In 1927, Baylis described Oxysomatium hylambatis parasitizing the large intestine of Leptopelis aubryi (Duméril, 1856) from Macenta, Guinea, Africa (Baylis, 1927). In that work, the author established a synonymy between the genera Oxysomatium and Aplectana; however, he did not question the validity of the genus Aplectana, stating that it included species without a gubernaculum (called at that time “accessory piece”).

Later, Travassos (1931) published a review of the family Cosmocercidae (Railliet, 1916) Travassos, 1925, where he established that the division between the 2 genera occurs mainly by the female reproductive system, which is prodelphic in the genus Aplectana and amphidelphic in Oxysomatium. In this way, it reproduces the description of the specimens made by Baylis (1927), but places them under the genus Aplectana due to the anterior location of the reproductive system of females.

Over the years, both genera have been studied in numerous works around the world. In some of them, these taxa were considered synonymous (Fotedar, 1960; Gutiérrez, 1945; Harwood, 1930; Hsü & Hoeppli, 1933; Skrjabin, 1910, 1951; Walton, 1940, 1941), whereas in others, they were separated on the basis of the size and shape of gubernaculum and spicules, the presence or absence of lateral alae, the number of cephalic papillae, the position of the vulva, and the stage of the eggs in utero (Bravo-Hollis, 1943), or on the basis of the presence or absence of gubernaculum in males, and prodelphic or amphidelphic condition in females (Ballesteros-Márquez, 1945).

Baker (1980) redescribes A. hylambatis (Baylis, 1927) Travassos, 1931 based on specimens collected from Sclerophrys mauritanica (Schlegel, 1841) (= Bufo mauritanicus), Leptopelis aubryi (Duméril, 1856), and Rhinella achalensis (Cei, 1972) (= Bufo achalensis). Subsequently, Baker and Vaucher (1986) synonymized Oxysomatium bonariensis (Gutiérrez, 1945) collected from Rhinella arenarum (Hensel, 1867) (= Bufo arenarum) in Argentina, and A. pudenda (Masi Pallarés and Maciel, 1974) collected from Rhinella diptycha (Cope, 1862) (= Bufo paracnemis), Rana pacybrachion (Steffen, 1815) (= Leptodactylus latrans), Boana raniceps (Cope, 1862), and Boana punctata (Schneider, 1799) (= Hyla punctata) in Paraguay, with A. hylambatis.

In South America, this cosmocercid has a wide geographic and host range; it was found in Argentina, Ecuador, Guyana, Paraguay, Peru, Brazil, and Uruguay (Campião et al., 2014; González, Hamann et al., 2021). In Argentina it is the nematode species of amphibians with the widest geographic distribution. There are records in 7 provinces, namely Buenos Aires, Córdoba, Corrientes, Chaco, Formosa, Salta, and San Juan, where it has been found parasitizing anurans of the families Bufonidae, Ceratophryidae, Hylidae, Leptodactylidae, Microhylidae, and Odontophrynidae (Draghi et al., 2015; González et al., 2019; González, Hamann et al., 2021).

Rhinella arenarum (Anura: Bufonidae) is distributed in Argentina, Bolivia, Brazil, and Uruguay (Frost, 2023). It is characterized by having terrestrial habits, reproducing in temporary or semi-permanent lentic and lotic environments where it deposits strings of pigmented eggs, and having a generalist diet (Babini et al., 2015; Bionda et al., 2011; de Oliveira et al., 2017; Quiroga et al., 2009). Leptodactylus mystacinus (Burmeister, 1861) (Anura: Leptodactylidae) is distributed in Argentina, Bolivia, Brazil, Paraguay, and Uruguay (Frost, 2023). It is a terrestrial anuran, with fossorial characteristics; it breeds on foam nests built in temporary or semi-permanent lentic and lotic environments, and has a generalist diet (Babini et al., 2015; De-Carvalho et al., 2008; Heyer et al., 2010; López et al., 2005). These amphibian species are categorized as “Not Threatened” according to Vaira et al. (2012) and as “Least Concern” according to IUCN (2023). Finally, the genus Odontophrynus Reinhardt and Lütken, 1862 (Anura: Odontophrynidae), which includes 11 species, is distributed in southern and eastern South America (Frost, 2023). Recently, Argentinean species of this genus were revised by Rosset et al. (2022). These authors synonymized Odontophrynus americanus (Duméril and Bibron, 1841) with O. occidentalis, and established its distribution area, restricted to the center, west and south of the country, in the ecoregions of Monte, Dry Chaco, Patagonian steppe, and in the coastal dunes of the Humid Pampa ecoregion. On the other hand, these authors also proposed to name the cryptic diploid and tetraploid species currently grouped as the Odontophrynus americanus species group, as Odontophrynus asper (Philippi, 1902), with distribution in extensive areas of central and northern Argentina, Uruguay, southern Paraguay, southeastern Brazil, and probably also Bolivia (Rosset et al., 2022).

Studies on parasitic helminths in these 3 hosts, both in the larval and adult stages, refer to descriptions of new species, intraspecific variations, or extensions of host or geographic range (Campião et al., 2014; da Graça, et al., 2017; da Silva et al., 2018; González et al., 2019; González, Duré et al., 2021; González, Hamann et al., 2021; Queiroz et al., 2020; Ramallo et al., 2020). Throughout its geographic range, the helminth fauna of R. arenarum has been studied in Argentina, Brazil, and Uruguay (Campião et al., 2014). Specifically, in Argentina, A. hylambatis has been reported from Buenos Aires and San Juan provinces; Rhabdias elegans Gutiérrez, 1945 from Buenos Aires and Tucumán provinces; Borrellostrongylus platensis Gutiérrez, 1945 (= Parabatrachostrongylus platensis Tantalean and Naupay, 1974) from Buenos Aires province; Oswaldocruzia proencai Ben Slimane and Durette-Desset, 1995, Ophidascaris sp., Aplectana tarija Ramallo, Bursey and Goldberg, 2007, and Cosmocercoides lilloi Ramallo, Bursey and Goldberg, 2007 from Salta province; and Bufonerakis andersoni Baker, 1980 has been found but its locality was not reported (González and Hamann, 2015; González et al., 2019; Ramallo et al., 2020). For L. mystacinus, previous reports about its helminths have been made in Brazil and Paraguay (Campião et al., 2014); so far, no helminths have been found in this amphibian in Argentina. The previous records of helminths that have been made in the country for amphibians of the genus Odontophrynus include the nematodes R. elegans, Cosmocerca podicipinus Baker and Vaucher, 1984, and C. parva Travassos, 1925, and the digeneans Echinostomatidae gen. sp., and Travtrema aff. stenocotyle for O. asper (= O. americanus) from Corrientes province; A. hylambatis and Falcaustra sanjuanensis González, Sanabria and Quiroga, 2013 for O. americanus (= O. occidentalis) from San Juan province, and A. hylambatis for O. lavillai Cei, 1985 from Formosa province (Campião et al., 2014; González, Hamann et al., 2021; Piñeiro-Gómez et al., 2023).

The aim of this work was to report the first record of the cosmocercid A. hylambatis from the province of San Luis, Argentina, in the anurans R. arenarum, L. mystacinus, and O. cf. asper.

Materials and methods

San Luis, Argentina is part of the Dry Chaco ecoregion, and has xerophytic deciduous shrub vegetation with patches of Larrea (Morello et al., 2012). The sampled sites were located on the Central Sierras of San Luis. In the extreme west, these mountain ranges constitute an orographic barrier to humid winds from the east, which causes greater rainfall on the eastern slopes and drier climates to the west (Torrella & Adámoli, 2006). From November 2019 to February 2023, through active sampling techniques of direct visual recording and auditory detection (Vaira et al., 2021), 25 anuran amphibians of 3 species, belonging to 3 families, were captured: R. arenarum (Bufonidae) (n = 6), L. mystacinus (Leptodactylidae) (n = 13), and O. cf. asper (Odontophrynidae) (n = 6) (Table 1). Secretaría de Medio Ambiente, San Luis Province, authorized the capture of the specimens for this study (Resolution N°295-PRN-2022). 

All organ systems were examined in the captured specimens, using helminthological techniques according to the criteria of Goater and Goater (2001) and González and Draghi (2021). Nematodes found in the large intestine, were counted, fixed with a hot 10% formalin solution, preserved in 70% alcohol, cleared with Amann’s lactophenol and then, were morphometrically analyzed. Illustrations were made using a Leica DM2500 optical microscope with the aid of a drawing tube. For examination with scanning electron microscope (SEM), nematodes were dehydrated through a series of ethanol and acetone, and critical point-dried using CO₂. The specimens were coated with gold-palladium and examined with a JEOL JSM-5800 scanning electron microscope. Measurements along the text are given in micrometers unless otherwise indicated and are presented as the mean ± 1 standard deviation, with the minimum and maximum value in parentheses. Nematodes were deposited in the Helminthological Collection of Museo de la Plata, La Plata, Argentina (MLP-He). The anuran specimens are stored in the Universidad Nacional de San Luis Herpetology Collection (CH-UNSL), San Luis, Argentina.

Results

Of the total amphibians analyzed, 11 were parasitized (total prevalence: 44%). In these, 338 specimens belonging to the species Aplectana hylambatis (Baylis, 1927) Travassos, 1931 (Nematoda, Cosmocercidae) were found (accession numbers: MLP-He 8044, 5 males and 5 females from O. cf. asper; MLP-He 8045, 5 males and 5 females from L. mystacinus; MLP-He 8046, 5 males and 5 females from R. arenarum). 

Description

Aplectana hylambatis (Baylis, 1927) Travassos, 1931 [= Oxysomatium bonariensis

Gutiérrez, 1945; = Aplectana pudenda Masi Pallarés and Maciel, 1974] 

(Figs. 1-3)

Table 1

Sample size (n), sex (F: females; M: males), collection date and accession numbers for 3 amphibian species from San Luis Province, Argentina.

General morphology (based on 82 adult specimens). Small nematodes, cuticle with transverse striations evenly distributed along the body. Lateral alae present in both sexes beginning at the level of the nerve ring and ending at the level of the anus in females and at the level of the first pairs of precloacal papillae in males. Triangular mouth surrounded by 3 lips, 1 dorsal with 2 cephalic papillae, and 2 ventral lips each with a papilla and an amphid (Fig. 1A). Well-differentiated esophageal bulb with chitinous valves. Very visible excretory pore, pre-bulbar and fringed (Fig. 2A). Prodelphic females with a highly developed ovijector (Fig. 2D, E); thin-membrane, morulated eggs and larvated eggs at the level of the vulva. Males with gubernaculum and equal spicules with characteristic articulation. The morphometric characters of males and females collected are provided in Table 2. 

Male (based on 40 adult specimens). Specimens with 3 typical groups of caudal papillae present in this species, precloacal, adcloacal, and postcloacal, with a large medial unpaired papilla anterior to the cloaca. In the analyzed specimens, thirteen pairs of caudal papillae were counted with the following arrangement: 4 pairs of ventral precloacal papillae; 5 pairs adcloacal (2 pairs ventrolateral, 3 pairs in anterior lip of the cloaca) and 1 unpaired papilla on anterior lip of the cloaca; 4 pairs of postcloacal papillae (2 pairs ventrolateral at the middle tail; 2 pairs close to its posterior end) (Figs. 1B-D; 2B, C). 

Table 2

Morphometric characters (mean ± SD and Min.-Max.) of males and females of Aplectana hylambatis collected in the 3 anuran species from San Luis Province, Argentina.

Figure 1. Aplectana hylambatis parasite of anurans from San Luis province, Argentina. A, Anterior end, apical view; B, male, posterior end, ventral view; C, detail of cloacal region, latero-ventral view; D, detail of paired papillae, latero-ventral view; E-F, female, mamelon-like cuticular, latero-ventral view. Abbreviations: a: amphid; ad: adcloacal papillae; fc: cuticular fringe; up: unpaired papilla; po: postcloacal papillae; pr: precloacal papillae; * cephalic papillae; closed arrows indicate paired papillae. A-D from O. cf. asper; E, F from L. mystacinus.

Figure 2. Aplectana hylambatis parasite of anurans from San Luis province, Argentina. A, Male, anterior end, ventral view; B, male, posterior end, ventral view; C, male, posterior end, lateral view; D, ovijector and 2 mamelon-like cuticular protuberances anterior to the vulva, ventral view; E, ovijector and 2 mamelon-like cuticular protuberances, 1 anterior and 1 posterior to the vulva, ventral view; F, female, posterior end, lateral view; G, gubernaculum, ventral view; H, spicules and gubernaculum, ventral view. Scale: A-D: 200 μm; E: 100 μm; F: 200 μm; G: 50 μm; H: 100 μm). A-C from R. arenarum; D-F from L. mystacinus; G, H from O. cf. asper.

Spicules and gubernaculum did not show morphological variation between specimens collected in different amphibian hosts and localities. Spicules showed the characteristic structure described for the species, long and chitinised, with a capitulum in the proximal end, and a pointed distal end that, when the spicules are outside the body, have a sickle or hockey stick appearance (Fig. 2H). Gubernaculum has an oval shape, chinitised, concave ventrally, with thickened edges, tapering towards the distal end (Fig. 2G).

Female (based on 42 adult specimens). Regarding the mamelon-like cuticular protuberances, most of the specimens analyzed showed between 1 large mamelon or 2 smaller mamelon-like protuberances located on the anterior lip of the vulva. However, in an analyzed specimen collected in L. mystacinus, 2 mamelon-like cuticular protuberances were observed, but with a different disposition than the one previously detailed; in this case, one protuberance was located anterior to the vulva and the other, posterior to it (Figs. 1E, F; 2D-F). 

Some specimens collected from R. arenarum (2 couples) and from L. mystacinus (2 couples) were found in copulation (Fig. 3A). In these cases, a detailed examination of these specimens showed that only the right spicule enters the female reproductive system. In it, the distal end presented the typical articulation described in previous works. This articulation is in the elbow of the ovijector. The spicule that remains inside the male reproductive system was fully stretched. Although the specimens were clarified, it was not possible to identify the gubernaculum in the specimens in copulation (Fig. 3B).

Figure 3. Aplectana hylambatis parasite of anurans from San Luis province, Argentina. A, Couples in copulation; B, detail of the arrangement of the spicules at the copulation time. Scale: A: 400 μm; B: 100 μm. A, B from L. mystacinus.

Discussion

The genus Aplectana is characterized by having lateral alae and somatic papillae in both sexes. Males lack plectanes or rosettes at the posterior end. Females have numerous thin-shelled eggs in the uterus and have prodelphic reproductive system (Gibbons, 2010). These characters were easily observable in the specimens studied here. The species of the genus Aplectana are grouped mainly according to the presence or absence of gubernaculum. Other features that distinguish these helminths are the number and arrangement of caudal papillae, and the size and morphology of the gubernaculum and spicules (Baker, 1980; Piñeiro-Gómez et al., 2017).

Morphological characters such as number and arrangement of caudal papillae in males, and mamelon-like cuticular protuberances on the anterior lip of the vulva in females, are presented in Table 3, and compared to those previously found in other hosts and other localities in Argentina. Regarding the number and arrangement of the caudal papillae, the males collected from the 3 host species presented a total of 13 pairs with the formula: 4:5:4+1. Compared with previous studies, only the specimens found in T. typhonius had a lower number of pairs of caudal papillae (12) (Draghi et al., 2015). Thirteen pairs of caudal papillae were also found in P. santafecinus and R. major from Taco Pozo (Table 3); however, their arrangement presented differences (P. santafecinus: 3:5:5; R. major: 4:4:5 vs. 4:5:4 in the present study). Finally, in males collected from other amphibians, a greater number of pairs of cloacal papillae were observed (up to 16 pairs in D. muelleri) (González et al., 2019) (Table 3).

Table 3

Some morphological characters of males and females of Aplectana hylambatis of the present study compared with specimens collected in different hosts in Argentina. n.e. = not specified. 

The mamelon-like cuticular protuberances are structures that were not included in the original description of the species by Baylis (1927); however, subsequent studies have detailed their number and arrangement in specimens collected from different hosts and locations (Baker, 1980; González et al., 2019; Gutiérrez, 1945). In this study, these structures of the females varied between 1 and 2, and were located on the anterior lip of the vulva. This character of the females was like that found in the specimens studied in R. arenarum from La Plata, R. major from Concepción del Bermejo and Las Lomitas, and L. bufonius from Las Lomitas (Table 3) (González et al., 2019). However, one female presented a protuberance posterior to the vulva; and to our knowledge this had not been reported until now. Finally, of all the morphological characters studied, mamelon-like cuticular protuberances varied the most among hosts and localities (González et al., 2019; Gutiérrez, 1945) (Table 3). 

In reference to the specimens in copulation, they were found with the typical arrangement of the male coiled by its posterior end to the female at the level of the vulva and forming an angle of approximately 90 degrees between them. In nematode mating, the spicules play an active role in copulation, while the gubernaculum, which is typically not everted during this process, acts as a guide for the spicules and prevents them from piercing the wall of the spicular pouch and cloaca when pressure is exerted on them (Chitwood & Chitwood, 1974).

In the 4 couples studied, it was observed that the right spicule is inserted into the vagina of the female and the articulation of the distal part is curved at the level of the ovijector’s elbow, while the left spicule remains fully elongated in the male without showing the articulation. Regarding this, the spicules serve to keep the vulva and vagina open. In trichostrongylids, the branching of the spicular lamina and the formation of twisted spicules seem to be adapted to this activity; in taxa with unequal spicules, the short spicule opens the vulva and the proximal region of the vagina, while the long one ensures sperm progression (Chitwood & Chitwood, 1974). In reference to the gubernaculum, unfortunately, we could not observe its location in the specimens studied. Thus, it is necessary to find new mating specimens to elucidate the position it occupies during copulation.

Intraspecific variations in parasitic nematodes of amphibians have been recorded for different families such as Molineidae (Skrjabin & Schulz, 1937) Durette-Desset and Chabaud, 1977, Onchocercidae (Leiper, 1911), Pharyngodonidae (Travassos, 1919), and Cosmocercidae (González et al., 2019; Kirillova et al., 2021; Kuzmin et al., 2023; Rhoden & Bolek, 2011; Vhora & Bolek, 2013; Willkiens et al., 2023). Specifically, in A. hylambatis, previous studies carried out in different hosts from different locations of the Neotropical realm showed variations in 2 of the characters studied here, namely number and arrangement of mamelon-like cuticular protuberances and number and arrangement of cloacal papillae, in addition to variations in the total length of individuals of both sexes (Baker, 1980; González et al., 2019; Gutiérrez, 1945; Masi-Pallarés & Maciel, 1974). In this regard, intraspecific variation may be attributed to a number of factors related to the host, such as size, age, physical condition, metabolic rate, and food supply, and factors related to the parasite, such as the time of penetration (first, second or subsequent infection) and the crowding of the specimens, or related to different study techniques, in addition to ecological and geographic factors (Baker, 1980; Chitwood, 1957; Perkins et al., 2011; Vhora & Bolek, 2013). 

The studies of helminth parasites of Argentine amphibians have focused on the Dry and Humid Chaco ecoregions; specifically, in the Dry Chaco the reports have been made in the northern provinces of the country, Chaco and Formosa. Until now, the southernmost records in this ecoregion correspond to Concepción del Bermejo in Chaco province (González, Duré et al., 2021; González, Hamann et al., 2021). In this way, the present study expands the knowledge on parasitic helminths from the South American Chaco by providing the southernmost record for the Dry Chaco ecoregion. Furthermore, it represents the first record of A. hylambatis in L. mystacinus and O. cf. asper from Argentina.

Acknowledgments

Financial support was received from Secretaría de Ciencia y Tecnología, Universidad Nacional de San Luis-UNSL (grant number PROICO 02-1520 to M.B.J.), from Universidad Nacional del Nordeste-UNNE (grant number PI21Q001 to C.E.G.), and from CONICET (PIP 11220200101582CO to C.E.G.). M.A. Villegas Ojeda was supported by Doctoral scholarship of CONICET. 

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Pedro Miguel Berrú-Paz * y Angelo Nizama-Chapoñan

Instituto del Mar del Perú, Sede Chimbote, Prolongación Los Pinos s/n Nueva Caleta Chimbote, Perú

*Autor para correspondencia: pberru@imarpe.gob.pe (P.M. Berrú-Paz) 

Recibido: 25 enero 2024; aceptado: 23 octubre 2024

Resumen

Se identificó por primera vez en la región Ancash y el Perú, el cangrejo ermitaño Isocheles aequimanus (Dana, 1852), recolectado en las áreas de Mar Brava (09°16’57.39” S, 78°30’44.16” O) y Colorado (09°40’02.19” S, 78°19’21.59” O). Se describen sus caracteres taxonómicos y se analiza la asociación que existe con I. aequimanus y 8 conchas de gasterópodos (Nassarius dentifer, Polinices uber, Solatia buccionides, Stramonita biserialis, Thaissella chocolata, Trophon peruvianus, Xanthochorus buxea y Sinum cymba) que utiliza como habitáculo. Se estudiaron 231 ejemplares de I. aequimanus cuyas tallas variaron de 3.19 a 9.21 mm de longitud del escudo, con peso medio de 0.35 g. El 33.6% de los individuos fueron machos y 66.4% hembras. Las conchas de gasterópodos con mayor frecuencia de ocupación fueron N. dentifer con 59.7%, P. uber con 25.5%, S. buccionides con 6.9% y S. biserialis con 5.6%. Isocheles aequimanus se reporta como registro nuevo para las costas de Perú, incrementando el conocimiento de la riqueza específica de cangrejos ermitaños, promoviendo la necesidad de aumentar el esfuerzo en el estudio de la biodiversidad local y nacional, con el fin de cubrir vacíos informativos y establecer conexión geográfica con otros países de la región.

Palabras clave: Isocheles aequimanus; Cangrejo ermitaño; Conchas; Selección de conchas

© 2025 Universidad Nacional Autónoma de México, Instituto de Biología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Isocheles aequimanus (Paguroidea: Diogenidae), a new record of hermit crab for Peru, with bioecological observations in natural banks of Ensis macha

Abstract

The hermit crab Isocheles aequimanus (Dana, 182), was first identified in the Ancash region of Peru, specifically in the Mar Brava (09°16’57.39” S, 78°30’44.16” W) and Colorado (09°40’02.19” S, 78°19’21.59” W) areas. This study describes its taxonomic characteristics and analyzes the association between I. aequimanus and eight gastropod shells (Nassarius dentifer, Polinices uber, Solatia buccionides, Stramonita biserialis, Thaissella chocolata, Trophon peruvianus, Xanthochorus buxea, and Sinum cymba) used as shelter. Two-hundred and thirty-one specimens of I. aequimanus were analized, ranging in size from 3.19 to 9.21 mm shield length, with an average weight of 0.35 g. Of the analyzed individuals, 33.6% were males and 66.4% were females. The gastropod shells with the highest occupancy frequency were N. dentifer with 59.7%, P. uber with 25.5%, S. buccionides with 6.9%, and S. biserialis with 5.6%. Isocheles aequimanus is reported as a new record from the coasts of Peru, increasing the knowledge of the specific richness of hermit crabs, promoting the need to increase efforts in the study of local and nationalbiodiversity, in order to cover information gaps and establish geographic connections with other countries in the region.

Keywords: Isocheles aequimanus;Hermit crab; Shells; Shell selection

Introducción

Los cangrejos ermitaños de la familia Diogenidae se conocen comúnmente como cangrejos zurdos, por tener el quelípedo izquierdo generalmente más grande que el derecho (Bijukumar y Reshmi, 2018). Esta familia se identifica por no presentar un espacio en la parte basal del tercer maxilípedo (Ingle, 1992). Comprende 429 especies existentes, lo que representa la segunda familia más grande de cangrejos ermitaños marinos, después de Paguridae (McLaughlin et al., 2010). Dentro de esta familia se encuentra el género Isocheles (Stimpson, 1858) caracterizado por presentar los quelípedos iguales o subiguales, con los dactilos abriéndose en un plano generalmente horizontal (McLaughlin, 2003; Sánchez y Campos, 1978). 

La forma de alimentarse puede darse captando partículas filtradas por las antenas, rastrillando la superficie de la arena con el tercer maxilípedo o utilizando las quelas para recoger alimento (Wicksten, 2012). Su distribución biogeográfica está restringida a aguas someras de las costas tropicales y subtropicales americanas (Mantelatto et al., 2006). 

Vera-Silva y Mantelatto (2022) señalan que existen grandes similitudes entre las especies de este género, por ello, recientemente realizaron una actualización de la clave taxonómica basada en la forma y ornamentación de los quelípedos y del número de dientes ubicados en el segundo artículo de la antena, validando estos caracteres a nivel genético, y como resultado han sido descritas 6 especies en la actualidad: Isocheles aequimanus (Dana, 1852) en Chile, I. pilosus (Holmes, 1900) e I. wurdemanni (Stimpson, 1859) en Estados Unidos, I. pacificus (Bouvier, 1907) en México y Ecuador, I. sawayai (Forest y de Saint Laurent, 1968) en Venezuela y Brasil, e I. ingowehrtmanni (Vera-Silva y Mantelatto, 2022) en Costa Rica. En Perú, Moscoso (2013) reportó la presencia de I. pacificus caracterizándose por la terminación aguda en las puntas de los dedos del quelípedo, sin embargo, esta descripción no es un indicador taxonómico. En este estudio, se describe por vez primera a Isocheles aequimanus (Dana, 1852) para el litoral peruano y se analiza el patrón de ocupación de conchas de gasterópodos, asociadas al banco natural de Ensis macha en el submareal arenoso de las playas de Mar Brava y Colorado en la región Ancash.

Materiales y métodos

Los muestreos biológicos se realizaron en el submareal arenoso en las ensenadas de Mar Brava y Colorado, ubicadas en las provincias de El Santa y Casma, respectivamente, en la región Ancash (fig. 1a, b). Los ejemplares fueron recolectados manualmente en 4 estaciones, empleando como referencia un cuadrante de 1 m², en el marco de las investigaciones propias que realiza el Laboratorio Costero de IMARPE Chimbote, durante julio del 2023.

Isocheles aequimanus fue identificado considerando principalmente: 1) la región dorsal y la forma del caparazón; 2) la forma y protuberancias en la vista dorsal de la quela izquierda; 3) el número de espinas en el segundo artículo de la antena izquierda en vista dorso mesial; y 4) la isometría de los quelípedos en forma y tamaño. La identificación específica se llevó a cabo utilizando las referencias bibliográficas de McLaughlin (2003), Guzmán (2004), Moscoso (2013) y Vera-Silva y Mantelatto (2022). Los especímenes están depositados en las colecciones del Instituto del Mar del Perú, sede Chimbote con el código IMARPE-LABCH 23-002. 

Se registró como medida referencial la longitud del escudo, considerada como la máxima distancia entre el rostro y el borde medio posterior del escudo empleando un estereoscopio y un amplificador de imagen. Los especímenes fueron extraídos de sus conchas y dimensionados en las siguientes variables: longitud del escudo (LE) y peso total húmedo con una balanza analítica de 0.0001 gr de precisión; el sexo se determinó según Lancaster (1988), y con base en la presencia de gonoporos conspicuos en el sector basal (coxa) del tercer par de pereiópodos en las hembras y quinto par de pereiópodos en los machos, además de la presencia de huevos en las hembras.

Figura 1. Estaciones de muestreo del material biológico en las áreas de Mar Brava (a) y Colorado (b). Julio del 2023. Mapa elaborado por P.M. Berrú-Paz.

Con respecto de las conchas de gasterópodos usadas como refugios temporales, éstas fueron identificadas, medidas y pesadas considerando la talla de cada ejemplar de I. aequimanus hospedado en ellas. Las relaciones somatométricas se estimaron para confrontar pares de variables cuantitativas como longitud vs. peso total de I. aequimanus, longitud I. aequimanus vs. longitud de la concha, longitud I. aequimanus vs. peso de la concha, entre otras. El grado de coherencia entre estas variables se midió mediante el coeficiente de correlación de Pearson.

Resultados

Se estudiaron un total de 231 ejemplares de I. aequimanus, con medidas que variaron de 3.19 a 9.21 mm de longitud del escudo y con peso medio de 0.35 g. El 33.6% de los individuos analizados fueron machos y 66.4% hembras, de las cuales 82.2% correspondieron a hembras grávidas (tabla 1).

Tabla 1

Algunas variables biológicas registradas en Isocheles aequimanus en las playas de Mar Brava y Colorado en la región Áncash, julio de 2023. 

Tabla 2

Dimensiones de conchas de gasterópodos empleadas como refugio por Isocheles aequimanus en las playas de Mar Brava y Colorado, junio de 2023.

Figura 2. Abundancia porcentual de la comunidad macro-bentónica en las áreas de estudio. Julio 2023.

La comunidad macrobentónica en las áreas de estudio estuvo representada por 27 taxones, de los cuales los crustáceos fueron los más abundantes con 62.25%, seguido por gasterópodos (13.28%), bivalvos (8.95%), poliquetos (3.61%) y otros (11.91%) (fig. 2). Dentro de los crustáceos, I. aequimanus correspondió al 97.3% de la abundancia, mientras que de los gasterópodos, Nassarius dentifer representó 80.8% de abundancia, y destacó la preferencia de I. aequimanus por las conchas de N. dentifer debido a la facilidad con que éstas se encuentran en el entorno.

Se identificaron 8 taxones de conchas de gasterópodos utilizadas como refugio por I. aequimanus, de los cuales N. dentifer (Powys, 1835), Polinices uber (Valenciennes, 1832), Solatia buccionoides (Sowerby, 1832) y Stramonita biserialis (Blainville, 1832) mostraron el mayor porcentaje de ocupación con 59.7, 25.5, 6.9 y 5.6%, respectivamente; mientras que Thaisella chocolata (Duclos, 1832), Trophon peruvianus (Lamarck, 1816), Xanthochorus buxeus (Broderip, 1833) y Sinum cymba (Menke, 1828) fueron los menos representados (tabla 2). 

La macrofauna asociada al grupo gasterópoda estuvo representada por 5 especies, con abundancia numérica de 80.8% en N. dentifer, 15.1% en S. buccinoides, 1.7% en S. cymba, 1.4% en X. buxea y 1.0% en A. unifasciata (tabla 3).

Tabla 3

Diversidad de gasterópodos registrados como fauna asociada en las playas de Mar Brava y Colorado, junio de 2023.

Diagnosis. Quelípedos de igual forma y tamaño, pilosos, con la superficie de color verde azulado en ejemplares vivos o frescos y anaranjados en individuos preservados con alcohol, presencia de tubérculos de diferentes tamaños distribuidos aleatoriamente (fig. 3).

Figura 3. Ejemplar macho (a) y hembra (b) de Isocheles aequimanus.

Márgenes laterales con espinas curvas de color dorado terminadas en el dedo fijo y móvil (fig. 4a). Segundo y tercer pereiópodo de gran tamaño a comparación del tercero y cuarto que son pequeños. Escudo más largo que ancho, con una coloración moteada entre azul, verde y gris, con presencia de mechones de setas largas y espinas pequeñas, proyecciones laterales terminadas con una espina (fig. 4b). Pedúnculo ocular de color azul grisáceo con pequeñas setas, una banda longitudinal de color marrón, córnea pequeña (fig. 4c). Acículos oculares con 3-4 espinas de color dorado (fig. 4d). Telson asimétrico, piloso, márgenes dorso laterales con espinas (fig. 4e). Urópodo izquierdo más grande que el derecho (fig. 4f).

Tabla 4

Valores medios de talla y peso de Isocheles aequimanus y distintos taxones de concha de gasterópodo habitada.

Figura 4. Isocheles aequimanus: a) quelípedos, b) escudo, c) pedúnculo ocular, d) acículos oculares, e) telson y f) urópodos.

Figura 5. Indicadores taxonómicos de Isocheles aequimanus: a) quela izquierda con presencia de tubérculos; b) segundo artículo izquierdo de la antena con 6 espinas.

El análisis de los especímenes de Isocheles aequimanus concuerda con las características mencionadas en la redescripción de Vera-Silva y Mantelatto (2022). Como información adicional, I. aequimanus se diferencia de sus congéneres por la distribución de tubérculos en la superficie dorsal de la quela, con 3 filas más prominentes que otras (fig. 5a), y por la presencia de 6-8 espinas en el margen mesial del segundo artículo de la antena (fig. 5b).

Relaciones somatométricas. La correlación entre la talla media de I. aequimanus y las tallas y pesos de las distintas conchas de gasterópodos habitadas, permitió estimar una correlación positiva con valores de r² de 0.7767 para talla vs. talla y de 0.9774 para talla vs. peso, lo que sugiere que los ejemplares de una talla determinada de I. aequimanus elijen proporcionalmente una talla y peso adecuado de la concha de gasterópodo a ocupar como refugio (tabla 4, fig. 6).

Figura 6. Curvas de correlación entre la talla de Isocheles aequimanus y talla (a) y peso (b) de distintos taxones de conchas de gasterópodos.

Figura 7. Correlación entre las variables talla y peso de Isocheles aequimanus.

Isocheles aequimanus vs. Nassarius dentifer. La correlación talla vs. peso, talla vs. talla y peso vs. peso entre I. aequimanus y concha de N. dentifer registró una asociación de tipo moderada con un coeficiente de correlación de Pearson estimado de 0.492, 0.481 y 0.436 para cada uno de los casos, respectivamente. En el caso de Polinices uber las correlaciones fueron más altas(0.791, 0.862 y 0.798, respectivamente); así como para Solatia buccionides (0.845, 0.630 y 0.770, respectivamente). 

Relación longitud-peso. La relación longitud-peso en I. aequimanus permitió encontrar un buen ajuste al modelo potencial, con valores del coeficiente de determinación (r²) de 0.745, un valor de la pendiente “b” de 1.080 que denota un crecimiento alométrico negativo y un valor de la constante “a” de 0.0596 (fig. 7).

Discusión

En el submareal costero de la región Ancash (norte-centro del Perú) fue recolectada, identificada y registrada por primera vez la especie Isocheles aequimanus, como parte de la macrofauna acompañante durante el estudio de un recurso infaunal conocido como “navaja” Ensis macha, sin haber reportes hasta la fecha sobre otros estudios relacionados con su taxonomía y/o biología en el país.

En otros países, I. aequimanus fue revalidada por Guzmán (2004) con ejemplares recolectados en las playas de Iquique y Copiapó en Chile y refiere, además, la descripción de 5 especies del género Isocheles: 2 para el océano Atlántico (I. wurdemanni, I. sawayai) y las otras 3 para el Pacífico e Indopacífico (I. pilosus, I. pacificus, I. aequimanus); considerando a I. pacificus como una variación de I. wurdemani, citada para el Perú por Chirichigno (1970). En una última clave para la identificación de crustáceos decápodos y estomatópodos del Perú (Moscoso, 2013), solo se cita a I. pacificus; mientrasque Vera-Silva y Mantelatto (2023) realizan una revisión taxonómica del género,utilizan datos morfológicos y multigénicos e incluyen a I. ingowehrtmanni como una nueva especie en el Pacífico oriental.

Dentro de los mecanismos de selección y las relaciones interespecíficas entre los pagúridos y la diversidad de gastrópodos, I. aequimanus se encontró habitando 8 conchas distintas de gasterópodos en las playas de Mar Brava y Colorado (fig. 8), con una presencia porcentual de 59.74% para N. dentifer, 25.54% para P. uber, 6.93% para S. buccionoides, 5.63% para S. biserialis, 0.87% para T. chocolata y 0.43% para T. peruvianus, X. buxea y S. cymba. Quiñe et al. (2003) encontraron para la zona de Pisco (sur de Perú), que 94% de la población de Pagurus villosus estuvo habitando la concha de N. gayi; mientras que Iannacone y Alvariño (2005), registraron en la bahía de Ancón (centro de Perú) la preferencia de 3 especies simpátricas de pagúridos (P. villosus, P. edwarsi, P. perlatus) por N. dentifer, lo cual coincide con la preferencia de I. aequimanus en el presente estudio.

Figura 8. Conchas de gasterópodos utilizados como refugio de I. aequimanus: a) Nassarius dentifer; b) Poliniceps uber; c) Solatia buccionoides; d) Stramonita biserialis; e) Thaisella chocolate; f) Trophon peruvianus; g) Xantochorus buxea y h) Sinum cymba.

El uso de conchas empleadas como refugios temporales por los cangrejos ermitaños es de vital importancia para su supervivencia y reproducción, y seguramente existe una coherencia entre el peso y talla del organismo ocupante y el peso y talla de la concha ocupada, que se traduce en el uso óptimo de la energía empleada por el pagúrido. La diversidad de conchas empleadas ha sido relacionada con el crecimiento, reproducción y protección de los depredadores, según Hazlett (1981). Así, conchas livianas y de mayor volumen interno (globosas) han sido relacionadas con un aumento en las tasas de crecimiento y tamaño de puestas (Bertness, 1981), patrón observado para otras especies como Pagurus bernhardus (Elwood et al., 1979). Sin embargo, en el presente estudio se registró un patrón local diferente, se observó una asociación más fuerte entre I. aequimanus y las conchas de N. dentifer, P. uber, S. buccionoides y S. biserialis; por otro lado, se notó una disminución en el grado de ocupación de T. chocolata, T. peruvianus, X. buxeus y S. cymba.

Figura 9. Importancia numérica del grupo gasterópoda en el área de muestreo.

El uso preferencial de I. aequimanus por las distintas conchas de gasterópodos en el presente estudio, contrario a lo sucedido en otras especies de pagúridos, estaría más bien relacionado con la abundancia relativa de cada uno de los taxones de gasterópodos en el área de muestreo, donde se registraron 5 especies en la macrofauna asociada. De éstas, N. dentifer y S. buccinoides presentaron la mayor abundancia numérica con 80.8 y 15.1%, respectivamente (fig. 9), valores muy correlacionados al grado de ocupación de I. aequimanus, estimado en 59.7% para N. dentifer y 25.5% para P. uber (tabla 2).

De los 8 taxones de conchillas de gasterópodos ocupadas por I. aequimanus, 4 se registraron como macrofauna viva, durante el muestreo, lo que supone que las conchas vacías de P. uber, S. biserialis, T. chocolata y T. peruvianus ocupadas por I. aequimanus reflejan un gran dinamismo de interacciones entre individuos de la misma especie y muestran un alto grado de oportunismo al ocupar conchillas con muy baja abundancia numérica, que pudieron ser transportadas desde el borde costero al submareal arenoso y terminar muriendo, muy fuera de su zona de confort.

Isocheles aequimanus constituye un nuevo registro para el litoral costero de Perú, incrementa el conocimiento de la riqueza de cangrejos ermitaños y refleja la necesidad de aumentar el esfuerzo para el estudio de la biodiversidad en general, con el fin de cubrir vacíos informativos y establecer conexión geográfica con otros países de la región.

En las áreas de estudio de Mar Brava y Colorado, I. aequimanus utiliza 8 conchas de gasterópodos como refugios temporales. El uso preferencial de I. aequimanus por las conchas de N. dentifer, está mayormente relacionado por su abundancia relativa en la composición macrobentónica de las áreas estudiadas. 

Agradecimientos

Los autores agradecen a Guillermo Guzmán, profesor de Recursos Naturales de la Universidad Nacional Arturo Prat, por sus aportes oportunos y la confirmación en la identificación taxonómica de Isocheles aequimanus.

Referencias 

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Bijukumar, A. y Reshmi, R. (2018). Taxonomy of common hermit crabs of India. En A.K. Jaiswar et al. (Eds.), Advances in finfish and shellfish taxonomy (pp. 131–181). Delhi: Narendra Publishing House.

Chirichigno, N. (1970). Lista de crustáceos del Perú (Decapoda y Stomatopoda) con datos de su distribución geográfica. Informe Instituto del Mar del Perú-Callao, Núm. 35. Instituto del Mar del Perú. https://hdl.handle.net/20.500.12958/263 

Elwood, R. W., Mclean, A. y Webb, L. (1979). The development of shell preferences by the hermit crab Pagurus bernhardus. Animal Behaviour, 27, 940–946. https://doi.org/10.1016/0003-3472(79)90032-0 

Guzmán, G. (2004). Isocheles aequimanus (Dana, 1852) (Decapoda, Anomura, Paguroidea): revalidación para la carcinofauna chilena. Investigaciones Marinas, Valparaíso, 32, 129–132.

Hazlett, B. A. (1981). The behavioral ecology of hermit crabs. Annual Review of Ecology, Evolution, and Systematics, 12, 1–22. https://doi.org/10.1146/annurev.es.12.110181.000245 

Iannacone, J. y Alvariño, L. (2005). Morfometría, proporción sexual y selectividad a conchillas de gasterópodos de tres especies simpátricas de cangrejos ermitaños Pagurus (Decapoda. Anomura, Paguridae) en la bahía de Ancón, Lima, Perú. Gayana, 69, 139–143. http://dx.doi.org/10.4067/S0717-65382005000100015 

Ingle, R. W. (1992). Hermit crabs of the northeastern Atlantic Ocean and Mediterranean Sea: an illustrated key. London, New York: Chapman y Hall.

Lancaster, I. (1988). Pagurus bernhardus (L.), an introduction to the natural history of hermit crabs. Field Studies, 7, 189–238. 

Mantelatto, F. L., Robles, R., Biagi, R. y Felder, D. L. (2006). Molecular analysis of the taxonomic and distributional status for the hermit crab genera Loxopagurus Forest, 1964 and Isocheles Stimpson, 1858 (Decapoda, Anomura, Diogenidae). Zoosystema, 28, 495–506. 

McLaughlin, P. A. (2003). Illustrated keys to families and genera of the superfamily Paguroidea (Crustacea: Decapoda: Anomura), with diagnoses of genera of Paguridae. Memoirs of Museum Victoria, 60, 111–144.

McLaughlin, P. A., Komai, T., Lemaitre, R. y Rahayu, D. L. (2010). Annotated checklist of Anomuran decapod crustaceans of the world (exclusive of the Kiwaoidea and families Chirostylidae and Galatheidae of the Galatheoidea) Part I – Lithodoidea, Lomisoidea and Paguroidea. Raffles Bulletin of Zoology Supplement, 23, 5–107.

Moscoso, V. (2013). Clave para identificación de crustáceos decápodos y estomatópodos del Perú. Boletín Instituto Mar Perú, 28, 8–135.

Quiñe, M., Tarazona, J. y Balapatiño, A. (2003). Características de la estructura poblacional de Pagurus villosus (Nicolet, 1849) en Bahía Independencia, Pisco, Perú durante 1999. Libro de Resúmenes XII Reunión Científica ICBAR. UNMSM. Lima, Perú.

Sánchez, H. y Campos, N. (1978). Los cangrejos ermitaños (Crustacea, Anomura, Paguridae) de la costa norte colombiana. Anales del Instituto de Investigaciones Marinas de Punta Betín, 10, 15–62.

Vera-Silva, A. L. y Mantelatto, F. L. (2022). Taxonomic revision of Isocheles Stimpson, 1858 and Loxopagurus Forest, 1964 (Decapoda: Anomura: Diogenidae) using morphological and multigene data, with the description of a new Eastern Pacific species. Journal of Crustacean Biology, 42, 1-25. https://doi.org/10.1093/jcbiol/ruac058 

Wicksten, M. K. (2012). Decapod Crustacea of the Californian and Oregonian zoogeographic provinces. Zootaxa, 3371, 1–307. https://doi.org/10.11646/zootaxa.3371.1.1

Miguel Ángel Mosqueda-Cabrera *, Diana Laura Desentis-Pérez y Tania Araceli Padilla-Bejarano

^a Centro de Investigación Científica y de Educación Superior de Ensenada, Departamento de Biología de la Conservación, Carretera Tijuana-Ensenada # 3918, Zona Playitas, 22860 Ensenada, Baja California, Mexico

^b Universidad Autónoma de Baja California, Facultad de Ciencias, Carretera Transpeninsular # 3917, Colonia Playitas, 22860 Ensenada, Baja California, Mexico

*Corresponding author: arteaga@cicese.mx (M.C. Arteaga)

Received: 28 February 2024; accepted: 01 July 2024

Resumen

Las larvas de tercer estadio avanzado (AdvL₃) de Gnathostoma sp. I aisladas de la musculatura de Dormitator latifrons y Rhamdia guatemalensis son morfológica y molecularmente iguales entre sí y se relacionan genéticamente con un juvenil aislado del hígado de Didelphis marsupialis, en la cuenca del río Ostuta, Oaxaca. Asimismo, son diferentes de las 3 especies de Gnathostoma descritas para México por el tamaño del cuerpo, por los ganchos del bulbo cefálico en la fase AdvL₃ y por la presencia de un prepucio cuticular en el extremo posterior de un macho juvenil. A través del marcador molecular COI, un análisis de distancias genéticas y la inferencia de la filogenia entre las especies del género, se concluye que Gnathostoma sp. I está estrechamente emparentada, pero taxonómicamente es diferente a G. turgidum y a las otras especies presentes en México y el mundo, aun cuando falta material para establecerla como especie nueva. Por otro lado, con base en características morfológicas se documenta el hallazgo de las AdvL₃ de G. lamothei (en Rhambdia guatemalensis y Lontra longicaudis) y la AdvL₃ de otra especie no identificada (en R. guatemalensis y Synbranchus marmoratus), pero distinta a las anteriores de acuerdo con evidencias morfológicas y moleculares.

Palabras clave: Gnathostoma spp.; Tercer estadio avanzado; Filogenia; México; Marsupiales

© 2025 Universidad Nacional Autónoma de México, Instituto de Biología. Este es un artículo Open Access bajo la licencia CC BY-NC-ND

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

The high morphological variation found in the immature stages of the nematode Gnathostoma sp. I is not supported by molecular information

Abstract

The third-stage advanced larvae (AdvL₃) of Gnathostoma sp. I isolated from the musculature of Dormitator latifrons and Rhamdia guatemalensis are identical to each other morphological and molecularly, and are genetically related to a juvenile isolated from the liver of Didelphis marsupialis, in the Ostuta River basin, Oaxaca. They are different from the 3 species of Gnathostoma described from Mexico by body size and the hooks of the cephalic bulb in the AdvL₃ stage, as well as, in the presence of a cuticular pouch at the posterior end of the juvenile male. Through the COI molecular marker, a genetic distance analysis, and phylogenetic inference among the species of the genus, we conclude that Gnathostoma sp. I is closely related to, but distinct from G. turgidum and from other species found in Mexico and worldwide, even though there is not enough material to establish it as a new species. Additionally, based on morphologic characteristics, we documented the discovery of AdvL₃ of G. lamothei (in Rhambdia guatemalensis and Lontra longicaudis) and AdvL₃ of another unidentified species (in R. guatemalensis and Synbranchus marmoratus), which is distinct to above according to morphologic and molecular evidence.

Keywords: Gnathostoma spp.; Advanced third stage; Phylogeny; Mexico; Marsupials

Introducción

Entre el enorme conjunto de especies de helmintos que se distribuyen en México se encuentra el género Gnathostoma Owen, 1836(Spirurida: Gnathostomatidae) conformado por 12 especies válidas (Almeyda-Artigas, 1991; Bertoni-Ruiz et al., 2005; Miyazaki, 1954). Tres de ellas se distribuyen en México: Gnathostoma binucleatum Almeyda-Artigas, 1991; Gnathostoma turgidum Stossich, 1902 y Gnathostoma lamothei Bertoni-Ruiz, García-Prieto, Osorio-Sarabia y León-Règagnon, 2005 (Gaspar-Navarro et al., 2013). Las especies presentan una alta especificidad hacia mamíferos carnívoros como sus hospederos definitivos; G. binucleatum (Canidae, Felidae, Suidae), G. lamothei (Procyonidae) y G. turgidum (Didelphidae) (Pérez-Álvarez et al., 2008). El hospedero definitivo adquiere la infección al alimentarse de los segundos hospederos intermediarios, peces dulceacuícolas para las 2 primeras especies (Almeyda-Artigas, 1991; Bertoni-Ruiz et al., 2005), ranas y accidentalmente peces para la última (Mosqueda-Cabrera et al., 2009, 2023).

El objetivo de la presente investigación fue describir un morfotipo de Gnathostoma que difiere morfológicamente de las especies conocidas para didélfidos de México, proveniente de una zona no explorada previamente, la cuenca del río Ostuta, Oaxaca. 

Materiales y métodos

La cuenca del río Ostuta forma parte de la porción oriente de la región hidrográfica Tehuantepec (RH22). Se localiza en la zona suroriente del estado de Oaxaca, que limita con Chiapas. La laguna Las Garzas (16°17’46” N, 94°27’17” O), es un cuerpo de agua semipermanente, remanente de un antiguo cauce de río ubicado en la región hidrográfica Costa de Chiapas (RH23) en la cuenca del mar Muerto, que colinda al oeste con la cuenca del río Ostuta (Conagua, 2021) y es irrigada por la misma durante lluvias torrenciales (fig. 1).

Esta investigación fue conducida debido al hallazgo fortuito de larvas tercer estadio avanzado (AdvL₃) en las heces de la nutria neotropical Lontra longicaudis de la cuenca del río Ostuta, Oaxaca durante abril y mayo de 2018. Posteriormente, durante el 2022 se realizó la búsqueda de larvas de Gnathostoma en peces de este río y en cuerpos de agua asociados a la región hidrográfica (RH23). Para su captura se utilizaron redes de pesca como atarrayas y chinchorros. La musculatura de los peces fue revisada a contraluz entre 2 vidrios y posteriormente digerida con pepsina artificial (16 gr de pepsina, 6 gr de NaCl y 8 ml de HCl en 1 L de agua). La búsqueda de las larvas se realizó con ayuda de un microscopio estereoscópico; fueron fijadas en alcohol etílico al 70% caliente y conservadas en alcohol etílico al 70% frío. Para su estudio fueron transparentadas con lactofenol de Amman y observadas bajo el microscopio compuesto. Todas las medidas se presentan en micras, se especifica el rango y entre paréntesis el promedio seguido de la desviación estándar y el número de observaciones. Los parámetros de la infección fueron calculados de acuerdo con Bush et al. (1997). Se tomaron fotografías con una cámara digital montada a un microscopio óptico y los dibujos fueron realizados con ayuda una cámara clara. La captura y el sacrificio del tlacuache común Didelphis marsupialis fue realizada de acuerdo con Almeyda-Artigas et al. (2010) e identificado por la morfología craneal siguiendo a Gardner (1973). El material de referencia fue depositado en la Colección Nacional de Helmintos (CNHE) del Instituto de Biología, Universidad Nacional Autónoma de México (IB-UNAM). La obtención de los organismos se realizó bajo el permiso de colecta SCPA/DGVS/03184/22 expedido por la Secretaría de Medio Ambiente y Recursos Naturales, México.

Figura 1. Cuenca del río Ostuta, Oaxaca en la región hidrográfica Tehuantepec (RH22). Laguna Las Garzas en la región hidrográfica Costa de Chiapas (RH23). Mapa elaborado por D.L. Desentis Pérez.

La extracción del DNA se llevó a cabo utilizando el kit de extracción DNeasy Blood & Tissue (QIAGEN). Se amplificó la región del segundo espaciador interno transcrito (ITS2) y la subunidad I del citocromo c oxidasa (COI) mediante la reacción en cadena de la polimerasa (PCR por sus siglas en inglés). La amplificación de las secuencias nucleotídicas parciales de los genes mencionados se llevó a cabo a partir de reacciones compuestas por una solución de 8.5 µl de H₂O, 3 µl de buffer 5x, 0.2 µl de cada oligonucleótido, 0.1 µl de enzima Taq polimerasa (Bioline) y 3 µl de DNA genómico en un volumen total de 15 µl. La región ITS2 se amplificó con los oligonucleótidos NEWS2 (forward) 5ʼ-TGTGTCGATGAAGAACGCAG-3ʼ e ITS2-RIXO (reverse) 5ʼ-TTCTATGCTTAAATTCAGGGG-3ʼ (Almeyda-Artigas et al., 2000a), con el siguiente perfil térmico: 1 ciclo de 94 °C por 1 min; 5 ciclos de 92 °C por 30 s, 45 °C por 30 s y 72 °C por 1 min; 35 ciclos de 92 °C por 30 s, 53 °C por 30 s y 72 °C por 1 min; elongación final a 72 °C por 4 min. Las secuencias parciales del gen mitocondrial COI se amplificaron con los cocteles de oligonucleótidos previamente preparados según Prosser et al. (2013), utilizando los siguientes oligonucleótidos: NemF1_t15ʼCRACWGTWAATCAYAARAATATTGG3-ʼ, NemF2_t1 5ʼ-ARAGATCTAATCAT
AAAGATATYGG3-ʼ, NemF3_t1 5ʼ-ARAGTTCTAATCATAARGATATTGG-3ʼ (forward) y NemR1_t1 5ʼ-AAACTTCWGGRTGACCAAAAAATCA-3ʼ, NemR2_t1 5ʼ-AWACYTCWGGRTGMCCAAAAAAYCA-3ʼ, NemR3_t1 5ʼAAACCTCWGGATGACCAAAAAATCA-3ʼ (reverse) implementando el siguiente perfil térmico: 1 ciclo de 94 °C por 1 min; 5 ciclos de 94 °C por 40 s, 45 °C por 40 s y 72 °C por 1 min; 35 ciclos de 94 °C por 40 s, 53 °C por 40 s y 72 °C por 1 min; elongación final a 72 °C por 5 min. Los productos de la PCR fueron purificados mediante el kit de purificación QUIAquick PCR (50) (QIAGEN). Las secuencias fueron obtenidas mediante el secuenciador de DNA automatizado ABI Prism 310 en el Laboratorio de Secuenciación Genómica del PABIO-UNAM. En el caso del ITS-2 se usaron los oligonucleótidos: NEWS2 e ITS2-RIXO para la secuenciación, en el caso del COI se usaron los oligonucleótidos M13F (5ʼ-TGTAAAACGACGGCCAGT-3ʼ) y M13R (5ʼ-CAGGAAACAGCTATGAC-3ʼ) (Messing, 1993).

Las secuencias obtenidas se alinearon en el programa MAFFT V7 (en línea) con sus homólogas (COI/ITS2) disponibles en el repositorio GenBank del NCBI (National Center for Biotechnology Information), correspondientes a especies nominales del género Gnathostoma en México y una especie del género Anisakis (A. pegreffi FJ907317/AY603531) como grupo externo: G. binucleatum (AB180103/EU915244), G. lamothei (KF648543), G. turgidum (KT894798/KF648548), G. spinigerum (AB037132/KF648553), G. nipponicum (JQ824059/JN408314), G. hispidum (JQ824056/JQ824057). A partir de los alineamientos múltiples de secuencias se realizó un análisis de distancias genéticas en el programa MEGA X 11.0.13 para establecer la similitud entre las distintas especies utilizando el modelo Kimura 2 parámetros (K2P) de acuerdo con Hebert et al. (2003). El análisis filogenético se realizó con los 2 marcadores moleculares (COI, ITS-2), de manera independiente y posteriormente fueron concatenados con un ajuste manual en el programa Mesquite v. 3.6 (Maddison y Maddison, 2019). La inferencia filogenética se realizó utilizando el criterio de inferencia bayesiana (IB) en el programa MrBayes v. 3.2.1 (Ronquist et al., 2012). Los resultados se ilustraron en un árbol filogenético construido en el programa FigTree v. 1.4.2 (Rambaut, 2006).

Descripción

Gnathostoma sp. I (figs. 2-4)

Juvenil. Bulbo cefálico con ganchos de una sola punta dispuestos en 10 hileras completas, mide 393.6 × 861, de largo y ancho, respectivamente. Dos papilas cervicales laterales, derecha e izquierda, ambas en la décima hilera. Ancho del cuerpo a la altura de la intersección esófago-intestino 1,479.60. Esófago de 14,586.39 de largo × 836.10 de ancho, cubre 58.3% respecto al ancho del cuerpo. Cuatro sacos cervicales se proyectan desde la base del bulbo cefálico, en promedio miden 2,379.69 de largo, 16.31% respecto al largo del esófago. Bursa con pequeñas espinas ventrales en sentido posteroanterior, presenta 4 pares de papilas pedunculadas; 2 pares preanales, un par adanal y 2 pares postanales; además, 4 pares de papilas no pedunculadas; un par preanal, 2 pares adanales y un par postanal. Dos espículas, la derecha 3,267.43 de largo × 142.68 de ancho; la izquierda 949.41 largo × 93.48 de ancho; proporción de 1:3.4; el extremo posterior con cutícula holgada más larga que el cuerpo, similar a un prepucio.

Espinas corporales presentes solo en la región anterior del cuerpo con variaciones en el número de puntas según la región; a, inmediatamente posteriores al bulbo cefálico, con 5-7 puntas son comunes las de 5, más largas (49.06) que anchas (22.08); b, en la región que ocupan las papilas cervicales, de 5-9 puntas son comunes las de 7 y 8, con 61.32 de largo y 31.89 de ancho; c, a la altura de los sacos cervicales, con 5 a 10, más largas (108.24) que anchas (75.80), con una a 3 puntas laterales y tronco central con 3 y 5 puntas; d, a la altura de la intersección esófago-intestino, espinas con 5 a 6 puntas más anchas que largas, 59.04 y 39.36, respectivamente, las puntas laterales generalmente más cortas y con 3 a 4 puntas centrales; e, posterior a la intersección esófago-intestino, cambian drásticamente de forma y tamaño, siendo más largas (59.04) que anchas (34.44) con un par de puntas laterales cortas y de 2 a 3 puntas centrales de igual tamaño; en la parte más posterior de la porción escamada del cuerpo se observa un gradiente donde las puntas centrales de las espinas van ensanchándose y desapareciendo las puntas laterales hasta terminar en espinas de una sola punta.

Datos morfológicos adicionales (basados en la observación directa de especímenes depositados en la CNHE 4739): macho obtenido del estómago de Didelphis marsupialis. Mide 46,000 de largo por 2,182.41 de ancho máximo. Esófago 6,625 por 542.52 de largo y ancho, respectivamente; cubre 14.4% respecto a la longitud del cuerpo. Dos papilas cervicales en 11 (izquierda) y 10 (derecha), sobre las hileras de espinas del cuerpo. Bulbo cefálico 442.80 por 939.72 de largo y ancho, respectivamente, con 9 hileras completas de ganchos con una sola punta de base gruesa y cónica. Extremo posterior con cutícula holgada corrugada y arreglo de las espinas en el cuerpo iguales a la forma juvenil que aquí se describe. Las espinas cubren 61% del cuerpo.

Resumen taxonómico

Hospedero: tlacuache común Didelphis marsupialis Linnaeus, 1758 (Didelphimorphia: Didelphidae).

Sitio de infección: hígado.

Localidad: inmediaciones del río Ostuta, San Francisco Ixhuatán, Oaxaca.

Depósito de especímenes: CNHE 12827.

Comentarios taxonómicos

Para propósitos comparativos, estudiamos especíme-
nes de G. turgidum de D. marsupialis (CNHE 4739) y G. turgidum de D. virginiana (CNHE 4261, 4740, 4736). La presencia de una cutícula holgada en el extremo posterior en la forma juvenil estudiada en este trabajo y su ausencia en el adulto de G. turgidum (CNHE 4739), así como el porcentaje espinado del cuerpo (61% vs. 40%, respectivamente), son las únicas características diferentes entre las especies. La relación entre las espículas del macho, el número de puntas y arreglo de las espinas en el cuerpo no mostraron diferencias entre ambos lotes de material.

Los fragmentos de gen amplificados, ITS-2 (480 pb) y COI (702 pb), se encuentran disponibles en GenBank con números de acceso PQ149238 y PQ143178, respectivamente, y pertenecen al segundo tercio del juvenil de Gnathostoma sp. I

Figura 2. Gnathostoma sp. I parásito de D. marsupialis. a) Espícula derecha y cutícula holgada del extremo posterior; b) patrón de espinación de la bursa con papilas ventrales y laterales.

Figura 3. Extremo posterior de juvenil de Gnathostoma sp. I. (a) Cutícula holgada; b) espícula derecha y papilas de la bursa. Escala de las barras = 200 μm.

Las notables diferencias morfológicas en el tamaño del cuerpo entre las AdvL₃ de Gnathostoma aisladas de los peces D. latifrons y las de G. turgidum (tabla 1), así como el tamaño de los ganchos en las 4 hileras del bulbo cefálico, nos condujeron a pensar que pudieran tratarse de especies independientes. El análisis de distancias genéticas de las secuencias (modelo K2P del ITS2) determinó que las AdvL₃ obtenidas de D. latifrons y el juvenil obtenido de D. marsupialis son iguales entre sí pero también muy similares a las secuencias de G. turgidum con valores de 0.0043 para el juvenil y 0.0046 para la AdvL₃. En cuanto al marcador molecular COI, en el análisis de distancias genéticas de las secuencias registramos una distancia genética de 0.0657 con respecto a las ya mencionadas y la secuencia de G. turgidum. Basados en estos datos, nuestro material podría representar una variante de G. turgidum o una especie cercanamente relacionada a ésta (fig. 5). Sin embargo, y pese a las diferencias morfológicas tan claras entre ambos taxones, preferimos adoptar una posición conservadora y no establecer a las larvas y al juvenil que recuperamos en el río Ostuta, Oaxaca, como una especie independiente hasta contar con una mayor cantidad de evidencias, tanto morfológicas (de hembras y machos adultos) como moleculares.

Figura 4. Arreglo de las espinas en el cuerpo de Gnathostoma sp. I. a) Primeras hileras del cuerpo; b) región de la papila cervical; c) región distal de los sacos cervicales; d) intersección esófago-intestino; e) región final de la superficie espinada. Escala de la barra = 100 μm.

Gnathostoma sp. I (figs. 6, 7)

Larva de tercer estadio avanzado. La siguiente descripción está basada en la observación de 23 larvas. El cuerpo mide 1,149-1,414 (1, 262 ± 90.86; 11) de largo y 94.71-129.77 (105.82 ± 12.59; 11) de ancho; está cubierto totalmente por hileras de espinas transversales, cuyo número oscila entre 180-223 (192.79 ± 11.82; 13). Bulbo cefálico 30.77-85.85 (44.69 ± 14.57; 13) de largo × 65.23-169.24 (91.90 ± 26.72; 13) de ancho; presenta espinas dispuestas en 4 hileras transversales: 27-37 (31.17 ± 2.59; 23), 28-44 (34.39 ± 3.78; 23), 30-44 (37.22 ± 3.42; 23) y 33-46 (41.26 ± 3.75; 23), de la primera a la cuarta, respectivamente. Miden: 4.44-6.22 (5.33 ± 0.68) × 1.77-2.66 (2.28 ± 0.31), 5.33-5.77 (5.64 ± 0.21) × 2.22-2.66 (2.35 ± 0.21), 5.33-6.22 (5.65 ± 0.34) × 2.66-3.55 (3.11 ± 0.26), 4.44-4.88 (4.82 ± 0.17) × 2.66-3.55 (3.11 ± 0.36), largo y ancho de la primera a la cuarta hilera, respectivamente. Cuatro sacos cervicales ocupan de 43.23 a 75.29% (58.99 ± 7.65; n = 10) de la longitud de esófago. El esófago abarca de 32 a 73.81% (40.30 ± 2.60; n = 10) del largo total y 51% de su ancho, a la altura de la intersección esófago-intestino. El poro excretor ventral, se ubica en las hileras 15-21 (18.73 ± 1.60; n = 15). Dos papilas cervicales laterales, la derecha situada en la hilera 9-13 (10.58 ± 1.20; n = 19) y la izquierda en la hilera 9-16 (11.11 ± 1.60; n = 18). Primordio genital ubicado en 65.61 a 69.67% (68.39 ± 0.56; n = 4) del cuerpo. Dos papilas caudales, la derecha anterior al primordio genital de 58.06 a 70.91% (62.82 ± 2.27; n = 11) y la izquierda inmediatamente posterior al primordio genital ubicada en 61.26 a 81.56% (72.85 ± 4.18; n = 11) del extremo anterior del cuerpo, respectivamente.

Tabla 1

Comparación morfométrica entre larvas de tercer estadio avanzado de las 3 especies de Gnathostoma en México. PCi = Papila cervical izquierda, PCd = papila cervical derecha, PE = poro excretor, Ea = ancho del esófago, Ca = ancho del cuerpo, — sin datos.

^a Larvas obtenidas experimentalmente de Lithobates heckscheri, ^b de una larva obtenida de la musculatura de M. albus,^c de 3 larvas encontradas en excretas de L. longicaudis, ^d de una larva obtenida de la musculatura de Rhamdia guatemalensis, ^e de 11 larvas obtenidas de la musculatura de R. sweifeli, ^f de 28 larvas obtenidas del hígado de Didelphis virginiana (S-L3), ^g de 5 larvas obtenidas de la musculatura de M. albus, ^h de una larva obtenida de la musculatura de Gobiomorus dormitor,ⁱ de 2 larvas obtenidas de la musculatura de Rhamdia guatemalensis, ^j de 23 larvas obtenidas de la musculatura de Dormitator latifrons, ^k de 30 larvas obtenidas de la musculatura de Petenia splendida, ^l de 8 larvas obtenidas de la musculatura de D. latifrons y Sciades guatemalensis, ^m de 3 larvas 2 obtenidas de la musculatura de S. marmoratus y una de la musculatura de R. guatemalensis. * Datos de Gaspar-Navarro et al. (2013). ** Datos de 2 larvas.

Figura 5. Relaciones filogenéticas entre Gnathostoma sp. I y algunas especies del género Gnathostoma inferidas mediante los marcadores moleculares (COI/ITS-2). Los valores de probabilidad bayesiana se indican en los nodos.

Resumen taxonómico

Localidad: laguna Las Garzas, San Francisco Ixhuatán, Oaxaca.

Hospedero: Dormitator latifrons (Eleotridae).

Otros hospederos: Rhamdia guatemalensis (Heptapteridae).

Sitio de infección: musculatura.

Parámetros de infección: prevalencia 30.6%, intensidad promedio 4.8, abundancia promedio 1.47.

Distribución en el hospedero: 51 larvas en 11 hospederos: 0 (25), 1 (3), 2 (2), 3 (1), 6 (2), 7 (2), 15 (1).

Depósito de especímenes: CNHE 11674.

Comentarios taxonómicos

Las AdvL₃ de Gnathostoma sp. Ison similares a las de G. turgidum (Mosqueda-Cabrera et al., 2009, 2023) en la cantidad de ganchos en las hileras del bulbo cefálico y en la ubicación de las papilas cervicales y el poro excretor. Sin embargo, difieren en el tamaño del cuerpo (tabla 1), en la forma de los ganchos de las hileras del bulbo cefálico, ligeramente redondos en los ejemplares que ahora describimos (fig. 7a) vs. rectangulares (fig. 1, Mosqueda-Cabrera et al., 2009, 2023; en ambos fig. 1) en G. turgidum; asimismo, las dimensiones de los ganchos de la segunda y tercera hileras son iguales y mayores que los de la primera y cuarta hileras en el material de la laguna Las Garzas mientras que en G. turgidum los ganchos de la cuarta hilera son menores con respecto a los de las 3 hileras precedentes.

Las AdvL₃ de Gnathostoma sp. I se diferencian de las de G. lamothei (Gaspar- Navarro et al., 2013) y G. binucleatum (Almeyda-Artigas, 1991) en el tamaño del cuerpo, ya que son más pequeñas; además, presentan menor cantidad promedio de ganchos en las hileras del bulbo cefálico. Finalmente, la posición del poro excretor se traslapa con la de Gnathostoma sp. II, y es diferente de G. lamothei y G. binucleatum (tabla 1).

Los fragmentos de gen amplificados, ITS-2 (496 pb) y COI (694 pb), se encuentran disponibles en el GenBank con número de acceso PQ149241 y PQ141296, respectivamente.

Los primeros reportes del material que describimos es este trabajo fueron realizados por Almeyda-Artigas et al. (2000a, b). Las secuencias del ITS2 del DNA ribosomal de las AdvL₃ obtenidas experimentalmente de D. marsupialis por estos autores fueron nombradas como Gnathostoma sp. III, y de acuerdo con ellos, no fueron distintas a las de G. turgidum, no obstante las diferencias morfológicas observadas ahora.

Figura 6. Gnathostoma sp. I. a) Larva de tercer estadio avanzado; b) ganchos en las hileras del bulbo cefálico; c) extremo anterior del cuerpo. Escala de las barras, a = 200 μm, b y c = 10 μm.

Gnathostoma sp. I (fig. 8)

Larva de tercer estadio temprano. Descripción basada en la observación de 3 larvas envueltas en un quiste. Longitud del cuerpo 494.90-676.20 (563.06 ± 99.36; 3) y 225.40-380.18 (314.56 ± 80.03; 3) del esófago. El esófago cubre el 45.54-73.81% (56.45 ± 15.20; 3) de la longitud del cuerpo. Cuatro sacos cervicales 223.21 (n = 1) ocupan 43.33% del largo del esófago. Bulbo cefálico con 4 hileras transversales de ganchos 29-33 (30.67 ± 2.08; 3), 29-36 (32.33 ± 3.51; 3), 31-38 (35.00 ± 3.61; 3) y 37-45 (39.67 ± 4.62; 3), respectivamente.

Resumen taxonómico

Localidad: laguna Las Garzas, San Francisco Ixhuatán, Oaxaca.

Hospedero: Dormitator latifrons (Eleotridae).

Sitio de infección: musculatura.

Parámetros de infección: prevalencia 2.8%, IP 3.0, AP 0.08.

Depósito de especímenes: CNHE 11826.

Comentarios taxonómicos

Las larvas presentan características propias de tercer estadio temprano (EaL₃) por el tamaño del cuerpo (563) y la forma de los ganchos (fig. 8b). Esta fase se desarrolla en copépodos y no existe registro de EaL₃ enquistadas en la musculatura de los segundos hospederos intermediarios como las que aquí se documentan. No pudo ser recuperado material molecular de las larvas.

Figura 8. Larva de tercer estadio temprano de Gnathostoma sp. I enquistada en la musculatura de Dormitator latifrons. a) Bulbo cefálico; b) ganchos en las hileras del bulbo cefálico. Escala de la barra = 10 μm.

Gnathostoma lamothei Bertoni-Ruiz, García-Prieto, Osorio-Sarabia y León-Règagnon (2005) (fig. 9)

Larva de tercer estadio avanzado. La siguiente descripción está basada en la observación de 3 larvas. El cuerpo mide 1,879.44-1,928.64 (1,906.36 ± 24.9) de largo y 245.28-260.76 (253.71 ± 7.8) de ancho. Cuatro sacos cervicales se proyectan desde el inicio del cuerpo y cubren 53.83-66.37% (59.32 ± 6.4), con respecto a la longitud del esófago. Cuerpo cubierto totalmente de diminutas espinas dispuestas en hileras transversales de 209-240 (220 ± 17.3). Bulbo cefálico 73.58-85.85 (81.48 ± 6.9) por 176.6-205 (192.61 ± 14.5) de largo y ancho, respectivamente. Bulbo cefálico con espinas dispuestas en 4 hileras transversales, 41-45 (42.33 ± 2.3), 43-45 (44.33 ± 1.2), 46-49 (47.33 ± 1.5) y 49-51 (50.00 ± 1.0), de la primera a la cuarta, respectivamente. Poro excretor ventral entre las hileras 20-23 (21.33 ± 1.5) del cuerpo. Dos papilas cervicales laterales, la izquierda entre las hileras 9-15 (11.33 ± 3.2) y la derecha 10-14 (11.33 ± 2.3; 3) del cuerpo. El esófago ocupa 38.48-42.51% (40.47 ± 2.0) del cuerpo, en promedio abarca 57% del ancho a la altura de la intersección esófago-intestino. Dos papilas caudales laterales, la izquierda a 130-144 (137.33 ± 7.0) hileras del cuerpo y la derecha a 123-149 (136.67 ± 13.1). Primordio genital no observado. Ano subterminal, a 122.64 (n = 1) del extremo posterior.

Figura 9. Larva de tercer estadio avanzado de Gnathostoma lamothei. a) Vista lateral del bulbo cefálico; b) vista lateral del poro excretor; c) vista frontal de la papila cervical; d) intersección esófago-intestino; e) cola. Escala de la barra = 25 μm.

Resumen taxonómico

Localidad: río Ostuta.

Hospedero: excreta de Lontra longicaudis (Mustelidae).

Prevalencia: (1/20) 5.0%.

Otros hospederos: musculatura de Rhamdia guatemalensis (Heptapteridae).

Depósito de especímenes: CNHE 11160.

Comentarios taxonómicos

La AdvL₃ de G. lamothei ha sido descrita únicamente con datos morfométricos a partir de hospederos infectados experimentalmente (Gaspar-Navarro et al., 2013). Ha sido identificada en 2 registros, a partir de datos sobre la cantidad de ganchos de una larva obtenida de Monopterus albus de agua abiertas de Florida en EUA (Cole et al., 2014), y el otro con datos moleculares de AdvL₃ obtenidas de Gobiomorus dormitor en Tabasco, México (Hernández-Gómez et al., 2010). La presente investigación describe por primera vez a la AdvL₃ de G. lamothei a partir de hospederos infectados de manera natural, descripción que coincide con la obtenida de infecciones experimentales (Gaspar-Navarro et al., 2013), pero difiere con la larva de Florida (Cole et al., 2014) por el menor número de ganchos, los datos taxonómicos de esta larva son más parecidos a G. turgidum, pero identificada con marcadores moleculares como G. lamothei (tabla 1). En la presente investigación, no pudo ser recuperado material molecular de las larvas por el método de fijación de las excretas de nutria usando formaldehído.

Gnathostoma sp. II (fig. 10).

Larva de tercer estadio avanzado. Descripción basada en la observación de 3 larvas. Larvas con fluido blastocelómico rojo. Cuerpo 1,918.80-1989.5 × 137.36- 144.72 de largo y ancho, respectivamente. Presenta 197-210 hileras transversales de espinas en todo el cuerpo. Bulbo cefálico 61.32-80.94 (67.86 ± 11.3) × 117.73-116.79 (134.90 ± 27.6), de largo por ancho, respectivamente. Cuatro hileras transversales de ganchos en el bulbo cefálico 33-37 (34.33 ± 2.3; 3), 35-43 (38.00 ± 4.4), 38-43 (40.67 ± 2.5), 41-47 (44.67 ± 3.2), de la primera a la cuarta, respectivamente. Poro excretor ventral entre las 17-25 (20.33 ± 4.2) hileras del cuerpo. Dos papilas cervicales laterales en la porción anterior del cuerpo, la derecha 10-15 (12.00 ± 2.6) y la izquierda 10-16 (12 ± 3.5), en las hileras de espinas del cuerpo. El esófago abarca 34.9-38.5% del total del cuerpo y cubre 58.9-59.3% del ancho del cuerpo, en la intersección esófago-intestino. Presenta 4 sacos cervicales, en promedio cubren 42.1-44.7% del largo del esófago. Primordio genital en 68.0-82.3% del cuerpo. Papilas caudales no observadas. Ano subterminal, a 29.43-34.34 del extremo posterior.

Resumen taxonómico

Localidad: río Ostuta, San Francisco Ixhuatán, Oaxaca.

Hospedero: Synbranchus marmoratus (Symbranchidae)

Otros hospederos: Rhamdia guatemalensis (Heptapteridae).

Sitio de infección: musculatura.

Depósito de especímenes: CNHE 11708, 11709.

Comentarios taxonómicos

Las AdvL₃ de Gnathostoma sp. II son diferentes a las de Gnathostoma sp. I descritas en este trabajo, G. turgidum (Mosqueda-Cabrera et al., 2009, 2023), G. lamothei (Gaspar-Navarro et al., 2013)y G. binucleatum (Almeyda-Artigas, 1991) en la forma y tamaño de los ganchos de las 4 hileras del bulbo cefálico; además, presentan ganchos más grandes, tanto en la base como en la hoja; son diferentes a Gnathostoma sp. I y G. turgidum por la mayor cantidad de ganchos en las hileras del bulbo cefálico y por el mayor tamaño del cuerpo (tabla 1); en este aspecto, son más pequeñas que G. binucleatum y G. lamothei, y diferentes a estas especies en la ubicación del poro excretor (tabla 1).

Los fragmentos de gen amplificado ITS-2 (367 pb), se encuentra disponible en el GenBank con número de acceso OR428675 (ITS-2) y pertenece al cuerpo (sin el bulbo cefálico) de una de las AdvL₃ (Gnathostoma sp. II) del presente estudio. El análisis molecular y filogenético sugiere que Gnathostoma sp. II representa una especie distinta; no obstante, el escaso material disponible imposibilita erigirla como especie nueva.

Discusión

Los ciclos de vida complejos como el que presentan las especies del género Gnathostoma determinan que su estudio sea fragmentado y no se tenga claridad en muchos aspectos. Esto es importante ya que la gnathostomiasis humana es una enfermedad parasitaria con alta incidencia principalmente en Asia y en Latinoamérica. En México, se ha demostrado que la especie causante de esta parasitosis humana es G. binucleatum (Almeyda-Artigas 1991; Álvarez-Guerrero y Alba-Hurtado, 2007); sin embargo, es necesario conocer con mayor detalle los ciclos de vida de las otras 2 especies distribuidas en el país debido a su potencial zoonótico. Mosqueda-Cabrera et al. (2009) descartaron el papel de los peces como hospederos intermediarios de G. turgidum debido a la falta de hallazgos naturales; sin embargo, recientemente, la AdvL₃ de esta especie fue aislada de la musculatura de peces (Gobiomorus dormitor) (Mosqueda-Cabrera et al., 2023), confirmando su potencial zoonótico. Los autores resaltaron la posibilidad de que en los estudios sobre gnatostomosis en peces, las larvas de G. turgidum pasaran desapercibidas por su tamaño y método de búsqueda (observación a contraluz de la musculatura entre 2 vidrios). Este también sería el caso de las larvas Gnathostoma sp. I, colectadas en el río Ostuta, ya que son aún más pequeñas, y solo fueron encontradas usando un microscopio estereoscópico; en consecuencia, es importante valorar la participación de éstas en la gnatostomiasis humana.

Figura 10. Larva de tercer estadio avanzado de Gnathostoma sp. II. a) Vista frontal del bulbo cefálico; b) poro excretor; c) papila cervical; d) intersección esófago-intestino; e) cola. Escala de la barra = 20 μm. 

Entre las especies de Gnathostoma, existe un complejo infectando diferentes especies de hospederos muy emparentados filogenéticamente. Se conoce que el mapache (Procyon lotor) es parasitado por 2 especies de Gnathostoma con alta especificidad hospedatoria: G. lamothei asociado a Procyon lotor hernandezii Wagler, 1831 y G. procyonis a la subespecie Procyon lotor lotor Linnaeus, 1758. Las formas adultas de ambas especies son muy parecidas entre sí, con diferencias únicamente en el tamaño de las espículas, el número de puntas de las escamas transversales del cuerpo en la región posterior al bulbo cefálico y a la altura de la papila, así como en la ausencia de escamas en la mitad posterior de cuerpo (presencia de “bosses”) en G. lamothei (Bertoni-Ruiz et al., 2005). Por otra parte, el género Didelphis Linnaeus, 1758 comprende 6 especies de marsupiales que se distribuyen en toda América (Gardner, 1973); entre éstas, D. marsupialis Linnaeus, 1758 es una especie con distribución neotropical y D. virginiana Kerr, 1792 con una neártica (Dias y Perini, 2018); ambas están presentes en México y se han reportado como hospederos definitivos de G. turgidum (Pérez-Álvarez et al., 2008). La evidencia de un complejo de diferentes especies de Gnathostoma en las 2 subespecies de prociónidos sugeriría la hipótesis de que cada especie de marsupial pueda actuar como hospedero definitivo de especies diferentes de Gnathostoma; D. virginiana para G. turgidum y D. marsupialis para el material descrito en este trabajo, ya que ambas especies son muy parecidas entre sí, y sus hospederos están estrechamente relacionados filogenéticamente. No obstante, como se señaló anteriormente, consideramos que es necesario obtener más información de campo y laboratorio para apuntalar esta hipótesis.

Agradecimientos

Agradecemos profundamente la colaboración de Luis García Prieto (CNHE) durante la colecta de peces y helmintos, además, por el préstamo de especímenes y literatura. A Edgar Uriel Montes de Oca y Andrés Velázquez Brito de la CNHE por su valiosa ayuda con los datos moleculares. A Gerardo Torres Carrera por la lectura crítica del manuscrito. Agradecemos el servicio del Laboratorio de Biología Molecular del Instituto de Biología de la UNAM, como parte del Laboratorio Nacional de Biodiversidad, y en particular el apoyo técnico de A. Jiménez-Marín, N. López y L. Márquez.

Referencias

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Almeyda-Artigas, R. J., Bargues, M. D. y Mas-Coma, S. (2000a). ITS-2 rDNA sequencing of Gnathostoma species (Nematoda) and elucidation of the species causing human gnathostomi-asis in the Americas. Journal of Parasitology, 86, 537–544. https://doi.org/10.1645/0022-3395(2000)086[0537:irsogs]2.0.co;2 

Almeyda-Artigas, R. J., Bargues, M. D. y Mas-Coma, S. (2000b). rDNA of Gnathostoma species (Nematoda): ITS-2 microsatellites and 5.8 S gene secondary structure. Research and Reviews in Parasitology, 60, 51–56.

Almeyda-Artigas, R. J., Mosqueda-Cabrera, M. Á. y Sánchez-Núñez, E. (2010). Precocity of Gnathostoma turgidum in naturally infected four-eyed opossum Philander opossum pallidus from Temascal, Oaxaca, Mexico. Parasitology Research, 106, 439–443. https://doi.org/10.1007/s00436-009-1682-8 

Álvarez-Guerrero, C. y Alba-Hurtado, F. (2007). Estuarine fish and turtles as intermediate and paratenic hosts of Gnathostoma binucleatum in Nayarit, Mexico. Parasitology Research, 102, 117–122. https://doi.org/10.1007/s00436-007-0738-x 

Bertoni-Ruiz, F., García-Prieto, L., Osorio-Sarabia, D. y León-Règagnon, V. (2005). A new species of Gnathostoma (Nematoda: Gnathostomatidae), parasite of Procyon lotor hernandezii from Mexico. Journal of Parasitology, 91, 1143–1149. https://doi.org/10.1645/ge-516r.1 

Bush, A. O., Lafferty, K. D., Lotz, J. M. y Shostak, A. W. (1997). Parasitology meets ecology on its own terms: Margolis et al. revisited. The Journal of Parasitology, 83, 575–583.

Cole, R. A., Choudhury, A., Nico, L. G. y Griffin, K. M. (2014). Gnathostoma spinigerum in live Asian swamp eels (Monopterus spp.) from food markets and wild populations, United States. Emerging Infectious Diseases, 20, 634–642. https://doi.org/10.3201/eid2004.131566 

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