Three new species of the spider genus Naphrys Edwards (Araneae, Salticidae) under morphology and molecular data with notes in the distribution of Naphrys acerba (Peckham & Peckham) from Mexico

Taxon sampling

The molecular analyses were carried out with a total of 110 specimens, including one undescribed species of Naphrys and three new Naphrys species described herein. Because this study it is not a phylogenetic analysis, we use only one outgroup taxon to root the trees, Corticattus latus Zhang & Maddison, 2012 which represents the genus most closely related to Naphrys according with Zhang & Maddison (2015) (Table 1).

DNA extraction, amplification, and sequencing

The DNA was isolated separately from all eight legs of 13 individual specimens, using proteinase K/phenol/chloroform following the protocol by Hillis et al. (1996). Briefly, all eight legs of a single spider were incubated at 60 °C for 24 h with a digestion buffer containing 400 µL saline solution, 45 µL of 1% SDS solution, and 5 uL of proteinase K. After digestion, 200 µL of Phenol and 200 µL of isoamyl chloroform was added and shaken vigorously. Afterwards, samples were centrifuged at 12,000 rpm for 10 min. Once finished, 400 µL of upper aqueous phase was recovered, repeating the phenol/chloroform washes once more. Once the phenol/chloroform washes were done, 200 µL of phenol was added to the mixture, shaken gently, and then centrifuged immediately at 12,000 rpm for 10 min. 300 µL of upper aqueous phase was recovered and 750 µL of cold (−20 °C) absolute ethanol was added. The mixture was then shaken gently and incubated for 12 h at −20 °C. Once incubated, it was centrifuged at 13,000 rpm for 20 min, and the ethanol was decanted by inversion, avoiding losing the bottom pellet. 600 µL of cold 70% ethanol (−20 °C) was then added and centrifuged at 13,000 rpm for 20 min, with ethanol decanting by inversion while avoiding losing the bottom pellet. Finally, drying in a vacuum centrifuge was performed at 60 °C for 10 min. Once the vial is dry, DNA is suspended in 50 µL of distilled water and stored at −20 °C. After DNA extraction, the mitochondrial gene Cytochrome Oxidase subunit 1 (COI), proposed by Folmer et al. (1994), was amplified (LCO1498 and HCO2198). Amplifications were carried out in a GeneAmp PCR System 2700 thermal cycler, in a total volume of 25.9 µL: 1.66 µL Buffer (5X), 1.5 µL MgCl2 (50 mM), 1.25 µL LCOI1498 (10 µM), 1.25 µL HCOI2198 (10 µM), 0.23 µL Taq (5U/µL), 0.875 µL dNTP’s (10 mM), 1 µL BSA (1.25 mg/µL), 16.135 µL H2O, 2 µL DNA. The PCR was set up as follows: an initial step for 1 min 30 s at 95 °C; 35 amplification cycles of 30 s at 94 °C (denaturation), 30 s at 50 °C (annealing), 45 s at 72 °C (elongation), and final elongation of 10 min at 72 °C. PCR products were checked via gel electrophoresis to analyze length and purity on 1% agarose gels with a molecular marker of 1,000 bp.

DNA extractions were carried out at the Laboratorio de Biología Acuática “J. Javier Alvarado Díaz”, while PCR amplifications were carried out at the Centro Multidiciplinario de Estudios en Biotecnología (CMEB), both at the Universidad Michoacana de San Nicolás de Hidalgo (UMSNH) in Morelia, Michoacán, Mexico. Purification and sanger sequencing in both directions were carried out in Psomagen, Maryland, United States.

Sequence editing and alignment

The sequences were visualized in Geneious Prime v.2023.2.1 (Geneious Prime, 2023) and then manually edited using the BioEdit v. 7.7.1 program (Hall, 1999). After saving in FASTA format (.fas), the sequences were aligned using MAFFT v. 7 (Katoh & Toh, 2008) with default parameters on the MAFFT online server (https://mafft.cbrc.jp/alignment/server/).

Molecular analysis and species delimitation

Four different molecular delimitation methods were employed using the corrected p-distances neighbor-joining (NJ) as initial criteria: (1) ASAP (Assemble Species by Automatic Partitioning) (Puillandre, Brouillet & Achaz, 2021), (2) GMYC (General Mixed Yule Coalescent) (Pons et al., 2006), (3) bPTP (Bayesian Poisson Tree Process) (Zhang et al., 2013), and (4) mPTP (multi-rate Poisson tree processes) (Kapli et al., 2017).

p-distances neighbor-joining (NJ) criteria

MEGA v.10.0.5 (Kumar et al., 2018) was used to construct the genetic distance tree, using the following parameters: number of replicates = 1,000, bootstrap support values = 1,000 (significant values ≥ 50%), substitution type = nucleotide, model = p-distance, substitutions to include = d: transitions + transversions, rates among sites = gamma distributed with invariant sites (G+I), missing data treatment = pairwise deletion.

Assemble species by automatic partitioning (ASAP)

This method is an ascending hierarchical clustering algorithm that analyzes single-locus DNA barcode datasets. It iteratively merges sequences with the highest pairwise similarity into progressively larger clusters. Additionally, ASAP retains information on all potential clustering steps, resulting in a comprehensive series of partitions representing putative species groupings within the data. Subsequently, ASAP calculates a probability score for each partition based on the within-group sequence similarity compared to between-group similarity. Finally, the method identifies the partitions with the highest probability scores as the most likely species-level groupings and constructs a species partition tree reflecting the hierarchical relationships among these putative species (Puillandre, Brouillet & Achaz, 2021). ASAP analyses were run on the online platform (https://bioinfo.mnhn.fr/abi/public/asap/) using Kimura (K80) distance matrices and configured under following parameters: substitution model = p-distances, probability = 0.01, best scores = 10, fixed seed value = −1.

General mixed yule coalescent (GMYC)

The GMYC method (Fujisawa & Barraclough, 2013) is a statistical framework employed for species delimitation using single-locus DNA barcode data. This approach utilizes single time thresholds to define species boundaries within a Maximum Likelihood context, relying on ultrametric trees as input (Ortiz & Francke, 2016; Nolasco & Valdez-Mondragón, 2022). Ultrametric trees were generated in this study through phylogenetic analyses performed in BEAUti and BEAST v.2.7.6 software (Bouckaert et al., 2019). A Yule Process tree prior was implemented during the analysis to account for lineage diversification patterns. Furthermore, an optimized relaxed molecular clock model was applied, incorporating the estimated evolutionary model for the COI gene (GTR + I + G). To ensure robustness of the phylogenetic inference, five independent BEAST analyses were executed, each running for 80 million iterations. Convergence of these analyses was subsequently evaluated using Tracer v1.6 (Rambaut & Drummond, 2003–2013), with a minimum threshold of 200 for the Effective Sample Sizes (ESS). Following this, Tree Annotator 2.6.0 (part of the BEAST package) was employed to generate maximum likelihood trees representing the most likely evolutionary histories. The first 25% of each independent run was discarded as burn-in to account for potential initial biases in the MCMC chains. Finally, the GMYC method was implemented through the online platform (https://species.h-its.org/gmyc/) (Fujisawa & Barraclough, 2013).

Bayesian Poisson tree processes (bPTP)

bTPT operates within a Bayesian framework, accounting for uncertainties in both the phylogenetic tree’s branch lengths and potential species assignments. This method assumes a Poisson process for speciation events along the tree branches and incorporates branch lengths reflecting sequence divergences. Considering this information and its inherent uncertainties, bPTP estimates posterior probabilities for various candidate species partitions within the data, which represent the likelihood of each partition accurately reflecting true species boundaries (Zhang et al., 2013). In this work, Bayesian and maximum likelihood variants were carried out on the online platform (https://species.h-its.org/ptp/), using following options: rooted tree, MCMC = 1000000, thinning = 100, burn-in = 0.1, seed = 123. The resulting trees were edited in FigTree 1.4.4 (Rambaut, 2018). Congruence integration criteria were employed to delimit different species. This approach compares evidence across multiple methods, resulting in more robust species delimitations and better supported species hypotheses (e.g., DeSalle, Egan & Siddall, 2005; Pons et al., 2006; Navarro-Rodríguez & Valdez-Mondragón, 2020; Valdez-Mondragón, 2020; Nolasco & Valdez-Mondragón, 2022).

Multi-rate Poisson tree processes

Multi-rate poisson tree processes (mPTP) uses a non-homogeneous Poisson process model. This approach allows for the estimation of distinct rate multipliers for individual branches within the phylogenetic tree, recognizing potential heterogeneity in evolutionary rates across lineages. ML tree estimation was used to identify branch-specific rate multipliers, and Markov chain Monte Carlo (MCMC) simulations were employed to integrate over the uncertainty associated with these estimates (Kapli et al., 2017). By identifying statistically significant shifts in diversification rates along the tree generated from our ML analysis, mPTP pinpoints potential species boundaries, specifically taking into account lineages that have undergone evolution at disparate paces. This analysis was carried out on the online platform (http://mptp.h-its.org/).

Zoobank

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN). Hence, the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information viewed through any standard web browser by appending the LSIDs to the prefix http://zoobank.org/. The LSIDs for this publication are: urn:lsid:zoobank.org:act:6CFF43A9-8C98-4027-A1DA-2838FE4D79F8; urn:lsid:zoobank.org:act:D67CCC72-E17D-450C-9193-231120527FDE; and urn:lsid:zoobank.org:act:3129A3DE-57E8-46CC-8036-86DC467EB056. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE, and CLOCKSS.

Molecular analysis of genetic distances

The corrected p-distances under NJ of COI recovered six putative species (Fig. 1). Genetic distance analyses recovered groups corresponding to one putative new species (with bootstrap support value below 50%), the two previously described species N. pulex and N. xerophila (with high bootstrap support value, 89%), and three new species described herein (with high bootstrap support value, 98%). Bootstrap support values for all species were high (>89%) (Fig. 1). The average genetic p-interspecific distances of Naphrys species was 14% (min: 11%, max: 18.1%) (Table 2). Average interspecific p-distance between previously known species (N. pulex and N. xerophila) was 11.8%. Between new species (N. echeri sp. nov., N. tecoxquin sp. nov., and N. tuuca sp. nov.) and previously known species, higher interspecific average p-distances were observed, between 12.9% and 14%. With average values above 15.1%, Naphrys sp. had the highest average interspecific p-distance. For most species, intraspecific distances were below 1.61%, except for Naphrys sp. that showed a higher value (Table 3).

Molecular methods for species delimitation

The ASAP delimitation analysis recovered all six species (N. echeri sp. nov., N. tecoxquin sp. nov., N. tuuca sp. nov., Naphrys sp., Naphrys pulex, and Naphrys xerophila) with high (>93%) bootstrap support value (Fig. 2) from the NJ tree. GMYC and mPTP methods recovered the three new species described herein and one putative new species, while N. pulex was not recovered as one species (Fig. 2). The most incongruent result was observed in bPTP, which delimited 42 and 50 putative species under ML and IB variants, respectively. Only N. tecoxquin sp. nov. and N. xerophila were recovered by the ML variant of bPTP.

Only N. xerophila was recovered under all methods and supported by a high bootstrap value (93%). Naphrys pulex shows the most incongruent results in all species delimitation methods, recovering 10 species in mPTP, 16 in GMYC, and 42 and 50 species in the ML and BI variants of bPTP method, respectively (Fig. 2). Nevertheless, N. pulex presents low intraspecific genetic distance (<2%) and high bootstrap support value (100%) (Table 3; Fig. 2).

Taxonomy

Emended diagnosis. After Richman (1981) and Edwards (2003). Naphrys species are characterized by their small size (2–6.1 mm) and dull, cryptic coloration (black and brown) (Figs. 3C–3D). Chelicera with one bicuspid promarginal tooth. Carapace high. First tibia has no more than two pairs of ventral macrosetae and leg III longer than leg IV (Tibia+Patella III >Tibia+Patella IV). Male palpal bulb is usually large with a proximal TL. Simple finger-like RTA and RSDL present. Also, palpal tibia with ventral apophysis (VTA). Embolar disk (ED) has a ventral conical projection. Embolus (E) possesses a J-shaped configuration, featuring a prolateral curvature in its distal part and an emerging projection in its proximal part (Figs. 4C–4H). Epigynum has a typical window structure with a median septum (Figs. 5C–5F). Copulatory openings (CO) are positioned along posterior, median (Figs. 5C–5F), or anteromedian edges of atria, with atrial rims intersecting them posteriorly. Rims fail to completely encircle the atria. Spermathecas (S) are nearly spherical, more or less contiguous medially, and half or more the diameter of the atria. They are positioned about halfway to entirely within the posterior part of atria as seen in ventral view (Figs. 5C–5F).

Current composition. Naphrys is composed of seven species: Naphrys acerba (Peckham & Peckham, 1909); Naphrys bufoides (Chamberlin & Ivie, 1944); Naphrys echeri sp. nov.; Naphrys pulex (Hentz, 1846); Naphrys tecoxquin sp. nov.; Naphrys tuuca sp. nov.; Naphrys xerophila (Richman, 1981).

Distribution. Canada, Mexico, and the United States.

Remarks. We emend the generic diagnosis based on copulatory organs of male and females.

Key to Naphrys species

Holotype: Holotype not assigned by author. Syntypes: several males and one female from Travis County, Austin, Texas, USA, and one male from Georgia, USA. NOT EXAMINED. Naphrys acerba Edwards, 2003 p. 69, figs. 5–8 (Transferred from Habrocestum)

Other material examined. MEXICO: Nuevo León: six females (CAFBUM88003, CAFBUM88004, CABUM84234, CAFBUM84242, CAFBUM84256, CAFBUM84257), along path to cable car, Cerro de la Silla, Guadalupe municipality (lat. 25.655501, long. -100.254415, 587 m), oak forest, ground hand collecting, J. Maldonado Carrizales, F. Morales Martínez, E. G. Fuentes Ortiz cols., 21/X/2023. Tamaulipas: three males (CAFBUM88005) and three immatures (CAFBUM880040), Mr. Sabino’s ranch, highway Ciudad Victoria-Tula km 28 (lat. 23.606512, long. 99.229572, 1,473 m), oak forest, ground hand collecting, J. Maldonado Carrizales, F. Morales Martínez, E. G. Fuentes Ortiz cols., 20/X/2023.

Emended diagnosis. After Peckham & Peckham (1909) and Richman (1981). Naphrys acerba resembles N. bufoides and N. xerophila by possessing white, round spots on dorsal abdomen (Figs. 3C, 4A, 5A). However, it differs from N. xerophila by lacking a medial longitudinal white stripe covering anterior portion. Additionally, N. acerba can be distinguished from N. bufoides by its thicker embolus, which is shorter than ED (Figs. 4C, 4E, 4F, 4H). In females, CO of N. acerba are located centrally within the epigynum, touching anterior edge of S (Fig. 5C, 5E). This contrasts with N. bufoides, where CO do not reach anterior edge of S, and N. tuuca, where CO are positioned in middle basal part of epigynum.

Distribution. UNITED STATES: Texas; MEXICO: Coahuila, Nuevo León, Tamaulipas [Richman, 1981; WSC, 2024], Jalisco, Michoacán and Nayarit [present data].

Natural history. According to Richman (1981), this species appears to be associated with oak and juniper woodlands. Specimens used in this study were collected from upper leaf litter layer of oak forests (Quercus sp.) at 1,473 m in Tamaulipas, Mexico, within known range of the species. This also included disturbed areas into Monterrey City (Figs. 3A–3D).

Type material: Male holotype, MEXICO: Michoacán, Cerro El Gigante, Jesús del Monte, Morelia (lat. 19.636605, long. -101.146877, 2,192 m), oak forest (Quercus sp.), ground hand collecting, J. Maldonado Carrizales, F. Morales Martínez, R. Cortés Santillán cols., 31/III/2023. (CARCIB-AR-047). Paratypes: one female (CARCIB-Ar-008), one male (CARCIB-Ar-0327) and one female (CARCIB-Ar-0328) with same collection data as for holotype. Jalisco: one male, one immature (CAFBUM84264) Piedras Bolas, Ahualulco de Mercado (lat. 20.653021, long. -104.057697, 1,907 m), oak forest (Quercus sp.), ground hand collecting, J. Maldonado Carrizales, G. L. López Solís, S. Montañez Hernández, N. Ruíz Hernández cols., 8/IV/2022. One female (CAFBUM88012) UMA Potrero de Mulas, San Sebastián del Oeste municipality (lat. 20.749852, long. -104.976763, 797 m) cloud forest, ground hand collecting, J. Maldonado Carrizales, E. G. Fuentes Ortiz cols., 13/XII/2022.

Other material examined. MEXICO: Jalisco: one female (CNAN-Ar011468) and one male (CNAN-Ar011467), beginning of the path to Cerro La Bufa, San Sebastián del Oeste municipality (lat. 20.758, long. -104.8438, 1,460 m), young pine forest, D. Guerrero, G. Contreras, C. Hutton, G.B. Edwards cols., 14/VI/2018. Three males, three immatures (CNAN-Ar011464), and one female (CNAN-Ar011462), Piedras Bolas, Ahualulco de Mercado municipality (lat. 20.64945, long. -104.05592, 1,863 m), oak forest (Quercus sp.), D. Guerrero, G. Contreras, C. Hutton and G.B. Edwards cols., 17/VI/2018.

Etymology. The species name “echeri” (/et eɾi/ native pronunciation) is a noun in apposition that means “land or soil” in the P’urépecha language, referring to the microhabitat where it inhabits. The P’urépecha state, which peaked in the 14th and 15th centuries before Spanish arrival, is known today as Michoacán, and represents the type locality of this species.

Diagnosis. Naphrys echeri sp. nov. resembles N. tuuca sp. nov. by males having an extended medial white longitudinal line on dorsal part of opisthosoma, which extends across the entire opisthosoma (Fig. 7A). However, N. echeri sp. nov. differs in possessing an ED that bears a well-developed triangular process (PED) next to embolus, clearly visible in retrolateral view (Figs. 7D, 7G). Naphrys echeri sp. nov. has a thick and straight E shorter than ED (Figs. 7C, 7E, 7F, 7h), whereas in N. pulex this is thick but curved, and in N. tuuca sp. nov. the E is thin and folds at midpoint forming a gentle curve, ultimately larger than ED. Naphrys echeri sp. nov. differs from N. tecoxquin sp. nov. and N. tuuca sp. nov. in morphology of its embolus apex, with N. echeri sp. nov. possessing a fine projection that abruptly narrows to a spine-like structure and is oriented towards the interior of the palp. Females of N. echeri sp. nov. share with N. tecoxquin sp. nov. the placement of CO on external lateral side of S, but differ in shape; in N. echeri sp. nov., S are circular (Figs. 8C–8F), while in N. tecoxquin sp. nov. they are pyriform.

Description. Male holotype (CARCIB-AR-047). Total length: 2.60. Prosoma 1.57 long and 1.22 wide. Darkish brown, with white setae forming a V-shaped mark, extending outwards from sides of PLE towards pedicel in cephalic region (Fig. 7A). Lower border covered with white seta forming a band. Ocular quadrangle (OQ) 0.30 long. Anterior eyes row (AER) 1.46 times wider than PER, AER 1.10 wide, PER 0.75 wide. Sternum reddish brown, 0.65 long, 0.50 wide. Labium reddish brown, as long as wide, 0.30 long, 0.30 wide. Endite 0.42 long, 0.17 wide, reddish brown, whitish anteriorly and square shaped (Fig. 7B). Opisthosoma 1.03 long and 0.95 wide; exhibiting a longitudinal band with white setae in dorsal view, covering more than half its width (Fig. 7A). Palp covered by white setae in dorsal view; in ventral view possesses a straight, short, and wide E that covers up to half distal part of cymbium (Figs. 7C, 7F, 9A, 10A). Ventral view of E with scales (Figs. 10A, 10C). A PED is present, easily seen in retrolateral view, triangular with fine projection on tip that abruptly narrows forming two spine-like structure (Figs. 7D, 7G, 9B, 10A–10C). Embolus apex and SP are oriented towards interior of palp (Figs. 9A, 10A–10B). Embolus apex presents one fine projection that abruptly narrows to a spine-like structure, while SP presents a multi-convex edge forming smooth ridges (Fig. 10D). Embolar disk (ED) completely rough and folded in anterior portion (Figs. 9A, 10A). Tegulum (T) yellow with darkish marks and wide RSDL occupying more than half of it, easily seen in retrolateral view (Figs. 7D, 7G). Furthermore, RSDL is divided in two, anterior loop is extremely curved forming a backwards “C” that extends from the middle of the T to its retrolateral edge. Posterior loop is curved anteriorly and straight in its most posterior part, forming a backwards “L” that does not touch retrolateral edge (Figs. 7D, 7G). Retrolateral tibial apophysis (RTA) wide at base, becoming smaller in distal part slightly anteriorly oriented (Figs. 7D, 7G, 9B, 9D). Ventral tibial apophysis (VTA) rounded with a large pit at the tip. It has faint lines running across its surface (Figs. 9A, 9C). Reddish brown legs with black bands. Legs I–II are pale with dark intersegmental markings, except for the joint between the metatarsus and tarsus. Legs III–IV exhibit dark intersegmental markings throughout. Leg formula 3412. Leg I 2.84 (0.90, 0.45, 0.60, 0.46, 0.42), Leg II 2.72 (0.92, 0.45, 0.52, 0.45, 0.38), Leg III 3.90 (1.20, 0.55, 0.82, 0.77, 0.47), Leg IV 3.80 (1.30, 0.50, 0.72, 0.82, 0.45).

Female (CARCIB-Ar-008). Paler coloration, less pronounced than that of the male. Total length: 5.10. Prosoma 2.50 long and 1.90 wide. Darkish brown, with white and orange setae anteriorly (Fig. 8A). Lower border covered with white setae forming a band. Ocular quadrangle (OQ) 0.60 long. Anterior eyes row (AER) 1.27 times wider than PER, AER 1.40 wide, PER 1.10 wide. Sternum reddish brown with dark marks, 1.67 long, 0.87 wide. Labium black slightly longer than wide, 0.37 long, 0.32 wide. Endite 0.25 long, 0.65 wide, reddish brown, whitish anteriorly and ovoid shaped (Fig. 8B). Opisthosoma 2.60 long and 2.50 wide; covered with coppery bronze setae across surface and exhibiting mottled pattern of faint translucent markings (Fig. 8A). Epigynum slightly wider than long, 0.40 long, 0.34 wide. Copulatory openings (CO) located on external lateral sides of S. Circular S and a unique loop in CD forms a D-shape in each side of epigynum (Figs. 8C–8F). Median septum (MS) and sides have a smooth, trident-shaped with grooves or ridges on anterior part (Fig. 10E). Windows of epigynum (W) mostly smooth, but striated centrally (Fig. 10E). Reddish brown legs with black marks. Leg formula 3412. Leg I 3.72 (1.12, 0.70, 0.85, 0.65, 0.40), Leg II 3.67 (1.30, 0.62, 0.67, 0.62, 0.45), Leg III 5.52 (1.85, 0.80, 1.25, 1.00, 0.62), Leg IV 4.40 (1.57, 0.67, 1.12, 0.52, 0.50).

Distribution. MEXICO: Michoacán and Jalisco.

Natural history. The specimens were collected from leaf litter in oak forest (Quercus sp.) and cloud forest. Adults were mainly found from March to November (Figs. 6A–6C).

Type material: Male holotype, MEXICO: Jalisco, Boca de Tomatlán, Cabo Corrientes (lat. 20.511861, long. -105.318, 36 m), tropical forest, ground hand collecting, J. Maldonado Carrizales, R. Cortés Santillán, E. G. Fuentes Ortiz cols., 13/IV/2023 (CARCIB-Ar-048). Paratypes: one female (CARCIB-Ar-009), one male (CARCIB-Ar-0329) and one female (CARCIB-Ar-0330) with same collection data as holotype; two males (CAFBUM84260-CAFBUM84261) and one female (CAFBUM84238) with same data as holotype.

Other material examined. MEXICO. Jalisco: one male (CAFBUM84232) and 12 immatures (CAFBUM84221), same collection data as holotype. 1 imm (CNAN-Ar011469), same collection data as paratype. One female (CNAN-Ar011471), Las Ánimas in same municipality as holotype (lat. 20.50002, long. -105.33869, 39 m), tropical forest, ground hand collecting, G. Contreras col., 6/IX/2018.

Etymology. The species name “tecoxquin” (/tek′o kin/ native pronunciation) is a noun in apposition in reference to the original native group that inhabited an extensive region covering the entire southern coast of Nayarit and neighboring coastal of Jalisco where type locality is found.

Diagnosis. Naphrys tecoxquin sp. nov. males possess bright, coppery bronze setae in the anterior part of the Prosoma (Figs. 11E, 12A), a light opisthosoma with four black spots in dorsal view, and upwards-pointing dark brown marks in posterior third (Fig. 12A). In contrast, N. echeri sp. nov. exhibits a dark opisthosoma covered with coppery bronze setae across its surface and displays a mottled pattern of faint translucent markings (Fig. 7A). Naphrys tecoxquin sp. nov. exhibits dark brown legs I–III (Fig. 11E), contrasting with the rest of species. Naphrys tecoxquin sp. nov. is similar to N. xerophila, but differs in having a thick and straight embolus (Fig. 12C–12H), in contrast to the curved embolus observed in N. xerophila and N. pulex. Naphrys tecoxquin sp. nov. differs from N. echeri sp. nov. and N. tuuca sp. nov. in morphology of its embolus apex, which is ventrally flat and dorsally convex, oriented towards the exterior of the pedipalp. The surface of the embolus apex in N. tecoxquin sp. nov. is sinuous with small projections. Additionally, N. tecoxquin sp. nov. lacks PED next to embolus, a characteristic of N. echeri sp. nov. (Figs. 7D, 7G, 9B, 10A–10C). In females of N. tecoxquin sp. nov., CO are located on external lateral side of S (Figs. 13C–13E). Naphrys tecoxquin sp. nov. differs to N. echeri sp. nov. in S shape, which is pyriform in N. tecoxquin sp. nov. (Figs. 13C–13F), but round in N. echeri sp. nov. (Figs. 8C–8F).

Description. Male holotype (CARCIB-Ar-048). Total length: 2.90. Prosoma 1.74 long and 1.26 wide. Darkish brown, with white setae forming a U-shaped mark, extending outwards from sides of PLE towards pedicel, anterior part is covered by bronze setae (Fig. 12A). Lower border covered with white setae forming a band. Ocular quadrangle (OQ), 0.60 long. Anterior eye row (AER) 1.31 times wider than PER, AER 1.18 wide, PER 0.90 wide. Sternum dark with faint yellow marks, 0.62 long, 0.46 wide. Labium dark, wider than long, 0.15 long, 0.22 wide. Endite 0.27 long, 0.25 wide, reddish brown, whitish anteriorly, and square shaped (Fig. 12B). Opisthosoma 1.16 long and 0.92 wide, exhibiting two straight longitudinal bands forming a “V” that cover almost half of anterior opisthosoma. In central part, there is a black mark in shape of three triangles joined at base. Additionally, a white diamond-shaped mark is present in distal part (Fig. 12A). Palp covered by white setae in dorsal view; in ventral view, a thick and straight E covers up to half of distal part of the cymbium (Figs. 12C, 12F). Embolus apex and SP are oriented towards exterior of the palp (Figs. 14A, 15A). Embolus apex is ventrally flat and dorsally convex, oriented towards the exterior of pedipalp. Surface of the embolus apex is sinuous with small projections (Figs. 15A–15B). Embolar disk (ED) possesses a slight fold anteriorly, with striations at center (Figs. 14A, 15A). Tegulum (T) dark with faint yellow and orange marks. RSDL wide and easily seen in retrolateral view (Figs. 12D, 12G). Furthermore, RSDL is divided in two, anterior loop is gently curved similar to a closed parentheses “)”, extended on retrolateral edge. Adjacent, the posterior loop shares the same shape, but does not touch retrolateral edge (Figs. 12D, 12G). Retrolateral tibial apophysis (RTA) exhibits a densely striated surface along entire length. This apophysis projects in a straight orientation, gradually attenuating distally. Notably, RTA displays a slight dorsal orientation relative to the palp (Figs. 14B, 14D). Ventral tibial apophysis (VTA) is rounded and smooth (Figs. 14A, 14C). Leg I, femur, patella, tibia and metatarsus are dark brown with faint reddish-brown marks. Legs II–III, femur, patella and tibia are dark brown with faint reddish-brown marks, metatarsus amber, and tarsus yellow. Leg IV, femur, metatarsus, and tarsus yellow, patella and tibia dark brown with faint blackish-brown marks. Leg formula 3412. Leg I 2.81 (0.82, 0.48, 0.62, 0.45, 0.42); leg II 2.86 (0.85, 0.47, 0.60, 0.52, 0.41); leg III 3.83 (1.25, 0.47, 0.77, 0.81, 0.52); leg IV 3.75 (1.27. 0.58. 0.78. 0.57. 0.52).

Female (CARCIB-Ar-009). Paler coloration, less pronounced than that of the male, particularly on the prosoma. Total length: 2.68. Prosoma 1.50 long and 1.10 wide, darkish brown, with anterior part covered with black and orange setae (Fig. 13A); lower border covered with white setae forming a band. Ocular quadrangle (OQ), 0.70 long. Anterior eyes row (AER) 1.50 times wider than PER, AER 1.08 wide, PER 0.72 wide. Sternum reddish brown with dark marks, 0.62 long, 0.46 wide. Labium black, wider than long, 0.22 long, 0.46 wide. Endite 0.28 long, 0.24 wide, reddish brown, and ovoid shaped (Fig. 13B). Opisthosoma 1.18 long and 0.92 wide; light with four black spots in dorsal view, along with dark brown upwards chevron marks in posterior last third (Fig. 13A). Epigynum longer than wide, 0.82 long, 0.46 wide. Copulatory openings (CO) are located on external lateral sides of S. Pyriform S and a unique loop in CD forms a D-shape on each side of the epigynum (Figs. 12C–13F). Median septum (MS) and sides smooth, trident-shaped, with grooves on anterior edges of W (Fig. 15C). Windows of epigynum (W) longer than wide, mostly smooth, but striated at center (Fig. 15C). Reddish brown legs with black marks. Leg formula 3412. Leg I 2.25 (0.67, 0.45, 0.47, 0.37, 0.27); leg II 2.12 (0.55, 0.40, 0.50, 0.35, 0.32); leg III 3.27 (1.05, 0.45, 0.70, 0.60, 0.47); leg IV 3.10 (1.00, 0.40, 0.67, 0.65, 0.37).

Distribution. MEXICO: Jalisco.

Natural history. The specimens were collected from leaf litter in tropical dry forests with broad-leaved trees. Adults were mainly found from April to July and from September to November (Fig. 11).

Type material: Male holotype, MEXICO: Nayarit, male from Cerro San Juan, Tepic (lat. 21.505877, long. -104.924464, 1,121 m), oak forest (Quercus sp.), ground hand collecting, J. Maldonado Carrizales, R. Cortés Santillán col., 24/V/2023 (CARCIB-Ar-049). Paratypes: one female (CARCIB-Ar-010), three males (CARCIB-Ar-0331; CAFBUM880039) and three females (CARCIB-Ar-0332; CAFBUM880021) with same collection data as holotype.

Other material examined. MEXICO. Nayarit: two males (CAFBUM880001; CAFBUM880002), one female (CAFBUM880075), same data as holotype. One male (CNAN-Ar011460), same data as holotype (CNAN-Ar011461). Three males and three females (CNAN-Ar011461), Ceboruco Volcano, Jala municipality (lat. 21.1149, long. -104.5014, 1,916 m), wet glen, D. Guerrero, G. Contreras, C. Hutton, and G.B. Edwards col., 16/V/2018.

Etymology. The species name “tuuca” (/ uuk’a/ native pronunciation) is a noun in apposition that means “spider” in the Wixárika language. Wixárika people are native to the Sierra Madre Occidental range in Nayarit state, where the type locality is found.

Diagnosis. Prosoma in dorsal view of N. tuuca sp. nov. has a unique characteristic white setae forming a Y-shaped mark, extending outwards from sides of PLE (Figs. 16C, 17A). In contrast, N. echeri sp. nov. exhibits white setae forming a V-shaped mark in this region (Fig. 7A). Naphrys tuuca sp. nov. has a dark opisthosoma covered with coppery-bronze setae across surface (Figs. 16C, 17A), similar to N. echeri sp. nov.; nevertheless, N. tuuca sp. nov. has a distinct mottled pattern of white markings and a medial longitudinal smooth white stripe that covers anterior portion of the opisthosoma (Figs. 16C, 17A). Males of N. tuuca sp. nov. possess a thin embolus (Figs. 17C–17H). Embolus is larger than ED and folds at midpoint, forming a gentle curve (Figs. 17E, 17H), in contrast to thin and straight embolus observed in N. bufoides. Similar to Naphrys tecoxquin sp. nov., embolus apex of N. tuuca sp. nov. is curved and oriented towards the exterior of palp. Surface of embolus apex in N. tuuca sp. nov. is smooth. Additionally, N. tuuca sp. nov. lacks PED, which is present in N. echeri sp. nov. Females of N. tuuca sp. nov. present CO located in middle basal part of epigynum (Fig. 18C, 18E), differing from central location of CO observed in N. acerba, N. bufoides and N. pulex.

Description. Male holotype (CARCIB-Ar-049). Total length: 2.48. Prosoma 1.42 long and 1.10 wide, dark with white setae forming a Y-shaped mark, extending outwards from sides of PLE towards pedicel (Figs. 16C, 17A). Lower border covered with white setae forming a band. Ocular quadrangle (OQ), 0.74 long. Anterior eye row (AER) 1.53 times wider than PER, AER 0.98 wide, PER 0.64 wide. Sternum dark with faint amber marks, 0.72 long, 0.50 wide. Labium dark, wider than long, 0.17 long, 0.25 wide. Endite 0.35 long, 0.27 wide, amber, and square-shaped (Fig. 17B). Opisthosoma 1.06 long and 0.88 wide, exhibiting a longitudinal band with white setae in dorsal view, covering one third of width (Figs. 16C, 17A). Palp covered by white setae in dorsal view, with a thin embolus in ventral view, larger than ED, which folds at midpoint, forming a gentle curve (Figs. 17C, 17E, 17F, 17H, 19A, 20A). Embolus apex exhibits a lateral flattening, resulting in a dorsally convex shape; oriented outwards from the main body of the palp. Embolus apex surface with smooth contours (Figs. 20A–20B). Embolar disk (ED) exhibits unfolded anterior margin, and central region displays a higher concentration of striations (Figs. 19A, 20A). Tegulum (T) dark with faint yellow and orange marks, RSDL wide, easily seen in retrolateral view (Figs. 17D, 17G). Furthermore, RSDL is divided in two, with anterior loop extremely curved, forming a backwards “C” that extends from middle of T to its retrolateral edge. Posterior loop is curved anteriorly and straight in its most posterior part, forming a hooked-shape that does not touch retrolateral edge (Figs. 17D, 17G). Retrolateral tibial apophysis (RTA) exhibits sparse striations along its entire length. This structure projects in a straight orientation, gradually attenuating distally and displaying a slight anterior orientation (Figs. 17D, 17G, 19B, 19D). Ventral tibial apophysis (VTA) presents a conical structure with a roughened surface texture and a small notch distally (Figs. 19A, 19C). Legs I–II are pale with dark intersegmental markings, except for the joint between the metatarsus and tarsus. Legs III–IV exhibit dark intersegmental markings throughout (Figs. 21D, 21H, 21L, 21P). Leg formula 3412. Leg I 2.71 (0.78, 0.47, 0.50, 0.49, 0.45); leg II 2.68 (0.96, 0.45, 0.51, 0.50, 0.24); leg III 3.91 (1.26, 0.65, 0.78, 0.74, 0.47); leg IV 3.60 (1.10, 0.45, 0.76, 0.87, 0.49).

Female (CARCIB-r-010). Paler coloration, less pronounced than that of the male. Total length: 3.64. Prosoma 1.64 long and 1.34 wide, darkish brown, anterior part covered with white and orange setae (Figs. 16D, 16E, 18A). Lower border covered with white setae forming a band. Ocular quadrangle (OQ) 0.68 long. Anterior eyes row (AER) 1.47 times wider than PER, AER 1.18 wide, PER 0.80 wide. Sternum reddish brown with dark marks, 0.67 long, 0.57 wide. Labium dark with faint amber marks, wider than long, 0.20 long, 0.27 wide. Endite 0.37 long, 0.25 wide, reddish brown, and ovoid shaped (Fig. 18B). Opisthosoma 2.00 long and 1.80 wide, dark, covered with coppery-bronze setae across surface, with a mottled pattern of white markings and a medial longitudinal smooth white stripe covering anterior portion (Fig. 18A). Epigynum slightly wider than long, 0.30 long, 0.34 wide. Copulatory openings (CO) located in middle basal part of epigynum. Circular S and a unique loop in CD form a D-shape in each side of epigynum (Figs. 18C–3F). Median septum (MS) exhibits a smooth surface texture, while anterior edges of W present grooves (Fig. 20C). Overall surface of W exhibits a slightly roughened texture. Windows of epigynum (W) as long as wide (Fig. 20C). Legs yellow with dark marks, metatarsus amber, and tarsus yellow. Legs III and IV yellow with dark bands near segment junctions. Leg formula 3412. Leg I 2.82 (0.88, 0.52, 0.56, 0.50, 0.36); leg II 2.86 (0.92, 0.44, 0.58, 0.58, 0.34); leg III 4.14 (1.34, 0.58, 0.82, 0.90, 0.50); leg IV 4.06 (1.30, 0.56, 0.80, 0.82, 0.58).

Distribution. MEXICO: Nayarit.

Natural history. The specimens were collected from leaf litter in oak forests (Quercus sp.). Adults were mainly found from May to September. This species was observed to prey on Collembola (Fig. 16D).