Research Article |
Corresponding author: Alejandro Valdez-Mondragón ( lat_mactans@yahoo.com.mx ) Academic editor: Cristina Rheims
© 2022 Samuel Nolasco, Alejandro Valdez-Mondragón.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Nolasco S, Valdez-Mondragón A (2022) To be or not to be… Integrative taxonomy and species delimitation in the daddy long-legs spiders of the genus Physocyclus (Araneae, Pholcidae) using DNA barcoding and morphology. ZooKeys 1135: 93-118. https://doi.org/10.3897/zookeys.1135.94628
|
Integrative taxonomy is crucial for discovery, recognition, and species delimitation, especially in underestimated species complex or cryptic species, by incorporating different sources of evidence to construct rigorous species hypotheses. The spider genus Physocyclus Simon, 1893 (Pholcidae, Arteminae) is composed of 37 species, mainly from North America. In this study, traditional morphology was compared with three DNA barcoding markers regarding their utility in species delimitation within the genus: 1) Cytochrome c Oxidase subunit 1 (CO1), 2) Internal Transcribed Spacer 2 (ITS2), and 3) Ribosomal large subunit (28S). The molecular species delimitation analyses were carried out using four methods under the corrected p-distances Neighbor-Joining (NJ) criteria: 1) Automatic Barcode Gap Discovery (ABGD), 2) Assemble Species by Automatic Partitioning (ASAP), 3) General Mixed Yule Coalescent model (GMYC), and 4) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 75 terminals from 22 putative species of Physocyclus. The average intraspecific genetic distance (p-distance) was found to be < 2%, whereas the average interspecific genetic distance was 20.6%. The ABGD, ASAP, and GMYC methods were the most congruent, delimiting 26 or 27 species, while the bPTP method delimited 33 species. The use of traditional morphology for species delimitation was congruent with most molecular methods, with the male palp, male chelicerae, and female genitalia shown to be robust characters that support species-level identification. The barcoding with CO1 and 28S had better resolution for species delimitation in comparison with ITS2. The concatenated matrix and traditional morphology were found to be more robust and informative for species delimitation within Physocyclus.
Arteminae, cellar spiders, molecular markers, molecular methods, North America
Species delimitation is the act of identifying species-level biological diversity (
The spiders of the family Pholcidae C. L. Koch, commonly known as cellar spiders or daddy long-legs spiders, is currently composed of 1,896 species in 97 genera (WSC 2022). Pholcidae is the ninth largest spider family in the World and the most diverse within the Synspermiata clade. The family is composed of five subfamilies: Arteminae Simon, 1893, Modisiminae Simon, 1893, Ninetinae Simon, 1890, Pholcinae C. L. Koch, 1850, and Smeringopinae Simon, 1893 (
Physocyclus comprises 37 described species, classified into two species groups proposed by
The most recent taxonomic revisions and morphological phylogenetic analyses (
The general morphology among the different genera of pholcid spiders is conservative, with slight differences in somatic structures. However, primary sexual structures such as male palps and female genitalia, as well as secondary sexual structures such as male chelicerae, are important features used for identification and species diagnosis due to the fact that genitalia evolve more rapidly than non-genital morphological features (
Spiders with generally simple genitalia, such as mygalomorphs and some araneomorphs (Synspermiata), are complicated cases for species delimitation and identification using morphology (
Due to the poor morphological variation and simple genitalia in some taxonomic groups, approximations based on DNA barcoding using mitochondrial data are often used to establish limits between species, detect species complexes, and/or discover new species in different spider groups (
In modern systematics, integrative taxonomy studies that combine different sources of evidence, such as molecular markers and morphological data, are commonly implemented to help delimit and diagnose new species or even identify cryptic species complexes (
From the first proposal for a DNA barcoding initiative using a single locus (the CO1 mitochondrial gene for animals) as a diagnostic for assigning species (
The aim of this study is to carry out different species delimitation methods within the spider genus Physocyclus under an integrative taxonomic approach. To carry out this, we use a combination of molecular markers (CO1, ITS2, and 28S) and traditional morphology of diagnostical features (e.g., male palps, male chelicerae, and female epigynes) to test the validation of the currently recognized species within the genus.
Specimens were provided by the Laboratory of Arachnology (LATLAX) IB-UNAM, Tlaxcala, Mexico; the Colección Nacional de Arácnidos (CNAN), Institute of Biology, Universidad Nacional Autónoma de México (
The molecular analyses were based on a total of 194 sequences of 23 putative species. Species used in the molecular analyses are listed in Table
Specimens sequenced for each species of Physocyclus, DNA voucher numbers, localities, and GenBank accession numbers for CO1, ITS2, and 28S. Mexican state abbreviations: BC, Baja California; BCS, Baja California Sur; COL, Colima; GRO, Guerrero; HGO, Hidalgo; JAL, Jalisco; MICH, Michoacán, OAX, Oaxaca; PUE, Puebla. **Non-Mexican localities.
Species | DNA Code LATLAX | Locality (Mexico) | CO1 | ITS2 | 28S |
---|---|---|---|---|---|
P. bicornis | Ara0394 | GRO: Copala | OP293157 | OP296540 | OP295410 |
P. bicornis | Ara0396 | GRO: Quechultenango | OP293158 | OP296538 | OP295411 |
P. bicornis | Ara0398 | GRO: Coyuca | OP293159 | OP296539 | |
P. bicornis | Ara0445 | GRO: Quechultenango | OP293160 | OP296541 | OP295412 |
P. brevicornus | Ara0515 | JAL: Cocula | OP293161 | OP296542 | OP295413 |
P. brevicornus | Ara0516 | MICH: Morelia | OP293162 | ||
P. brevicornus | Ara0518 | MICH: Morelia | OP293163 | OP296543 | OP295414 |
P. cornutus | Ara0405 | BCS: Los Cabos | OP293164 | OP295415 | |
P. cornutus | Ara0406 | BCS: Los Cabos | OP293165 | OP295416 | |
P. dugesi | Ara0597 | HGO: Tula | OP293166 | OP296544 | OP295417 |
P. dugesi | Costa Rica** | AY560787 | AY560750 | ||
P. enaulus | Ara0391 | COA: Saltillo | OP293167 | OP296545 | OP295418 |
P. enaulus | Ara0392 | COA: Saltillo | OP293168 | OP296546 | OP295419 |
P. enaulus | Ara0393 | COA: Saltillo | OP293169 | OP296547 | OP295420 |
P. enaulus | U.S.A.** | MG268722 | |||
P. franckei | Ara0378 | HGO: Tolantongo | OP293170 | OP296548 | |
P. franckei | Ara0379 | HGO: Cárdenas | OP293171 | OP296549 | OP295421 |
P. franckei | Ara0381 | HGO: Cardonal | OP293172 | OP296550 | |
P. franckei | Ara0382 | HGO: Cardonal | OP293173 | OP296551 | |
P. gertschi | Ara0575 | GRO: José Azueta | OP293174 | OP296552 | OP295422 |
P. gertschi | Ara0576 | GRO: José Azueta | OP293175 | OP296553 | OP295423 |
P. gertschi | Ara0577 | GRO: José Azueta | OP293176 | OP296554 | OP295424 |
P. globosus | Ara0473 | COL: Coquimatlán | OP293177 | OP296555 | OP295425 |
P. globosus | Ara0533 | BCS: Comundú | OP293178 | ||
P. globosus | Ara0535 | GRO: Técpan | OP293179 | OP296556 | OP295426 |
P. globosus | Quintana Roo | MT888253 | |||
P. globosus | Cuba** | AY560788 | AY560751 | ||
P. lautus | Ara0459 | MICH: Cárdenas | OP293180 | OP296557 | OP295427 |
P. lautus | Ara0579 | MICH: Coahuayana | OP293181 | OP296558 | OP295428 |
P. lautus | Ara0583 | JAL: La Huerta | OP293182 | OP295429 | |
P. lyncis | Ara0437 | JAL: Zapopan | OP293183 | OP296559 | OP295430 |
P. lyncis | Ara0754 | JAL: Zapopan | OP293184 | OP296560 | OP295431 |
P. mariachi | Ara0745 | JAL: Hostotipaquillo | OP293185 | OP296561 | OP295432 |
P. mariachi | Ara0746 | JAL: Hostotipaquillo | OP293186 | OP296562 | OP295433 |
P. mariachi | Ara0748 | JAL: Plan de Barrancas | OP293187 | OP296564 | OP295434 |
P. merus | Ara0898 | SLP: Villa de Reyes | OP293188 | OP296565 | OP295435 |
P. merus | Ara0915 | SLP: Villa de Reyes | OP293189 | OP295436 | |
P. merus | Ara0916 | SLP: Villa de Reyes | OP293190 | OP296566 | OP295437 |
P. merus | Ara0917 | SLP: Villa de Reyes | OP293191 | OP296567 | OP295438 |
P. merus | Ara0918 | SLP: Villa de Reyes | OP293192 | OP295438 | |
P. michoacanus | Ara0585 | MICH: Tzitzio | OP293193 | OP296568 | OP295440 |
P. michoacanus | Ara0586 | MICH: Tzitzio | OP293194 | OP296569 | OP295441 |
P. michoacanus | Ara0598 | JAL: Jilotlán | OP293195 | OP295442 | |
P. modestus | Ara0467 | PUE: Miahuatlán | OP293196 | OP296570 | OP295443 |
P. modestus | Ara0469 | GRO: Tepecoacuilco | OP293197 | OP296571 | OP295444 |
P. modestus | Ara0480 | GRO: Escudero | OP293198 | OP295445 | |
P. modestus | Ara0482 | GRO: Quechultenango | OP293199 | ||
P. mysticus | Ara0450 | BC: Ensenada | OP293200 | OP296572 | |
P. mysticus | Ara0451 | BC: Ensenada | OP293201 | OP296573 | |
P. mysticus | Ara0452 | BCS: Mulegé | OP293202 | OP295446 | |
P. mysticus | Ara0453 | BCS: Mulegé | OP293203 | OP295447 | |
P. mysticus | Ara0524 | BC: Ensenada | OP293204 | ||
P. paredesi | Ara0483 | OAX: Tadela | OP293205 | OP296574 | OP295448 |
P. paredesi | Ara0484 | OAX: Tadela | OP293206 | OP296575 | OP295449 |
P. paredesi | Ara0485 | OAX: Totolapa | OP293207 | OP296576 | OP295450 |
P. paredesi | Ara0486 | OAX: Totolapa | OP293208 | OP295451 | |
P. pocamadre | Ara0371 | BCS: Mulegé | OP293209 | ||
P. reddelli | Ara0487 | HGO: Araya | OP293210 | OP296577 | OP295452 |
P. reddelli | Ara0488 | HGO: Araya | OP293211 | OP296578 | |
P. rothi | Ara0383 | BCS: Comundú | OP293212 | OP296579 | OP295453 |
P. rothi | Ara0384 | BCS: Comundú | OP293213 | OP296580 | OP295456 |
P. rothi | Ara0386 | BCS: La Paz | OP293214 | OP295454 | |
P. rothi | Ara0387 | BCS: La Paz | OP293215 | OP295455 | |
P. sikuapu | Ara0749 | MICH: Costa Aquila | OP293216 | OP296581 | OP295457 |
P. sikuapu | Ara0750 | MICH: Costa Aquila | OP293217 | OP296582 | OP295458 |
P. sikuapu | Ara0751 | MICH: Costa Aquila | OP293218 | OP296583 | OP295459 |
P. sikuapu | Ara0752 | MICH: Costa Aquila | OP293219 | OP296584 | OP295460 |
P. validus | Ara0502 | COL: Coquimatlán | OP293220 | OP296585 | |
P. validus | Ara0503 | GRO: Eduardo Neri | OP293221 | OP296586 | OP295461 |
P. xerophilus | Ara0372 | BCS: Mulegé | OP293222 | OP296587 | OP295464 |
P. xerophilus | Ara0373 | BCS: Mulegé | OP293223 | OP296588 | OP295462 |
P. xerophilus | Ara0374 | BCS: Mulegé | OP293224 | OP296589 | OP295465 |
P. xerophilus | Ara0375 | BCS: Mulegé | OP293225 | OP296590 | |
P. xerophilus | Ara0376 | BCS: Mulegé | OP293226 | OP296591 | OP295463 |
P. xerophilus | Ara0377 | BCS: Mulegé | OP293227 | OP296592 | |
Chisosa sp. | Ara0454 | PUE: Miahuatlán | OP293228 | OP296593 | OP295466 |
Chisosa sp. | Ara0455 | PUE: Miahuatlán | OP293229 | OP296594 | OP295467 |
For DNA extraction, we used the Qiagen DNeasy extraction kit, following the modifications suggested by
Molecular marker | Primer | Sequence (5’-3’) | Author |
---|---|---|---|
CO1 | HCO2198 | TAAACTTCAGGGTGACCAAAAAATC |
|
LCO1490 | GGTCAACAAATCATAAAGATATTGG | ||
HCO-JJ | AWACTTCVGGRTGCVCAAARAATCA |
|
|
LCO-JJ | CHACWAAYCATAAAGATATYGG | ||
ITS2 | 5.8S | CGCCTGTTTATCAAAAACAT |
|
CAS28sB1d | TTC TTT TCC TCC SCT TAY TRA TAT GCT TAA | ||
28S | 28S-B1 | GACCGATAGCAAACAAGTACCG | |
28S-B2 | CACGGGTCGATGAAGAACGC |
Both forward and reverse DNA strands were sequenced. DNA sequences were edited in Geneious v. 8.1.9 (
Four different molecular delimitation methods were used under the corrected p-distances Neighbor-Joining (NJ) criteria: 1) ABGD (Automatic Barcode Gap discovery) (
The genetic distance tree was reconstructed with MEGA v. 10.1.7 (
This method aims to find gaps in genetic divergence, considering that intraspecific genetic variation is theoretically smaller than interspecific divergences. It first generates a prior 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 on 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.
This is an ascending hierarchical clustering method. Sequences are merged into groups that are successively merged further until all sequences form a single group. Partitions are equivalent to each sequence merge step. The software analyzes all partitions and scores the most probable groups into a tree (
This approach applies single (
This method is similar to GMYC, but does not require an ultrametric tree as input because the models of speciation rate are implemented directly using the number of substitutions calculated from branch lengths. The Bayesian and Maximum Likelihood variants were carried out on the online platform (https://species.h-its.org/ptp/), with the following options: Rooted tree, MCMC = 1000000, Thinning = 100, Burn-in = 0.1, Seed = 123. The resulting trees were edited with the iTOL online version (https://itol.embl.de/) (
The corrected p-distances under NJ using the CO1 matrix recovered 22 species of Physocyclus. This is concordant with the morphology analysis of features commonly used to identify and diagnose at the species level (
Neighbor-Joining (NJ) with corrected p-distances tree constructed with CO1 barcode sequences from different species of Physocyclus. Branch colors indicate putative species. Male chelicerae, male palps, and female epigynes are shown for each species. Numbers above branches represent significant Bootstrap support values (> 50%).
Species | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1. P. bicornis | 0.7 | |||||||||||||||||||||
2. P. brevicornus | 18.4 | 0.3 | ||||||||||||||||||||
3. P. cornutus | 16.4 | 14.1 | 0.2 | |||||||||||||||||||
4. P. dugesi | 20.3 | 17.1 | 18.5 | 0.5 | ||||||||||||||||||
5. P. enaulus | 17.6 | 17.3 | 15.3 | 17.6 | 4.5 | |||||||||||||||||
6. P. franckei | 20.3 | 17.7 | 17.9 | 18.2 | 17.4 | 0.3 | ||||||||||||||||
7. P. gertschi | 13.8 | 18.3 | 17.1 | 20.1 | 19.7 | 19.4 | 0.2 | |||||||||||||||
8. P. globosus | 18.3 | 19.5 | 18.4 | 18.4 | 16.5 | 18.9 | 16.8 | 0.4 | ||||||||||||||
9. P. lautus | 12.9 | 17.4 | 15.9 | 17.8 | 16.9 | 18.4 | 14.3 | 17.1 | 1.4 | |||||||||||||
10. P. lyncis | 18.0 | 12.7 | 15.8 | 17.3 | 16.2 | 19.1 | 18.3 | 18.3 | 17.3 | 1.1 | ||||||||||||
11. P. mariachi | 16.4 | 20.1 | 20.1 | 18.4 | 20.0 | 19.2 | 16.5 | 19.6 | 15.6 | 18.3 | 1.5 | |||||||||||
12. P. merus | 17.6 | 17.2 | 15.8 | 17.9 | 11.0 | 19.7 | 19.1 | 17.9 | 16.9 | 15.5 | 19.4 | 0.9 | ||||||||||
13. P. michoacanus | 18.3 | 17.0 | 17.2 | 14.7 | 15.0 | 16.6 | 17.4 | 16.7 | 15.3 | 16.3 | 20.3 | 17.0 | 1.9 | |||||||||
14. P. modestus | 15.6 | 19.1 | 17.6 | 18.8 | 16.4 | 18.5 | 16.1 | 15.8 | 14.1 | 18.9 | 17.9 | 17.9 | 17.1 | 5.8 | ||||||||
15. P. mysticus | 18.2 | 14.3 | 12.8 | 16.0 | 15.0 | 16.9 | 17.4 | 16.9 | 16.2 | 15.3 | 17.8 | 14.6 | 15.8 | 16.9 | 5.7 | |||||||
16. P. paredesi | 18.0 | 19.5 | 19.2 | 18.5 | 17.0 | 19.4 | 17.0 | 17.5 | 16.3 | 18.0 | 18.9 | 18.2 | 19.8 | 17.8 | 17.6 | 1.3 | ||||||
17. P. pocamadre | 15.8 | 18.7 | 16.5 | 19.6 | 16.6 | 19.8 | 16.3 | 16.9 | 13.8 | 16.1 | 15.8 | 16.4 | 17.9 | 15.1 | 15.4 | 16.4 | 0.0 | |||||
18. P. reddelli | 19.2 | 17.5 | 18.5 | 17.1 | 16.6 | 13.0 | 19.2 | 19.4 | 17.5 | 16.8 | 18.6 | 17.0 | 17.1 | 18.4 | 15.8 | 17.7 | 16.9 | 0.0 | ||||
19. P. rothi | 16.0 | 13.0 | 9.7 | 16.4 | 14.3 | 14.6 | 14.2 | 17.0 | 15.6 | 14.6 | 17.3 | 15.5 | 14.9 | 16.6 | 11.0 | 17.8 | 16.5 | 17.5 | 0.8 | |||
20. P. sikuapu | 16.6 | 17.9 | 18.5 | 18.3 | 18.2 | 19.1 | 14.5 | 18.3 | 15.7 | 17.1 | 15.1 | 18.1 | 19.0 | 16.0 | 16.8 | 16.8 | 15.6 | 16.6 | 16.6 | 0.0 | ||
21. P. validus | 16.7 | 20.0 | 19.5 | 20.4 | 18.9 | 19.5 | 16.1 | 17.4 | 16.9 | 18.8 | 19.0 | 18.3 | 17.8 | 16.8 | 18.9 | 17.6 | 16.2 | 18.9 | 19.5 | 16.8 | 0.0 | |
22. P. xerophilus | 15.9 | 18.6 | 17.7 | 19.3 | 17.1 | 17.9 | 14.3 | 15.8 | 13.9 | 18.2 | 15.7 | 16.4 | 18.1 | 14.8 | 16.9 | 15.8 | 13.7 | 16.7 | 17.4 | 15.3 | 15.2 | 4.0 |
The Maximum Likelihood (ML) tree of the concatenated matrix (CO1+ITS2+28S) (Fig.
Maximum Likelihood (ML) tree of Physocyclus (log likelihood: -3749.87) constructed with the concatenated matrix (CO1+ITS2+28S). Bar colors represent putative species in the tree and in the columns, which represent the different species delimitation methods analyzed. Branch colors represent species groups: globosus (blue) and dugesi (red). Numbers below the columns represent the species recovered in each species delimitation method (not considering Chisosa sp.). Numbers above branches represent Bootstrap support values for ML (> 50% significant). Column abbreviations: Morphology (M); ABGD with initial (IP) and recursive (RP) partitions; ASAP; GMYC with single (SN) and multi (MT) thresholds; bPTP with Maximum Likelihood (ML) and Bayesian Inference (IB) variants.
The most congruent methods with morphology were the barcoding method ABGD, ASAP, and GMYC, which delimited 26 (ABGD IP and RP, ASAP, and GMYC MT) and 27 (GMYC SN) putative species, respectively. The most incongruent result of the analyses was bPTP, which delimited 33 putative species under ML and IB variants (Fig.
Both species groups (globosus and dugesi) were recovered in the ML analysis using the concatenated matrix (CO1+ITS2+28S) (Fig.
Two different approaches (DNA taxonomy and DNA barcoding) were proposed by
In the genetic distance analyses performed with independent matrices of CO1, ITS2, and 28S, all the species terminals were recovered. The genetic intraspecific distances for ITS2 were found to be relatively high in the majority of species (over 2%). However, the average intraspecific genetic distances using the CO1 and 28S markers were lower in the majority of species (< 2%).
When looking at CO1, Pholcid spiders generally show high genetic divergences among species (
With regards to molecular delimitation methods, ABGD used to be sensitive to sampling effect and tended to moderately over-split, as demonstrated in the mygalomorph genera Aphonopelma Pocock, 1901 by
The most congruent methods that delimited a similar number of species in this study were ABGD, ASAP, and GMYC, which was corroborated by traditional morphology (Fig.
In such cases of incongruency between the molecular methods and morphology, as in Physocyclus enaulus, P. modestus, P. mysticus, and P. xerophilus, no significant morphological differences were found within individuals of each species. However, P. enaulus was the exception, where
According with
As
Although the number of described species in the genus Physocyclus has doubled in the last decade (
In regard to the molecular methods used herein, each one presents its advantages and disadvantages. Barcoding methods (ABGD) can distinguish within-population differences caused by species divergences, analyzing the gaps of a data set and using it as a barcode to recognize different species (
The coalescence method (GMYC) is robust because it uses an a priori ultrametric species tree, taking into account the groups formed in the topology. However, this method assumes that the lineages in each population coalesce before any speciation event occurs, implying the absence of incomplete lineage sorting and ignoring the coalescent process within populations of ancestral species (
In conclusion, CO1 and 28S provide robust evidence for species-level delimitation in the genus Physocyclus, with high congruence among all methods. The genetic variability of ITS2 makes it an unreliable molecular marker for species delimitation on its own, however, it provides good information when used in combination with others mitochondrial and nuclear markers. Sexual morphological characters (male palps, male chelicerae, and female epigyne) are robust features for identifying and diagnosing Physocyclus species. However, in some cases, morphology alone is not enough to detect sister species, cryptic species, or even species complexes.
The first author thanks the Doctorate Program of the Centro Tlaxcala de Biología de la Conducta (CTBC), Universidad Autónoma de Tlaxcala, Tlaxcala City. The first author also thanks to the American Arachnological Society (AAS) and the Student Research Grants (2022) for financial support for field work. The second author thanks the program “Jóvenes investigadores por México (Cátedras CONACyT)” and the Consejo Nacional de Ciencia y Tecnología (CONACyT) for scientific support for the project No. 59: “Laboratorio Regional de Biodiversidad y Cultivo de Tejidos Vegetales (LBCTV), Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), sede Tlaxcala”. The second author also thanks SEP-CONACyT for financial support of the project of Basic Science (Ciencia Básica) 2016, No. 282834. We thank Dr. Edmundo González Santillán and Dr. Oscar F. Francke (ex-curator) of the Colección Nacional de Arácnidos (CNAN), Instituto de Biología, UNAM, for providing specimen loans, MSc. Laura Márquez Valdelamar for the molecular sequencing of the samples, Brett O. Butler for the English language review of the manuscript, and the reviewers for their comments and suggestions that improved the manuscript. We also thank the students of the Laboratory of Arachnology (LATLAX), IBUNAM, Tlaxcala, for their help in the field and processing of the material in the laboratory. Specimens were collected under Scientific Collector Permit FAUT-0309 from the Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT) provided to Dr. Alejandro Valdez-Mondragón.