Research Article
Research Article
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
expand article infoSamuel Nolasco§, Alejandro Valdez-Mondragón§
‡ Universidad Autónoma de Tlaxcala, Tlaxcala, Mexico
§ Universidad Nacional Autónoma de México, Tlaxcala, Mexico
Open Access


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 (Carstens et al. 2013). It arises from the need to classify, identify, and establish the limits between species (DeSalle et al. 2005; Carstens et al. 2013; Rannala and Yang 2020). The assignment of individual organisms into pre-existing species or higher-level categories (e.g., genus, family, order, etc.) and the designation of new species with proper diagnoses to distinguish them were, for a long-time, roles performed by taxonomist using only traditional morphology and/or somatic characters (Jörger and Schrödl 2013; Rannala and Yang 2020). However, the presence of plastic characters (i.e., characters with high morphological variation), relatively few distinctive morphological traits among species, and morphological stasis due to environmental selection in some groups of organisms (Bickford et al. 2007; Carstens et al. 2013), often makes species delimitation using only morphological evidence extremely difficult or impossible (DeSalle et al. 2005; Rannala and Yang 2020). Spiders are no exception, and due to the complications of using only morphology to identify and delimit species in some groups of araneomorphs and mygalomorphs spiders, different molecular methods have been applied to delimit spider species (Huber et al. 2005; Galtier et al. 2009; Hamilton et al. 2011; Ortiz and Francke 2016; Candia-Ramírez and Francke 2020; Navarro-Rodríguez and Valdez-Mondragón 2020; Valdez-Mondragón 2020; Hazzi and Hormiga 2021). Studies using DNA and RNA have been successful in classifying difficult groups and uncovering underestimated biodiversity (Fox et al. 1977; Wayne et al. 1987; Wilson 1995; Bond 2004).

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 (Huber 2011; Dimitrov et al. 2013; Eberle et al. 2018; Huber and Carvalho 2019). Arteminae includes 107 species distributed in nine genera: Artema Walckenaer, 1837, Aucana Huber, 2000, Chisosa Huber, 2000, Holocneminus Berland, 1942, Nita Huber & El-Hennawy, 2007, Pholcitrichocyclus Ceccolini & Cianferoni, 2022, Physocyclus Simon, 1893, Tibetia Zhang, Zhu & Song, 2006, and Wugigarra Huber, 2001 (WSC 2022).

Physocyclus comprises 37 described species, classified into two species groups proposed by Valdez-Mondragón (2013, 2014). To date, the globosus-group includes 15 species, while 22 species are recognized in the dugesi-group (Valdez-Mondragón 2013, 2014; Jiménez and Palacios-Cardiel 2013; Nolasco and Valdez-Mondragón 2020, 2022). Recently, five species from Mexico were described by Nolasco and Valdez-Mondragón (2020, 2022) based only on traditional morphology, four from the globosus-group and one of the dugesi-group. The genus is distributed mainly in arid and semiarid ecosystems, as well as tropical dry forests, mostly in North America, with some species in Central America (Valdez-Mondragón 2010, 2013, 2014; Jiménez and Palacios-Cardiel 2013; Nolasco and Valdez-Mondragón 2020, 2022). Most species in the genus are found under 1900 m a.s.l., with the exception of Physocyclus dugesi Simon 1893 and Physocyclus globosus (Taczanowski, 1874), whose distributions are influenced by synanthropic activities that have allowed them to occupy higher elevations in association with human dwellings (Valdez-Mondragón 2010). Physocyclus globosus is considered a cosmopolitan species, with records from North and Central America, the Caribbean Islands, the Pacific Islands, South America, Asia, Africa, and Oceania (Valdez-Mondragón 2010; WSC 2022).

The most recent taxonomic revisions and morphological phylogenetic analyses (Valdez-Mondragón 2010, 2013, 2014), as well as new species descriptions using traditional morphology (Jiménez and Palacios-Cardiel, 2013; Nolasco and Valdez-Mondragón 2020, 2022) have revealed 20 new species from Mexico, which represents 54% of the known species diversity in Physocyclus.

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 (Huber 2003; Huber et al. 2018; Valdez-Mondragón 2020). Speciation in arthropods is associated with marked changes in genital morphology, which explains the usefulness of genitalia in distinguishing closely related species (Huber et al. 2005). Sexual structures have different intraspecific and interspecific variation rates in spiders (Eberhard 1985; Eberhard et al. 1998). However, some groups show an overlapping of characters due to minimal morphological variation, making the identification and delimitation of species difficult (Hamilton et al. 2011; Zhang and Li 2014; Ortiz and Francke 2016; Tyagi et al. 2019; Valdez-Mondragón et al. 2019; Candia-Ramírez and Francke 2020; Navarro-Rodríguez and Valdez-Mondragón 2020; Hazzi and Hormiga 2021). Additionally, speciation without changes in genital shape has been recorded in some pholcid spiders, as demonstrated by Huber et al. (2005) with two species of “Psilochorus” from South America that showed the same genitalia shape but extreme differences in size and coloration pattern.

Spiders with generally simple genitalia, such as mygalomorphs and some araneomorphs (Synspermiata), are complicated cases for species delimitation and identification using morphology (Huber et al. 2005; Hamilton et al. 2011; Huber and Dimitrov 2014; Valdez-Mondragón 2013, 2014, 2020; Nolasco and Valdez-Mondragón 2022). Additionally, intrasexual polymorphism has been described among females in some pholcids species. Huber & Pérez-González (2001a, b) described two different epigyne morphotypes in females of Ciboneya antraia. In the same way, Valdez-Mondragón (2010) described discontinued interspecific variation in the epigyne shape in females of Physocyclus enaulus Crosby, 1926, with three distinct morphotypes.

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 (Barrett and Hebert 2005; DeSalle et al. 2005; Bickford et al. 2007; Galtier et al. 2009; Carstens et al. 2013; Navarro-Rodríguez and Valdez-Mondragón 2020; Rannala and Yang 2020; Valdez-Mondragón 2020). Barcoding based on cytochrome c oxidase subunit 1 (CO1) is the common standard in animal Barcoding (including spiders), and its effectivity and resolution has been tested in many studies (Astrin et al. 2006; Planas and Ribera 2015; Ortiz and Francke 2016; Valdez-Mondragón et al. 2019; Navarro-Rodríguez and Valdez-Mondragón 2020; Valdez-Mondragón 2020). While the mitochondrial marker CO1 seems to be suitable for DNA barcoding, it is susceptible to over- and underestimating the diversity in some cases (Astrin et al. 2006; Ortiz and Francke 2016). As such, it is preferable to complement the use of the CO1 marker with other informative mitochondrial (16S) or nuclear (ITS1 or ITS2) markers, as well as morphological evidence, to obtain better resolution (Astrin et al. 2006; Agnarsson 2010; Planas and Ribera 2015; Ortiz and Francke 2016; Valdez-Mondragón et al. 2019; Navarro-Rodríguez and Valdez-Mondragón 2020). The nuclear molecular marker 28S has been used mainly in phylogenetic analyses, providing high resolution for basal clades (Bruvo-Mađarić et al. 2005; Álvarez-Padilla et al. 2009; Arnedo et al. 2009; Dimitrov and Hormiga 2011; Dimitrov et al. 2013). Due to its low substitution rate, 28S is useful for estimating phylogenetic hypotheses of taxa with very old divergence times (Hillis and Dixon 1991). However, this marker has never been tested in species delimitation analyses within the family Pholcidae.

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 (Astrin and Stueben 2008; Álvarez-Padilla et al. 2009; Correa-Ramírez et al. 2010; Hamilton et al. 2011; Montes de Oca et al. 2015; Planas and Ribera 2015; Valdez-Mondragón and Francke 2015; Cao et al. 2016; Valdez-Mondragón et al. 2019; Newton et al. 2020; Valdez-Mondragón and Cortez-Roldán 2021). Recent publications on spider taxonomy have additionally used other sources of information, such as Ecological Niche Modeling (ENM), lineal morphology, and even geometric morphology to characterize species (Valdez-Mondragón et al. 2019; Navarro-Rodríguez and Valdez-Mondragón 2020; Solís-Catalán 2020).

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 (Hebert et al. 2003), the field of species delimitation has been refined and improved, incorporating different theories (e.g., coalescence) and methods (DeSalle et al. 2005; Hickerson et al. 2006; Pons et al. 2006; Puillandre et al. 2012, 2021; Carstens et al. 2013; Rannala and Yang 2020). Species delimitation analyses using DNA Barcoding, initially with only a single locus (CO1), is limited as a diagnostic for assigning species based on the fact that intraspecific and interspecific sequence distances may be similar in large populations (Hebert et al. 2003; Rannala and Yang 2020). Therefore, many studies have opted for a multi-locus approach, combining CO1 with other molecular markers to give additional robust evidence for species delimitation (Astrin et al. 2006; Agnarsson 2010; Planas and Ribera 2015; Ortiz and Francke 2016; Ballesteros and Hormiga 2018; Navarro-Rodríguez and Valdez-Mondragón 2020; Hazzi and Hormiga 2021).

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.

Materials and methods

Biological material

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 (IB-UNAM), Mexico City; Centro de Investigaciones Biológicas de Noreste (CIBNOR), La Paz, Baja California Sur, Mexico; and Colección Aracnológica de la Facultad de Biología de la Universidad Michoacana de San Nicolás de Hidalgo (CAFBUM), Michoacan, Mexico. Specimens were preserved in 80% ethanol for morphological studies and 96% ethanol for molecular studies. Female sexual structures (epigyne) were dissected in 80% ethanol and cleaned with potassium hydroxide (10% KOH). This to remove all soft tissue and observe with clarity the taxonomically important internal structures, such as the pore plates. The left male palps were dissected and observed in 80% ethanol. Structures were photographed submerged in commercial gel alcohol to hold them in the appropriate position, while the preparation was done with structures completely covered with 80% ethanol. Specimen observations and identifications were carried out using a Zeiss Discovery V8 stereo microscope. A Zeiss Axiocam 506 color camera attached to a Zeiss AXIO Zoom V16 stereo microscope was used to photograph focal structures (male palps, female epigynes, and male chelicerae). Digital images of morphological structures were edited in Adobe Photoshop CS6.

Taxon sampling

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 1. The ingroup includes 188 sequences of 22 species of Physocyclus previously described by Valdez-Mondragón (2010, 2013, 2014) and Nolasco and Valdez-Mondragón (2020, 2022). The CO1 sequence matrix is composed of 75 sequences (22 species), ITS2 with 55 sequences (20 species), and 28S with 58 sequences (21 species). Four different partitions were used in the analyses: 1) CO1: 642 bp, 2) ITS2: 505 bp, 3) 28S: 891 bp, and 4) the concatenated matrix CO1+ITS2+28S: 2038 bp. Since this study is focused solely on species delimitation within Physocyclus and not on the molecular dating and phylogenetic relationships within Arteminae, only Chisosa sp. (Arteminae) was used as an outgroup, to root the trees in the various analyses.

Table 1.

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

DNA extraction, amplification, and sequencing

For DNA extraction, we used the Qiagen DNeasy extraction kit, following the modifications suggested by Valdez-Mondragón and Francke (2015) and Valdez-Mondragón (2020). Three legs from each specimen were used for DNA extraction, using males, females, and juveniles, depending on the available specimens per species. The criterion for selecting tissues was based on tissue antiquity, considering only specimens collected in the last five years in order to extract high DNA quality. Amplification of the CO1 locus was carried out using two different primer sets: LCO1490/HCO2198 and LCO-JJ/HCO-JJ; for ITS2, the primer set 5.8S and CAS28SB1d was used; and for 28S, 28S-B1 and 28S-B2 (Table 2). Polymerase chain reactions (PCR) were carried out in a Verity-Applied Biosystems 96 Well Thermal Cycler. The final volume of each PCR tube was 20 µl: 2.3 µl injectable H2O, 2.0 µl Q-solution, 10 µl Multiplex-Mix PCR, 1.6 µl of each primer (forward and reverse), and 2.5 µl of extracted DNA sample. The cycles and optimal temperatures for CO1 and ITS2 amplification were as follows: Initial heating phase of 15 min at 95 °C, 35 amplification cycles of 35 s at 94 °C (denaturing), 1 min 30 s at 40 °C (alignment), and 1 min 30 s at 72 °C (elongation), with a final elongation of 10 min at 72 °C. Two different protocols were used to amplify the 28S region. The first followed Eberle et al. (2018): initial heating phase of 15 min at 95 °C, 35 amplification cycles of 35 s at 95 °C (denaturing), 1 min at 51 °C (alignment), and 1 min at 72 °C (elongation), with a final elongation of 10 min at 72 °C. The second protocol is a modification of Eberle et al. (2018): initial heating phase of 15 min at 95 °C, 35 amplification cycles of 35s at 94 °C (denaturing), 1 min 30 s at 59 °C (alignment), and 1 min 30 s at 72 °C (elongation), with a final elongation of 10 min at 72 °C. Gel electrophoresis was carried out with 0.5% agarose using the molecular weight marker Perfect DNA 100 bp Ladder Novage to calculate fragment size of amplifications. Gels were visualized in a photodoc BioDoc-It2 Imager 315 Imaging System LMS-20 Transilluminator. PCR products were purified using a QIAquick Qiagen purification kit. Tissue selection, DNA extraction, amplification, and purification were performed at the Laboratory of Molecular Biology at Laboratorio Regional de Biodiversidad y Cultivo de Tejidos Vegetales (LBCTV), IB-UNAM, Tlaxcala City. Sanger sequencing was done at the Laboratory of Molecular Biology and Health, IB-UNAM, Mexico City.

Table 2.

Primer sets used in this study for PCR amplification.

Molecular marker Primer Sequence (5’-3’) Author
ITS2 5.8S CGCCTGTTTATCAAAAACAT Ji et al. (2003); Planas and Ribera (2014)
28S 28S-B1 GACCGATAGCAAACAAGTACCG Bruvo-Mađarić et al. (2005)

DNA sequence alignment and editing

Both forward and reverse DNA strands were sequenced. DNA sequences were edited in Geneious v. 8.1.9 (Rozen and Skaletsky 2000). Multiple alignment of sequences was implemented using MAFFT v. 7 (Katoh and Toh 2008) through online platform (, with the following commands: Auto (FFT-NS-2, FFTNS-I or L-INS-I, depending on data size). In some cases, alignment was done manually and edited with BioEdit v. (Hall 1999). The concatenated matrix (COI+ITS2+28S) was built in Geneious v. 8.1.9. The aligned matrices were subsequently used in the molecular analyses.

Molecular analysis and species delimitation

Four different molecular delimitation methods were used under the corrected p-distances Neighbor-Joining (NJ) criteria: 1) ABGD (Automatic Barcode Gap discovery) (Puillandre et al. 2012), 2) ASAP (Assemble Species by Automatic Partitioning) (Puillandre et al. 2021), 3) GMYC (General Mixed Yule Coalescent) (Pons et al. 2006), and 4) bPTP (Bayesian Poisson Tree Process) (Zhang et al. 2013; Kapli et al. 2017).

p-distances under Neighbor-Joining (NJ)

The genetic distance tree was reconstructed with MEGA v. 10.1.7 (Kumar et al. 2016) under the following parameters: Number of replicates = 1000, Bootstrap support values = 1000 (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 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 ( 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. 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 (Puillandre et al. 2021). ASAP analyzes were run on the online platform ( using Kimura (K80) distance matrices and configured under following parameters: Substitution model = p-distances, Probability = 0.01, Best scores = 10, Fixed seed value = -.

General Mixed Yule Coalescent (GMYC)

This approach 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 as input (Ortiz and Francke 2016). Ultrametric trees were generated with phylogenetic analyses in the BEAUti and BEAST v. 1.10.4 software (Drummond et al. 2002–2018) using a coalescent (constant population) tree prior. An independent log normal uncorrelated clock was applied to each partition with their respective evolution model and substitution rates (CO1: GTR + I + G; ITS2: K2P; 28S: GTR + I + G). Five independent analyses were run, each with 40 million iterations. Tracer 1.6 (Rambaut and Drummond 2003–2013) was used to evaluate convergence values, with the ESS (Effective Sample Size) > 200. Tree Annotator 2.6.0 (a BEAST package) was used to construct maximum credibility of clades trees, after discarding the first 25% of each independent run as burn-in. Finally, the GMYC method was implemented in the web platform (, which uses the original R implementation of the GMYC model (Fujisawa and Barraclough 2013).

Bayesian Poisson Tree Processes (bPTP)

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 (, 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 ( (Letunic and Bork 2021) and Photoshop CS6. To delimit different species, we used the congruence integration criteria. It is based on the correspondence among the different molecular methods to generate a highly supported species hypothesis (DeSalle et al. 2005; Hamilton et al. 2011; Navarro-Rodríguez and Valdez-Mondragón 2020; Valdez-Mondragón 2020).


Molecular analyses of genetic distances

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 (Valdez-Mondragón 2013, 2020; Valdez-Mondragón and Francke 2015) (Fig. 1). Both the morphology and all genetic distance analyses using CO1 recovered groups that correspond with a described species. The NJ analyses using the ITS2 marker recovered 20 species (Fig. 2), whereas analyses with 28S recovered 21 species (Fig. 3). The average genetic p-distances among Physocyclus species were 16.4% (min: 14.89%, max: 17.96%) for CO1, 29.4% (min: 25.37%, max: 38.59%) for ITS2, and 14.4% (min: 14.15%, max: 17.87%) for 28S (Figs 13, Table 3). The average intraspecific distances using CO1 were below 2% for most species (18/22). However, four species (P. enaulus Crosby, 1926, P. modestus Gertsch, 1971, P. mysticus Chamberlin, 1924, and P. xerophilus Nolasco & Valdez-Mondragón, 2020) showed average intraspecific genetic distances between 4–6% (Table 3), with high Bootstrap support in each case (> 94%) (Fig. 1). The Bootstrap support values for all species were high (100%) (Fig. 1). The globosus and dugesi species groups were not recovered in the CO1 topology. In the ITS2 tree, six species (P. bicornis Gertsch, 1971, P. lautus Gertsch, 1971, P. lyncis Nolasco & Valdez-Mondragón, 2022, P. modestus, P. mysticus, and P. validus Gertsch, 1971) had average intraspecific genetic distances below 2%, while the rest showed average intraspecific genetic distances over 2% (Fig. 2). Bootstrap support values for species in the ITS2 topology were significant (over 89%), except for P. franckei (51%). Using the 28S marker, average intraspecific genetic distances for all species were below 2% (Fig. 3) with high Bootstrap support values (> 95%). The 28S tree was the only one to recover both the globosus and dugesi species groups with high Bootstrap support values (100% for the dugesi group and 91% for the globosus group) (Fig. 3).

Figure 1. 

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%).

Figure 2. 

Neighbor-Joining (NJ) with corrected p-distances tree constructed with ITS2 barcode sequences from different specimens and species of Physocyclus. Branch colors indicate putative species. Numbers above branches represent significant Bootstrap support values (> 50%).

Figure 3. 

Neighbor-Joining (NJ) with corrected p-distances tree constructed with 28S barcode sequences from different specimens and species of Physocyclus. Branch colors indicate putative species. Numbers above branches represent significant Bootstrap support values (> 50%).

Table 3.

Average CO1 genetic distances (p-distances) among Physocyclus species.

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

Molecular methods for species delimitation

The Maximum Likelihood (ML) tree of the concatenated matrix (CO1+ITS2+28S) (Fig. 4) found congruence among the four different molecular species delimitation methods and using traditional morphology. This was found for 15 species: Physocyclus bicornis, P. brevicornus, P. cornutus, P. dugesi, P. gertschi, P. globosus, P. lautus, P. lyncis, P. merus, P. paredesi, P. pocamadre, P. reddelli, P. rothi, P. sikuapu, and P. validus (Fig. 4). Physocyclus mariachi and P. michoacanus are recovered as distinct species by most methods except for bPTP, whereas P. franckei is not recovered under the bPTP and GMYC methods in the single threshold (SN) (Fig. 4). Physocyclus enaulus, P. modestus, P. mysticus, and P. xerophilus were found to contain more than two species (2–4) by most of the species delimitation methods (Fig. 4).

Figure 4. 

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. 4).

Both species groups (globosus and dugesi) were recovered in the ML analysis using the concatenated matrix (CO1+ITS2+28S) (Fig. 4). The dugesi group was recovered with significant Bootstrap support (100%), whereas the globosus group had Bootstrap support value of 51%.


Two different approaches (DNA taxonomy and DNA barcoding) were proposed by DeSalle et al. (2005) to overcome several weaknesses of traditional morphology-based taxonomic systematics, and to resolve the crucial need for accurate and rapid species identification tools (Hebert et al. 2003; Tautz et al. 2003). DNA barcoding is useful for recognizing cryptic species (two or more distinct species that are erroneously classified as the same species due to similar morphology) (Bickford et al. 2007). Even only with CO1, DNA barcoding is helpful in species diagnosis due to the fact that sequence divergences are ordinarily much lower among individuals of the same species than between closely related species (Hebert et al. 2004). For some groups of arachnids, traditional morphology fails to recognize and delineate species boundaries. Also, identify sister or cryptic species requires other types or evidence such as molecular data, ecological niche modeling, morphometric morphology, haplotype networks, and biogeographical approximations (Hebert et al. 2003, 2004; Hamilton et al. 2011, 2014; Montes de Oca et al. 2015; Ortiz and Francke 2016; Cruz-López et al. 2019; Valdez-Mondragón et al. 2019; Newton et al. 2020; Valdez-Mondragón and Cortez-Roldán 2021). However, as demonstrated herein, spiders of the genus Physocyclus have robust morphology for diagnosis and identification at the species level, mainly of primary and secondary sexual characters, such as chelicerae and palps (males) or epigynes (females).

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%). Agnarsson (2010), Valdez-Mondragón et al. (2019), and Navarro-Rodríguez and Valdez-Mondragón (2020) mention that ITS2 is inadequate for resolving relationships between closely related species of spiders. Our data corroborate this, showing the unreliability of this gene for genetic distance (and species delimitation) analyzes when used on its own. However, the use of a concatenated matrix of nuclear (28S and ITS2) and mitochondrial (CO1) markers provides better results with more robust evidence for delimiting species based on molecular data (Astrin et al. 2006; Agnarsson 2010; Planas and Ribera 2015; Ortiz and Francke 2016; Navarro-Rodríguez and Valdez-Mondragón 2020).

When looking at CO1, Pholcid spiders generally show high genetic divergences among species (Astrin et al. 2006). In contrast to reports for spiders in general that show values of 14.4% (Hebert et al. 2003) and 16.4% (Barrett and Hebert 2005), Pholcids’ average interspecific genetic distance for CO1 is 19.8% (Astrin et al. 2006). Our results found a value of average interspecific distance at the CO1 marker observed in Physocyclus of 16.4% (Fig. 1, Table 3). This value fits within the average limits of other spiders; however, this tendency in the Pholcidae family is not always the case. The average interspecific genetic distance for CO1 in other genera such as Ixchela Huber, 2000 (Modisiminae) were found to be lower, 12% (Valdez-Mondragón 2020). Although most species were included in the analyses (22 of 37), missing species might have an effect and overestimate the average interspecific distances, because perhaps the sister species of each species is not found in the data set. However, the results and topologies were consistent along the analyzes.

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 Hamilton et al. (2014) and Bonnetina Vol, 2000 by Ortiz and Francke (2016) and Candia-Ramírez and Francke (2020). Similar results were observed in Loxosceles Heineken & Lowe, 1832 (Valdez-Mondragón et al. 2019; Navarro-Rodríguez and Valdez-Mondragón 2020), where the ABGD method generated an inflated number of delimited species. However, within the pholcid genus Ixchela, it was observed by Valdez-Mondragón (2020) that the most incongruent method was bPTP (both ML and IB variants), as was also found in the molecular analyses herein with the genus Physocyclus.

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. 4). The bPTP method delimited a higher number of species in comparison with morphology. These results contrast with those found by Ortiz and Francke (2016), Valdez-Mondragón et al. (2019), and Navarro-Rodríguez and Valdez-Mondragón (2020), in which the bPTP and GMYC analyses were the most congruent methods with the morphology in spiders. This may be due to the inclusion of the 28S marker, which might be causing tree-based analyzes (bPTP) to generate an overestimation in the number of putative species recovered. According to Luo et al. (2018), GMYC and bPTP are negatively influenced by gene flow and are sensitive to the ratio of population size to divergence time, reflecting the important impact of incomplete lineage sorting on species delimitation.

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 Valdez-Mondragón (2010) recorded three different morphotypes of epigyne shape. Unfortunately, we could not to get sequences of all the morphotypes. As Valdez-Mondragón (2010, 2014) mentions, the morphology of somatic and sexual characters in the genus Physocyclus is usually highly conserved. Similarly, morphological changes might not be correlated with species boundaries, or may not be useful for delimiting species if interspecific recognition occurs in non-visual signs (chemical or mating calls), or even biogeographical traits (microhabitats). Furthermore, it is possible that the species may be under stabilizing selection that promotes morphological stasis (Bickford et al. 2007).

According with Hamilton et al. (2011), species delimitation based only on molecular data can rarely be achieved, and additional types of evidence such as biogeographical information is needed. In the case of the multiple species inferred within P. mysticus, no significative morphological or habitat differences are apparent. All specimens are from xerophytic scrub and present similar microhabitats of living under big boulders on the ground. Sympatric speciation may be possibly, as other species of the genus Physocyclus have been collected in the same locality (Valdez-Mondragón 2010, 2013). Physocyclus modestus presents a similar case, being one widespread species from Guerrero, Morelos, Oaxaca, and Puebla. However, habitat differences exist among the different populations of this species. Some specimens are from xerophytic scrub, while others are found in lowland forest. Microhabitat likely has a direct influence on the diversification of pholcids spiders, as was demonstrated by Eberle et al. (2018). Therefore, the morphological and molecular evidence suggest that P. modestus might be a species complex, as observed in P. mysticus. However, more male and female specimens from different populations are needed to confirm this assumption.

As Carstens et al. (2013) suggested, probing different methods or lines of evidence is necessary for properly implementing species delimitation. When the information and results are incongruent, it is better to be conservative about assumptions of species delimitation. In the case of some species in this study (e.g., P. enaulus and P. xerophilus), more detailed analyses of the morphological structures are necessary. Maybe including ultra-morphology, lineal morphology, or geometric morphometry in somatic features such as length legs, carapace shape, or even in sexual structures (Valdez-Mondragón et al. 2019). Lineal and geometric morphology has provided strong evidence for splitting species in cases where traditional morphology fails to delimit species. This has been demonstrated in araneomorph spiders (Planas and Ribera 2015; Valdez-Mondragón et al. 2019) and in brown recluse spiders of the genus Loxosceles (Solís-Catalán 2020). In this works, significant differences were found in carapace length, male palp shape, and length leg I among different species from the Canary Islands and central Mexico. Physocyclus enaulus is a widespread species from northern Mexico and southern United States. Valdez-Mondragón (2010) reported three different types of ventral apophyses in the female epigyne, which suggests that it might comprise a species complex rather than wide intraspecific morphological variation.

Although the number of described species in the genus Physocyclus has doubled in the last decade (Valdez-Mondragón 2010, 2013, 2014; Jiménez and Palacios-Cardiel 2013; Nolasco and Valdez-Mondragón 2020, 2022), the diversity of this genus in Mexico is still poorly known. Provinces such as the Sonoran and Chihuahuan Deserts have been poorly sampled, despite their arid and semiarid ecosystems being common habitats for the genus Physocyclus. Furthermore, cave habitats have been virtually unexplored in this genus, and will likely produce new troglophilic species (Valdez-Mondragón 2010, 2020; Huber et al. 2018).

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 (Puillandre et al. 2012; Rannala 2015; Rannala and Yang 2020). However, this method does not consider the rates of intra- and interspecific variation as an initial parameter, causing barcode values to vary among species groups and, in some cases, generating over splitting (Hickerson et al. 2006; Rannala 2015; Rannala and Yang 2020). The hierarchical integration of the ASAP method allows for many possible clusterings of terminals to be tested (Puillandre et al. 2021). However, it can offer different delimitation species hypothesis with similar asap-scores. In the case of Physocyclus spiders here, both methods offered a high congruence between themselves and the morphological evidence.

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 (Rannala and Yang 2020). The bPTP method accommodates the use of large data sets with thousands of species and considers the rates of intra- and interspecific genetic variation. However, it does not take into account the stochastic fluctuations in the coalescent process among the different loci in large multi-locus analyses (Rannala and Yang 2020). The coalescent analyses for species delimitation (GMYC, bPTP) do not require reciprocal monophyly to delimit species (Knowles and Carstens 2007) and can incorporate statistical uncertainty in gene trees. They are based on Maximum Likelihood and Bayesian Inferences and not only on corrected genetic distances.

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.


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