Under an integrative taxonomic approach: the description of a new species of the genus Loxosceles (Araneae, Sicariidae) from Mexico City

Abstract A new species of the spider genus Loxosceles Heineken & Lowe, 1832, Loxosceles tenochtitlan Valdez-Mondragón & Navarro-Rodríguez, sp. nov., is described based on adult male and female specimens from the states of Mexico City, Estado de Mexico and Tlaxcala. Integrative taxonomy including traditional morphology, geometric and lineal morphology, and molecules (DNA barcodes of cytochrome c oxidase subunit 1 (CO1) and internal transcribed spacer 2 (ITS2)), were used as evidence to delimit the new species. Four methods were used for molecular analyses and species delimitation: 1) corrected p-distances under neighbor joining (NJ), 2) automatic barcode gap discovery (ABGD), 3) general mixed yule coalescent model (GMYC), and 4) poisson tree processes (bPTP). All molecular methods, traditional, geometric and lineal morphology were consistent in delimiting and recognizing the new species. Loxosceles tenochtitlansp. nov. is closely related to L. misteca based on molecular data. Although both species are morphologically similar, the average p-distance from CO1 data was 13.8% and 4.2% for ITS2 data. The molecular species delimitation methods recovered well-supported monophyletic clusters for samples of L. tenochtitlansp. nov. from Mexico City + Tlaxcala and for samples of L. misteca from Guerrero. Loxosceles tenochtitlansp. nov. is considered a unique species for three reasons: (1) it can be distinguished by morphological characters (genitalic and somatic); (2) the four different molecular species delimitation methods were congruent to separate both species; and (3) there is variation in leg I length of males between both species, with the males of L. misteca having longer legs than males of L. tenochtitlansp. nov., also morphometrically, the shape of tibiae of the palp between males of both species is different.

Modern taxonomy uses multiple lines of evidence for species recognition, identification, diagnosis and delimitation. Several recently developed molecular delimitation methods have highlighted the extensive inconsistency in classical morphological taxonomy (Ortiz and Francke 2016). Molecular methods have provided a new way to resolve species delimitation problems by using the infra-specific genealogical information in DNA markers which provides objective implementation of modern species concepts (e.g., biological, phylogenetic, genotypic cluster). The appropriate way to species delimitation research is to analyze the data with a wide variety of methods and different lines of evidence to delimit lineages that are consistent across the results, understanding the behavior of the molecular species delimitation methods and contributing in this way to integrative taxonomy (Carstens et al. 2013, Luo et al. 2018. Currently, there are two separate tasks to which DNA barcodes are being applied in modern systematics. The first is distinguishing between species (equivalent to species identification or species diagnosis), and the second is the use of DNA data to discover new species (equivalent to species delimitation and species description) (DeSalle et al. 2005). For some groups of organisms, including some groups of spiders, morphology alone cannot determine species boundaries, and identifying morphologically inseparable cryptic or sibling species requires a new set of taxonomic tools, including the analysis of molecular data (Jarman and Elliott 2000;Witt and Hebert 2000;Proudlove and Wood 2003;Hebert et al. 2003Hebert et al. , 2004Bickford et al. 2007;Hamilton et al. 2011Hamilton et al. , 2014Hamilton et al. , 2016Ortiz and Francke 2016). The spider genus Loxosceles is no exception. Recent studies based on molecular evidence have suggested that the known diversity within the genus could be highly underestimated (Binford et al. 2008;Duncan et al. 2010;Ribera 2014, 2015;Tahami et al. 2017). One important factor leading to the underestimation is widespread intraspecific variation in sexual structures, mainly in the seminal receptacles of females, something noted previously by Brignoli (1968) and Gertsch and Ennik (1983) and recently by Valdez-Mondragón et al. (2018b) in the case of the species from Mexico.
The primary aim of this study is to use an integrative taxonomic approach for the delimitation and description of a new species of Loxosceles from Mexico City. We analyzed DNA barcodes and used traditional morphology, ultra-morphology, geometric and linear morphometrics, biogeography, and ecological niche modeling for species delimitation. This is the first-time multiple lines of evidence have been used in the taxonomy of the genus.

Biological material
The specimens of the new species were collected and deposited in 80% ethanol and labeled with their complete field data. The type specimens and additional examined material are deposited with their collection codes in the Laboratory of Arachnology (LAT-LAX), Laboratorio Regional de Biodiversidad y Cultivo de Tejidos Vegetales (LBCTV), Institute of Biology, Universidad Nacional Autónoma de México (IBUNAM), Tlaxcala City. The male holotype of Loxosceles misteca Gertsch, 1958 was examined and is deposited at the American Museum of Natural History (AMNH). The descriptions and observations of the specimens were done using a Zeiss Discovery V8 stereoscope. A Zeiss Axiocam 506 color camera attached to a Zeiss AXIO Zoom V16 stereoscope was used to photograph the different structures of specimens. The female seminal receptacles and male palps were dissected in ethanol (80%) and cleaned in potassium hydroxide (KOH-10%) for 5 to 10 min. Habitus, seminal receptacles and palps were submerged in 96% gel alcohol and covered with a thin layer of liquid ethanol (80%) to minimize diffraction during photography (Valdez-Mondragón and Francke 2015). For the photomicrographs, the morphological structures were dissected and cleaned with an ultrasonic cleaner at 20-40 kHz; subsequently, they were critical-point dried, and examined at low vacuum in a Hitachi S-2460N Scanning Electron Microscope (SEM). The descriptions follow Valdez-Mondragón et al. (2018b). All measurements in the descriptions are in millimeters (mm). Scale measurements on photomicrographs are in micrometers (μm). The distribution map was made using QGIS v.

DNA extraction, amplification and sequencing
Specimens for DNA extraction were preserved in ethanol (96%) and kept at -20 °C. DNA was isolated from legs, prosoma or complete specimens in the case of im-  (Table 1).

DNA sequence alignment and editing
Sequences were edited with the programs BioEdit v. 7.0.5.3 (Hall 1999) and Geneious v. 10.2.3 (Kearse et al. 2012). Sequences were aligned online using the default gap opening penalty of 1.53 in MAFFT (Multiple sequence alignment based on Fast Fourier Transform) v. 7 (Katoh and Toh 2008) using the following alignment strategy: Auto (FFT-NS-2, FFTNS-i or L-INS-i; depending on data size). These aligned matrices were subsequently used in analyses.

Molecular analyses, species delimitation and haplotypes networks
For molecular species delimitation four methods were used for analyzing the concatenated CO1+ITS2 matrix (1091 characters): 1) p-distances under neighbor joining (NJ) using MEGA v. 7.0, 2) automatic barcode gap discovery (ABGD) online version (Puillandre et al. 2012) using both uncorrected and K2P distance matrices. 3) general mixed yule coalescent model (GMYC) (Pons et al. 2006) using GMYC web server (https://species.h-its.org/gmyc/), and 4) Bayesian Poisson tree process (bPTP) (Zhang et al. 2013, Kapli et al. 2017) using web server (https://species.h-its.org/ptp/). The models of sequence evolution were selected using the Akaike information criterion (AIC) in jModelTest v.  (Ronquist and Huelsenbeck 2003) were implemented, and the analysis recognizes monophyletic cluster by searching differential intra-and inter-specific branching patterns (Ortiz and Francke 2016). The ML analysis was calculated with the parameters for CO1 and ITS2: Number of replicates = 1000, Bootstrap support values = 1000 (significant values ≥ 50%), Models of sequence evolution selected using jModelTest = GTR, Rates among sites = G+I, No. of discrete Gamma Categories = 6, Gaps Data Treatment = Complete deletion, Select Codon Position = 1 st +2 nd +3 rd +Noncoding Sites, ML Heuristic Method = Subtree-Pruning-Regrafting -Extensive (SPR level 5), Initial Tree for ML = Make initial tree automatically (Default -NJ/BioNJ). The BI analyses were run with four parallel Markov chains with the following parameters: MCMC (Markov Chain Monte Carlo) generations = 20000000, sampling frequency = 1000, print frequency = 1000, number of runs = 2, number of chains = 4, MCMC burnin = 2500, sumt burnin = 2500, sump burnin = 2500, Models of sequence evolution selected using jModelTest = GTR, Rates among sites = G+I, Select Codon Position = 1 st , 2 nd , and 3 rd . TRACER v. 1.6 Drummond 2003-2009) was used to analyze the parameters and the effective sample size (ESS) of the MCMC to ensure the runs converged. FigTree v. 1.4.3 was used to visualize the topology of the tree with the posterior probability values (PP) at nodes. The ABGD species delimitation method uses recursive partitioning with a range of prior intraspecific divergence and relative gap widths, estimating the threshold between intra-and interspecific genetic variation, generating species-level groupings (Ortiz and Francke 2016). ABGD analyses were conducted using both uncorrected and K2P distance matrices with default options: Pmin = 0.001, Pmax = 0.1, Steps = 10, Relative gap width (X) = 1, Nb bins = 20. The GMYC species delimitation method applies single (Pons et al. 2006) or multiple (Monaghan et al. 2009) time thresholds to delimit species in a Maximum Likelihood context, using ultrametric trees (Ortiz and Francke 2016). Phylogenetic analyses were run in BEAST v. 2.6.0 ) using a coalescent (constant population) tree prior. Independent lognormal relaxed clock was applied to each partition, for analyses of 20×10 6 generations were run. Convergence was assessed with TRACER v. 1.6 (Rambaut and Drummond 2014). TREEANNOTATOR v. 2.6.0 (BEAST package) was used to build maximum clade credibility trees, after discarding the first 25% of generations by burn-in. Following gene tree inference, GMYC was implemented in the web interface for single and multiple threshold GMYC (https://species.h-its.org/gmyc/) the backend of this web server runs the original R implementation of the GMYC model authored by Fujisawa and Barraclough (2013). A single threshold was used for the concatenated matrix. The PTP species delimitation method (Zhang et al. 2013) is similar to GMYC, but uses substitution calibrated (not ultrametric) trees to avoid the potential flaws in constructing time calibrated phylogenies (Zhang et al. 2013, Ortiz andFrancke 2016). We employed the Bayesian variant of the method (bPTP) on the online version (https://species.h-its.org/ ptp/). It was run on the Bayesian gene trees with default options: rooted tree, MCMC generations = 100000, Thinning = 100, Burnin = 0.1, Seed = 123. Haplotypes network for CO1 was constructed to visualize the mutations among haplotypes of species using the TCS algorithm (Clement et al. 2000) in PopArt v. 1.7 (Leigh and Bryant 2015).

Geometric and linear morphometry and sexual dimorphism
For the morphometric studies, tibiae of adult males in retrolateral views of L. tenochtitlan sp. nov. (N = 12) and L. misteca (N = 9) were analyzed using Make Fan 8 v. 1.0 software (Sheets and Zelditch 2014), performing brand and semi-brand protocols. Using TPsUtil v. 1.76 software (Rohlf 2015) the file was formatted (.tps) to perform the digitalization of the landmarks and semi-landmarks of the contours in the tpsDig v. 2.31 software (Rohlf 2015). In the CordGen8 v. 1.0 software (Sheets and Zelditch 2014), a "Procrustes" alignment was made for the brands and with the Semi Land option included in the CordGen8 v. 1.0 software (Sheets and Zelditch 2014). Posteriorly, the alignment of the semi-landmarks was carried out. To analyze the formation of groups in relation to the tibia shape, an analysis of canonical variables (CVA) was performed with the CVA Gen 8 v. 1.0 software. To analyze sexual dimorphism and variation in the new species, a T-test was performed to evaluate if the females and males have significant statistical differences in: 1) leg I length, 2) carapace length, and 3) carapace width. Also, leg I length was used to test if differences exist between the new species and Loxosceles misteca Gertsch, 1958; species that appears to be closely related to L. tenochtitlan sp. nov. morphologically. Forty specimens of Loxosceles tenochtitlan sp. nov. (24 females and 16 males) and 22 specimens of L. misteca (11 females and 11 males) were measured ( Table 5). The statistical analysis was carried out and graphics were made with R studio v.1.1.463 software.

Ecological niche modeling (ENM)
For georeferencing and corroboration of localities, two programs were used:  Etymology. The species is a noun in apposition dedicated to Tenochtitlán (Nahuatl language) city, a large Mexica city-state in what is now Mexico City where the type locality is located. Tenochtitlán was built on an island in what was then Lake Texcoco in the Valley of Mexico, being the capital of the expanding Aztec Empire in the 15 th century.
Chelicerae: Wider than in the male. Slightly dark reddish brown, with stridulatory lines laterally. Fangs dark reddish orange.
Variation. MALES. Mexico City: Males from Coyoacán are light brown, legs slightly darker than the carapace, males from Tlalpan are light brown, legs slightly darker than the carapace. Tlaxcala: Males from Santiago Tlacochcalco Municipality of Tepeyanco are light brown, legs slightly darker than the carapace and light brown, legs slightly darker than the carapace. Males from Huamantla are dark brown, legs slightly darker than the carapace. Mexico City: Coyoacán (N = 3): Tibia I 5.9-6.5 (x = 6.1); carapace length (CL) 2.6-3.1 (x = 2.9); carapace width (CW) 2.4-2.7 (x = 2.5). Tlalpan (N = 3): Tibia I 6.0-7.6 (x = 5.8); carapace length (CL) 2.2-3.2 (x = 2.8); carapace width (CW) 2.5-2.7 (x = 2.6). Tlaxcala: Santiago Tlacochcalco Municipality of Tepeyanco (N = 7): Tibia I 3.8-6.6 (x = 5.0); carapace length (CL) 2.5-4.2 (x = 3.1); carapace width (CW) 2.2-3.2 (x = 2.7). Huamantla (N = 3): Tibia I 5.0-6.5 (x = 5.8); carapace length (CL) 3.2-3.3 (x = 3.2); carapace width (CW) 2.7-2.9 (x = 2.8). FE-MALES. Mexico City: Females from Coyoacán are dark brown, legs the same color as the carapace. Females from Tlalpan are dark brown, legs the same color as the carapace. Estado de Mexico: Female from San Mateo Ixtacalco, Municipality Cuautitlán Izcalli is dark brown, legs slightly darker than the carapace. Tlaxcala: Females from Santiago Tlacochcalco, Municipality of Tepeyanco are light brown, legs slightly darker than the carapace. Females from Huamantla are dark brown, legs the same color as the carapace and light brown, legs the same color as the carapace and light brown. A female from the Trinidad Tenexyecac, Municipality of Ixtacuixtla is light brown, legs the same color There is little variation in the shape of the male palps, even those of specimens from different populations (Figs 48-55). The shape of the embolus varies little; the specimens from Tlaxcala have the embolus slightly more curved than the specimens from Mexico City (Figs 52-55). Also, the specimens from Tlaxcala have a slightly thinner palpal tibia than specimens from Mexico City (Figs 48-51). The seminal receptacles of females are asymmetrical, and although all they are all S-shaped with rounded or oval lobes apically, they are highly variable (Figs 56-61). The small accessory lobes of the receptacles on each side vary in width among specimens (Figs  56-61). The internal part of the bases of the seminal receptacles is round, wide and slightly sclerotized in all specimens, with the distance between them equal to their height (Figs 56-61).
Natural history. The specimens of L. tenochtitlan sp. nov. (Figs 1-9, 11-17) were collected in urban areas in houses and buildings (Figs 10, 18). The specimens from Mexico City were collected in houses, on doors, storage boxes, drawers, under chairs and tables (Figs 7-13). The specimens from Tlaxcala were collected in houses behind doors, behind decorative items on the wall, under beds, under chairs and tables, among wooden boards for construction, under wardrobes, and between ornamental artificial plants, and under stored items (Figs 14-18). Even the first record from Tlaxcala (Trinidad Tenexyecac) was a female specimen collected among construction debris close to a football/soccer field. Some specimens from Huamantla, Tlaxcala were collected inside an abandoned house, mainly under stored items, behind doors and under wardrobes; other specimens were collected outside of a house in spaces and cracks in a wall (Figs 14-17).

Molecular analyses and species delimitation
The analyzed matrices include 52 individuals of 11 species of Loxosceles, 39 individuals for the CO1 data set and 34 individuals for ITS2 (Table 1, Figs 70, 71). Specimens used in this study, GenBank accession numbers and localities of the specimens are listed in Table 1. Analyses of the concatenated matrix indicated that the four different methods used to delimit species with molecular data (CO1+ITS2) were consistent with morphology, recovering ten species (Fig. 72). Only the ABGD species delimi-  tation method under recursive partitions (RP) recovered 12 species (Fig. 72). Even, Loxosceles malintzi, the last species described from Mexico by Valdez-Mondragón et al. (2018) by only morphological characters, was recovered with molecular data under the different species delimitation methods (Fig. 70-72). The average genetic p-distance among analyzed species was of 17% for CO1 and 7.6% for ITS2 (Figs 70, 71). Corrected p-distances from the CO1 data recovered ten species of Loxosceles (Fig. 70), whereas nine species were recovered with ITS2 (Fig. 71) both with high statistical support. Based on molecular evidence, L. tenochtitlan sp. nov. is closely related to L. misteca (Figs 70-72), the average p-distances between both species for CO1 was 13.8% (Table 3) and 4.2% for ITS2 (Table 4). The haplotype network analysis with CO1 data is concordant with the results of the different species delimitation analyses (Fig. 73). There were more than ten mutations between haplotypes of CO1 for all the species (Fig. 73). Regarding L. tenochtitlan sp. nov. and L. misteca, the haplotype network was concordant with the delimitation of both species, showed 49 mutations between haplotypes under CO1 (Fig. 73).

Geometric and linear morphometry and sexual dimorphism
The analysis of canonical variables CVA shows a significant difference (χ 2 = 10.2555, df = 2, p = 0.00593003, λ = 0.5988) between both species, which indicates the formation of two groups according to the tibiae shape of the palps of the males (Fig.  74). The differences on the tibiae can be observed in the deformation rack, where a deformation is shown mainly in the ventral-basal and the dorsal-apical parts (Fig.  75). In this way, the tibiae of L. tenochtitlan sp. nov. is thinner in ventral-basal part (Fig. 77), whereas in L. misteca the ventral-basal part is wider and slightly less curved in the dorsal-apical part (Fig. 76). To analyze sexual dimorphism and variation in the new species, a T-test showed that between the males and females of L. tenochtitlan sp. nov., there are no statistically significant differences in leg I length (t = -1.3106, p = 0.1981, df = 37, α = 0.05), carapace length (t = 1.498, p = 0.142, df = 38, α = 0.05), and carapace width (t = 0.6955, p = 0.4912, df = 36, α = 0.05) (Figs 78-80). Therefore, there is no secondary sexual dimorphism between males and females of the new species (Table 5, Figs 78-81). However, a T-test showed that there is secondary sexual dimorphism between males and females of L. misteca in leg I length (t = 3.1086, p = 0.0038, df = 21, α = 0.05) (Fig. 81). A T-test indicated that there table 3. Genetic p-distance matrix from the CO1 data between Loxosceles tenochtitlan sp. nov. and Loxosceles misteca. Average p-distance = 13.8%.  are statistically significant differences between the new species and L. misteca in leg I length of males (t = 3.6174, p = 0.00331, df = 13, α = 0.05) with the longest legs occurring in L. misteca (Table 5, Fig. 81). There was no statistical support for significant differences in leg I length between females of each species (t = 0.274, p = 0.787, df = 17, α = 0.05) ( Table 5, Fig. 81).

Ecological niche modeling (ENM)
To analyze the potential distribution of L. tenochtitlan sp. nov., ENM was performed for the new species, with a total of 34 records from Mexico City, Estado de Mexico and Tlaxcala (Figs 82-84). The highest contribution to the model came from Vegetation Type (CON01) with 42% and Mean Temperature of Wettest Quarter (BIO10) with 28.5% (Table 6). Additionally, the Area Under the Curve (AUC) demonstrated good performance AUC= 0.993. Following the biogeographic scheme for Mexico proposed by Morrone (2004Morrone ( , 2005, the highest probability of the presence of L. tenochtitlan sp. nov. (0.75-1.0) was markedly toward the biogeographical province of the Transmexican Volcanic Belt (TVB), with a potential distribution including Mexico City, north of Estado de Mexico, west of Puebla, most of Tlaxcala, and a small portion of Hidalgo and Queretaro (Fig. 84).

Discussion
The first record of Loxosceles from Mexico City was by Gertsch (1958), who reported a female of Loxosceles nahuana Gertsch, 1958, a native species from Zimapán, Hidalgo; however, this record is a misidentification because posteriorly Gertsch and Ennik (1983) did not consider this record in their taxonomic revision of Loxosceles from North America. Hoffmann (1976) includes the same record of L. nahuana in her preliminary list of Mexican spiders, but she did not mention other species. Francke et al. (2009), Durán-Barrón and Pérez-Ortíz (2016) and Durán-Barrón and Ayala-Islas (2007) reported two species from Mexico City, L. misteca and one unidentified species of Loxosceles, comprising a single female, two males and two immature specimens. Surprisingly, the authors never identified it to species level. Unfortunately, we did not have access to those collections; therefore, we do not know whether there are two species or only one from Mexico City. In this way, L. nahuana is a valid and different species as the species delimitation methods and different topologies showed , even this species is not closely related with the new species described herein neither with L. misteca (Figs 70-72). In the present work, all the specimens reviewed belong to Loxosceles tenochtitlan sp. nov., therefore we can assume that the previous records of L. misteca belong to the new species described herein, and that L. misteca is not found in Mexico City or the rest of the states where the new species has been recorded (Estado de Mexico and Tlaxcala). Recently, Valdez-Mondragón et al. (2018a, b) mentioned that L. misteca from Mexico City and Tlaxcala was an introduced species, however this was an incorrect interpretation. Loxosceles misteca is a species from Guerrero and Morelos, whereas the records of L. misteca from Mexico City and Tlaxcala belong to L. tenochtitlan sp. nov., a native species of the region (Fig.  82-84 and the Middle East (Gertsch 1958(Gertsch , 1973Gertsch and Ennik 1983;Nentwig et al. 2017;Tahami et al. 2017;Valdez-Mondragón et al. 2018a, b;WSC 2019).
As was mentioned previously, recent taxonomic studies based on molecular analyses using mitochondrial markers have suggested that the known diversity within the genus Loxosceles could be greatly underestimated (Binford et al. 2008;Duncan et al. 2010;Ribera 2014, 2015;Tahami et al. 2017). Additionally, it has been decades since a revision of the North American species has been conducted, and given the intraspecific variation in sexual structures, primarily in the seminal receptacles in the females (Brignoli 1968, Gertsch andEnnik 1983) this can be very difficult. Despite this, the male palps remain a good character for species identification because there is little morphological variation in comparison with seminal receptacles as was showed by Valdez-Mondragón et al. (2018b) recently in the description of Loxosceles malintzi.
Although DNA barcodes are being applied in modern systematics as a useful tool to resolve species delimitation problems, modern taxonomy includes many different sources of evidence, such as traditional morphology, ecology, reproduction, and biogeography. Traditional morphology alone cannot determine species boundaries in some cases, and the genus Loxosceles is no exception. Identifying morphologically inseparable cryptic or sibling species requires a new set of taxonomic tools, including DNA and additional sources of evidence (integrative taxonomy) (Jarman and Elliott 2000;Witt and Hebert 2000;DeSalle et al. 2005;Hebert et al. 2003Hebert et al. , 2004Bickford et al. 2007;Hamilton et al. 2011Hamilton et al. , 2014Hamilton et al. , 2016Ortiz and Francke 2016). The researchers should apply different range of species delimitation method at the same time to their data and place their truth in delimitation that are congruent across methods (Carstens et al. 2013). Using several species delimitation methods, incongruence across the different results is evidence of either a difference in the power to detect cryptic lineages across one or more of the approaches used to delimit species and could indicate that assump- tions of one or more of the methods have been violated, in this cases the assumptions for species delimitations should be conservative (Carstens et al. 2013). In this work, the four different molecular species delimitation methods were congruent and consistent to separate L. tenochtitlan sp. nov and L. misteca (Fig. 72).
Although morphologically L. tenochtitlan sp. nov is quite similar to L. misteca in the seminal receptacles of the females and the male palps, there are some subtle morphological differences that allow diagnosis of the new species as was mentioned in the description section. Multiple lines of robust evidence are able to clearly separate it as a new species. These methods are genetic differences, geometric and linear morphometry and different biogeographical distribution patterns. Strictly, cryptic species are those that cannot be differentiated based on their morphology or external appearance and are reproductively isolated. The present genetic divergence indicates the two species are independent lineages (Bickford et al. 2007;Hebert et al. 2004;Struck and Cerca 2019).
The species separation based on corrected genetic distances indicates that CO1 performed better for species delimitation than ITS2 (Figs 70, 71). This result confirms the utility of DNA barcoding as a fast and reliable tool for the identification and species delimitation of the Loxosceles from the reclusa group of North America. Similar results have also been found in other molecular studies of Loxosceles. Ribera (2014, 2015) found genetic distances between species from the Canary Islands to be > 12% using COI, whereas Tahami et al. (2017) found genetic distances between species from the Middle East ranged for CO1 from 17.5 to 20.6%. Additionally, CO1 haplotypes network also corroborated the distinctiveness of the different species (Fig. 73). The approaches for analyzing DNA barcode data, using p-distances for CO1 and ITS2 and tree-based delimitation with ML and BI (CO1+ITS2), recovered a monophyletic cluster with high support values for the samples of L. tenochtitlan sp. nov from Mexico City + Tlaxcala (Figs 70-72), as well as another monophyletic cluster of the samples of L. misteca from Guerrero, where some samples were collected near the type locality of the species as well as localities previously reported by Gertsch (1958) and Gertsch and Ennik (1983) (Figs 41, 62-69). Sexual characters in spiders are robust and important morphological characters that are still used to separate species and to provide a diagnosis. This means that genitalia evolve, on average, more rapidly than non-genital morphological traits (Huber, 2003;Huber and Dimitrov 2014). Also, the somatic characters are useful as additional evidence to separate species in some groups of spiders; coloration, color pattern, body proportions, and even extreme size differences are useful traits for species separation (Huber et al. 2005;Huber and Dimitrov 2014). As additional evidence for the separation between L. tenochtitlan sp. nov. and L. misteca, geometric and linear morphometric variation was statistically significant for tibia shape of the palp of males and leg I length between males of both species, where the males of L. misteca have longer legs than the males of the new species (Table 5, Fig. 81). We do not know whether these differences in leg lengths between males of both species correspond to the microhabitat of each species or why this morphological difference only occurs in males. Loxosceles tenochtitlan sp. nov. only has been collected in urban areas (Figs 7-18), whereas L. misteca are common in caves and have been collected from caves in Guerrero and Estado de Mexico. Some studies have demonstrated how microhabitat plays an important role in driving spider diversification. Eberle et al. (2018) analyzed diversification in pholcids based on the framework of the largest molecular phylogeny of the spider family Pholcidae to date, analyzed their diversification and found that diversity may be caused by microhabitat changes. Planas and Ribera (2014) and Souza and Ferreira (2018) mentioned that Loxosceles are generally considered troglophiles because of their abundance in caves. In other animals, long legs are considered a hallmark of troglomorphism. Further research of North American species of Loxosceles is required to address a correlation between leg length and microhabitat.
ENM is a powerful approach to understand how abiotic factors (e.g., temperature, precipitation, and seasonality) impact the geographic limits of the species (Graham et al. 2004a;Wiens and Graham 2005). The integration of genetic and ecological approaches in the study of mechanisms driving geographic distributions of organisms is becoming more common (Hugall et al. 2002;Johnson and Cicero 2002;Graham et al. 2004;Lapointe and Rissler 2005;Rissler and Apodaca 2007;Raxworthy et al. 2007). In the ENM, following the biogeographical provinces proposed by Morrone   (2004,2005), vegetation type plays an important role in the ecological niche of the species (Fig. 84). ENM showed that the highest probability of presence (0.75-1.0) for L. tenochtitlan sp. nov. is strongly limited towards the Transmexican Volcanic Belt (TVB) (Fig. 84), characterized by high mountains and a temperate climate, with pine, oak or oak-pine forest. Although ENM calculated a potential distribution to the south of states of Puebla, south and north of the Estado de Mexico, and small regions of the states of Michoacan, Guanajuato and Queretaro, this can be explained as an over-prediction, and other species of Loxosceles might occur there (Fig. 84) (Valdez-Mondragón et al. 2018: figs 75-77). Although L. tenochtitlan sp. nov. is distributed widely in urban areas of Mexico City, Estado de Mexico and Tlaxcala, this species can be considered a native of this region and the urbanization process has not affected its establishment in such areas. However, the species has never been collected in natural areas in the state (Valdez-Mondragón et al. 2018a, b). In 2017, four collectors collected around 40 specimens of L. tenochtitlan sp. nov. in two hours from a house in the state of Tlaxcala, Mexico (Valdez-Mondragón et al. 2018a, b). As has been demonstrated for other species of the genus as Loxosceles reclusa from the United States, the partial synanthropy of some species of the brown recluse spiders is not the dominant influence on distributional patterns (Saupe et al. 2011). Although the species may be able to expand beyond their distribution with the aid of the anthropogenic activities, the species analyzed  Morrone (2004Morrone ( , 2005. herein does not have widespread distribution due to historical or biological barriers or their limited dispersion potential, where the vegetation type plays an important role to delimitation of their distribution (Table 6, Fig. 84).
Despite the similarity between L. tenochtitlan sp. nov. and L. misteca, we consider them different species for three main reasons: (1) they can be distinguished by morphological characters (genitalic and somatic); and the new species can be diagnosed morphologically; (2) molecular data from multiple genes analyzed with multiple methods consistently separate them (congruence among methods); and (3) statistically significant geometric and linear morphometric variation in tibias shape of the palp of the male and leg I length of males respectively.

Acknowledgments
The first author thanks the program "Cátedras CONACyT", Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico; for scientific support for the project No. 59: "Laboratorio Regional de Biodiversidad y Cultivo de Tejidos Vegetales (LBCTV) del Institute of Biology, Universidad Nacional Autónoma de México (IBUNAM), sede Tlaxcala". The first author also thanks SEP-CONACyT for the financial support of the project of Basic Science (Ciencia Básica) 2016, No. 282834: Arañas de Importancia Médica: Taxonomía integrativa basada en evidencia molecular y morfológica para la delimitación de las especies mexicanas de arañas violinistas del género Loxosceles Heineken & Lowe (Araneae, Sicariidae)-Etapa 1. The second and third authors thank Posgrado en Ciencias Biológicas of Centro Tlaxcala de Biología de la Conducta (CTBC), Universidad Autónoma de Tlaxcala (UATx) for educational support, and CONACyT for scholarship support during the Master's degree of Biological Science and to the Secretaría de Fomento Agropecuario del Estado de Tlaxcala (SEFOA) and the Government of the state of Tlaxcala for the facilities and support to conduct this research. We thank Dr. Oscar F. Francke, Curator of the Colección Nacional de Arácnidos (CNAN), IBUNAM, Mexico City, for the loan of the collection of Loxosceles specimens of the CNAN, Dr. Lazaro Guevara López for his suggestions and comments that improved the manuscript. We also thank the students of the CNAN and Laboratory of Arachnology (LATLAX), IBUNAM, Tlaxcala, for their donation of specimens, help in the field, and processing of the material in the laboratory, M. Sc. Berenit Mendoza Gárfias for the SEM photographs, Jared Lacayo-Ramírez for his helping photograph live specimens. A special thanks to the Biól. and friend Martin Sánchez Vílchis and his family for the disposition and the donation of the specimens collected in their house, which were selected as types of the species (¡gracias carnal!). We thank the family Juárez-Sánchez and José A. Castilla-Vázquez from Tlaxcala for allowing us to collect in their houses, Ignacio Beltrán for his help in the Grutas de Cacahuamilpa, Guerrero, Dr. Sarah Crews for the English language review of the manuscript, José C. Valerdi-Tlachi ( † may he rest in peace). The specimens were collected under Scientific Collector Permit FAUT-0309 from the Secretaría de Medio Ambiente y Recursos Naturales (SEMARNAT) to AVM.