Chromosomal Study on Selected Small Araneomorph Spiders from Brazil, Including the First Records in Palpimanidae and Theridiosomatidae (Araneae, Araneomorphae)

© 2023 Academia Sinica, Taiwan Open Access Chromosomal Study on Selected Small Araneomorph Spiders from Brazil, Including the First Records in Palpimanidae and Theridiosomatidae (Araneae, Araneomorphae) Débora Duarte Dutra1, Antônio Domingos Brescovit2, and Douglas Araujo1,* 1Laboratório de Citotaxonomia e Evolução Cromossômica Animal, Programa de Pós-Graduação em Biologia Animal, Instituto de Biociências, Universidade Federal de Mato Grosso do Sul, Avenida Costa e Silva, s/n, Campo Grande, Mato Grosso do Sul, Brazil, Caixa Postal 549, CEP 79070-900. Correspondence: E-mail: [email protected] (Araujo) E-mail: [email protected] (Dutra) 2Laboratório de Coleções Zoológicas, Instituto Butantan, Av. Vital Brasil, 1500, São Paulo, São Paulo, Brazil, CEP 05503-900. E-mail: [email protected].br (Brescovit) Received 24 November 2022 / Accepted 19 June 2023 / Published 11 August 2023 Communicated by John Wang Among the 50,474 spider species, only 849 have chromosomal data available in the literature. Fifty spider families remain unknown from a cytogenetic perspective. The aim of this study was to analyze chromosomally selected araneomorph spiders from Brazil, to contribute to the cytotaxonomy and chromosome evolution of this group. The karyotypes of 12 species belonging to families Corinnidae, Linyphiidae, Oonopidae, Palpimanidae, Theridiidae, Theridiosomatidae, Trachelidae, and Zodariidae were analyzed, including the first chromosomal record for the first two families. Specimens (with the abdomen content partially exposed by perforation) were subjected to colchicine, hypotonization, and fixation. In most cases, the total content of the abdomen was used to prepare slides by spreading of cell suspension and subsequent Giemsa staining. The results were as follows: Cinetomorpha simplex Simon, 1892 (Oonopidae) 2n♂ = 9, X0; Neotrops sp. and Neoxyphinus termitophilus (Oonopidae), 2n♂ = 7, X0; Otiothops birabeni (Palpimanidae); Agyneta sp. (Linyphiidae), 2n♂ = 24, X1X20; Coleosoma floridanum, Thymoites sp.1 and Thymoites sp.2 (Theridiidae), 2n♂ = 22, X1X20; Naatlo sp. (Theridiosomatidae), 2n♂ = 30, X1X20; Orthobula sp. (Trachelidae) 2n♂ = 21, X0; Falconina sp. (Corinnidae), 2n♂ = 28, X1X20; Epicratinus sp. (Zodariidae) 2n♂ = 42, X1X20. The chromosomal morphology was determined for all the samples except for Oonopidae. Most species exhibited telocentric chromosomes, with the exception of Palpimanidae and Theridiosomatidae. The main findings: 1) support an hypothesis on ancestral karyotype of Zodariidae and Oonopidae; 2) reveal a relatively high chromosome number in Palpimanidae that supports an idea on relatively high ancestral chromosome number (2n♂ = 42) of entelegyne spiders (Palpimanoidea is sister group of entelegyne spiders); 3) show that the karyotype found in Theridiosomatidae is exceptional within the Araneoidea. Key words: Mitosis, Meiosis, Diploid number, Sex chromosome system Citation: Dutra DD, Brescovit AD, Araujo D. 2023. Chromosomal study on selected small araneomorph spiders from Brazil, including the first records in Palpimanidae and Theridiosomatidae (Araneae, Araneomorphae). Zool Stud 62:42. doi:10.6620/ZS.2023.62-42. BACKGROUND Although 50,474 spider species placed in 132 families have been described (World Spider Catalog 2022), only 849 species belonging to 80 families have chromosomal data available (Araujo et al. 2022). This scarcity of cytogenetic data makes difficult analysis of chromosomal evolution in most groups of this order, Zoological Studies 62:42 (2023) doi:10.6620/ZS.2023.62-42 1 © 2023 Academia Sinica, Taiwan including families of spiders with small body sizes, which are often neglected because of the difficulties in collecting, dissecting, and obtaining chromosome plates. Chromosomal studies of leaf litter spiders are also rare due to specific methodology, which is required for their collection. Many spiders from this environment are tiny, difficult to determine with the naked eye (Indicatti and Villarreal 2016). In the present study, we analyzed species of spiders that are less than one centimeter in body length. Twelve species were evaluated, four of which belonged to families with exclusively minute species (Oonopidae and Theridiosomatidae) and eight belonged to families that also included larger species (Corinnidae, Linyphiidae, Palpimanidae, Theridiidae, Trachelidae, and Zodariidae). Most of these families are not closely related (Wheeler et al. 2017). Cytogenetic studies on most of these families are rare or do not exist. We recorded the first karyotype data for Palpimanidae and Theridiosomatidae and documented the first karyotype data for all other studied genera to fill gaps and expand the data for analysis of cytotaxonomy and karyotype evolution in araneomorph spiders. MATERIALS AND METHODS The specimens of Naatlo sp. (Theridiosomatidae) were collected over night. The remaining specimens were obtained during the day or night through litter sieving, according to Coddington et al. (1991), with some modifications. After the exclusion of leaves and larger particles with the aid of a metallic sieve with a mesh of 1 cm, the material was slowly spread in a plastic tray, and individuals were searched by tossing the sieved litter using tweezers under a strong light source, allowing the acquisition of living specimens, which are necessary for cytogenetic studies. Samples were collected exclusively in the State of Mato Grosso do Sul, Brazil, between March 2019 and September 2021. After dissection of the total abdomen content or the gonads, the specimens were identified and deposited at the Laboratory of Zoological Collections, Instituto Butantan, São Paulo, Brazil. The collection permit was provided by the Instituto Chico Mendes de Conservação da Biodiversidade, ICMBio, Ministério do Meio Ambiente, MMA (number 15382-3). We dissected 218 specimens of spiders. Thirtyeight showed dividing cells and were used in this study. We analyzed 12 species, namely Otiothops birabeni Mello-Leitão, 1945 (Palpimanidae), Naatlo sp. (Theridiosomatidae), Agyneta sp. (Linyphiidae), Coleosoma floridanum Banks, 1900, Thymoites sp.1 and Thymoites sp.2 (Theridiidae), Falconina sp. (Corinnidae), Orthobula sp. (Trachelidae), Epicratinus sp. (Zodariidae), and three Oonopidae species: Cinetomorpha simplex Simon, 1892, Neotrops sp., and Neoxyphinus termitophilus (Bristowe, 1938). Table 1 provides the details of the analyzed individuals. Chromosomes were prepared using the cell suspension technique and 3% Giemsa staining, according to Araujo et al. (2008), with some modifications. Due to the small size of the spiders, the whole specimens were transferred to the treatment after the perforation of the abdomen to allow the action of the reagents. The whole content of the abdomen was used to prepare slides, with the exception of Falconina sp. and Epicratinus sp. whose gonads were dissected due to their relatively large size. The suitable plates were photographed using an AxioImager D2 (Zeiss) microscope, at 1000× magnification. If possible, the chromosomal morphology was determined using the plugin LEVAN (Sakamoto and Zacaro 2009) from ImageJ software (Rasband 1997–2021), considering the criteria proposed by Levan et al. (1964) and Green and Sessions (1991). To calculate the relative length of the X chromosome in Orthobula sp., chromosomes of two oogonial and two spermatogonial metaphases were measured using ImageJ software (Rasband 1997–2021). RESULTS Synspermiata, Dysderoidea Oonopidae Mitotic metaphases of Cinetomorpha simplex consisted of 9(♂) or 10(♀) chromosomes, respectively (Fig. 1A–B). In diplotene and metaphase I, spermatocytes I showed four autosomal bivalents and one univalent that corresponded to the X chromosome (Fig. 1C). Therefore, the karyotype of this species is 2n♂ = 9 (8+X0) and 2n♀ = 10 (8+XX). The spermatogonial and oogonial metaphases of Neotrops sp. contained 7 (♂) or 8 (♀) chromosomes, respectively (Fig. 1D–E). In Neoxyphinus termitophilus, oogonial metaphases contained 8 chromosomes (Fig. 1F), and diplotene and metaphase I of spermatocytes I had three autosomal bivalents and one sex chromosome univalent (Fig. 1G). Based on these data, we determined the karyotype of these two species as 2n♂ = 7 (6+X0) and 2n♀ = 8 (6+XX), respectively. We were not able to determine the chromosomal morphology of oonopids which is described as holocentric (Král et al. 2019), because: 1) the few mitotic metaphases found did not allow us to determine page 2 of 9Zoological Studies 62:42 (2023) © 2023 Academia Sinica, Taiwan Table 1. Collection site, numbers of specimens (males and females), number of analyzed cells, diploid number, and sex chromosome system (SCS) in species analyzed Family/Higher classification Species Collection site Specimens Analyzed cells Diploid number ♂SCS ♂ Oonopidae (Dysderoidea) Cinetomorpha simplex 1 2♂/1♀9 9 X0 Neotrops sp. 3 2♂/1♀9 7 X0 Neoxyphinus termitophilus 1 1♂/1♀12 7 X0 Palpimanidae (Palpimanoidea) Otiothops birabeni 1 1♀3 36 (♀) - Linyphiidae (Entelegynae, Araneoidea) Agyneta sp. 1 2♂/1♀15 24 X1X20 Theridiidae (Entelegynae, Araneoidea) Coleosoma floridanum 3 3♂/3♀17 22 X1X20 Thymoites sp.1 1 3♂/2♀9 22 X1X20 3 Thymoites sp.2 1 1♂/1♀6 22 X1X20 Theridiosomatidae (Entelegynae, Araneoidea) Naatlo sp. 2 3♂22 30 X1X20 Trachelidae (Entelegynae, Clade RTA, Dionycha A) Orthobula sp. 1 3♂/2♀8 21 X0 2 3 Corinnidae (Entelegynae, Clade RTA, Dionycha B) Falconina sp. 3 2♂74 28 X1X20 Zodariidae (Entelegynae, Clade RTA, Zodarioidea) Epicratinus sp. 3 2♂/1♀36 42 X1X20 Collection sites: 1 - Fazenda Palmeiras II, city of Coxim, State of Mato Grosso do Sul, Brazil (18°21'45"S, 54°36'56"W) – Three field trips: 04/24/2020, 10/05//2020 and 03/09/2021. 2 - Morro do Paxixi, city of Aquidauana, State of Mato Grosso do Sul, Brazil (20°27'00"S, 55°37'18.2"W) - Two field trips: 03/19/2019 and 05/05/2020. 3 - Reserva Particular do Patrimônio Natural - RPPN/UFMS, city of Campo Grande, State of Mato Grosso do Sul, Brazil (20°30'36"S, 54°36'54"W) - Six field trips: 07/22/2020, 11/03/2020, 02/22/2021, 05/28/2021, 07/27/2021 and 09/13/2021. Fig. 1. Chromosomes of Oonopidae (Dysderoidea). Cinetomorpha simplex (A–C), Neotrops sp. (D–E) and Neoxyphinus termitophilus (F–G). A. Mitotic spermatogonial metaphase, 2n♂ = 9. B. Mitotic oogonial metaphase, 2n♀ = 10. C. Spermatocyte I, metaphase I showing four autosomal bivalents and a sex chromosome univalent (4II+X). D. Mitotic spermatogonial metaphase, 2n♂ = 7. E–F. Mitotic oogonial metaphase, 2n♀ = 8. G. Spermatocyte I, diplotene consisting of three autosomal bivalents and a sex chromosome univalent (3II+X). Scale bars = 5 µm. page 3 of 9Zoological Studies 62:42 (2023) © 2023 Academia Sinica, Taiwan the morphology, 2) metaphase II, which could help to determine chromosome morphology, was not found, and 3) our analyzes included colchicine, which reduced the number of cells showing segregation behavior during mitotic anaphase. Palpimanidae (Palpimanoidea) Oogonial metaphases of Otiothops birabeni consist of 36 chromosomes. The morphology of particular chromosome pairs was not determined, but the karyotype includes metacentric, submetacentric, subtelocentric, and telocentric elements (Fig. 2). It was impossible to determine the sex chromosome system (SCS) because males were not found. Entelegynae, Araneoidea Linyphiidae Analysis of the spermatogonial and oogonial metaphases of Agyneta sp. revealed 2n♂ = 24 and 2n♀ = 26, respectively. Chromosomes were exclusively telocentric (Fig. 3A–B). Spermatocytes I showed 11 autosomal bivalents and two sexual univalents (X1X2) in early diplotene. Sex chromosomes formed a positively heteropycnotic mass (Fig. 3C). In late diplotene and metaphase I, it was possible to observe 11 autosomal bivalents (the majority with only one chiasma) and two separated sex chromosome univalents (Fig. 3D), which allowed to determine the male karyotype of the species as 2n♂ = 24 (22+X1X20). Theridiidae Analysis of mitotic metaphases revealed 2n♂ = 22 in Coleosoma floridanum and Thymoites sp.1 (Fig. 3E, H) and 2n♀ = 24 in C. floridanum, Thymoites sp.1, and Thymoites sp.2 (Fig. 3F, I, K). Plates were composed exclusively by telocentric chromosomes (Fig. 3E–F, H– I, K). In these three species, spermatocytes I showed 10 autosomal bivalents and two sex chromosome univalents (X1X2) in diplotene and metaphase I. The bivalents showed only one chiasma (Fig. 3G, J), except for the early diplotenes, which showed some bivalents with two chiasmata (Fig. 3L). The sex chromosome univalents were placed close to each other (Fig. 3G) or side-by-side and showed positive heteropycnosis (Fig. 3J, L), depending on the stage of diplotene/metaphase I. Based on analyzed plates, we determined the karyotype formulas of these species as 2n♂ = 22 (20+X1X20) and 2n♀ = 24 (20+X1X1X2X2). Theridiosomatidae Male mitotic metaphases consisted of 30 chromosomes (Fig. 3M). Spermatocytes I in diplotene and metaphase I contained 14 autosomal bivalents and two X chromosome univalents (X1X2) (Fig. 3N). We found two types of metaphases II of spermatocytes II, one with n = 14 and another one with n = 16 (14+X1X2) (Fig. 3O–P). In some diplotenes and metaphases II, the sex chromosomes were positively heteropyknotic. Thus, the karyotype formula of Naatlo sp. was 2n♂ = 30 (28+X1X20). Despite many chromosome overlaps in mitotic metaphases, which did not allow detailed analysis of chromosome morphology, the observation of mitotic plates and metaphases II indicated that all or almost all the chromosomes were biarmed (meta/ submeta/subtelocentric) (Fig. 3O–P). Entelegynae, RTA clade Dionycha part A Trachelidae Analysis of mitotic metaphases of Orthobula sp. revealed 2n♂ = 21 and 2n♀ = 22 chromosomes, which exhibited telocentric morphology (Fig. 4A–B). Diplotene of spermatocytes I showed 10 autosomal bivalents with one or two chiasmata and a positively heteropycnotic univalent X (Fig. 4C). Thus, the karyotype was 2n♂ = 21 (20+X0) and 2n♀ = 22 (20+XX). The X chromosome was the largest element Fig. 2. Mitotic oogonial metaphase of Otiothops birabeni (Palpimanidae), 2n♀ = 36. Scale bar = 5 µm. page 4 of 9Zoological Studies 62:42 (2023) © 2023 Academia Sinica, Taiwan of the complement (Fig. 4A–B), corresponding to approximately 7% of the total chromosome length of diploid karyotype in males. Dionycha part B Corinnidae Spermatogonial metaphases of Falconina sp. showed 28 telocentric chromosomes (Fig. 4D). Spermatocytes I showed 13 autosomal bivalents and Fig. 3. Chromosomes of Linyphiidae (A–D), Theridiidae (E–L) and Theridiosomatidae (Araneoidea). Agyneta sp. (A–D), Coleosoma floridanum (E–G), Thymoites sp.1 (H–J), Thymoites sp.2 (K–L) and Naatlo sp. (M-P): A: Mitotic spermatogonial metaphase composed of 24 telocentric chromosomes. B: Mitotic oogonial metaphase with 26 telocentric chromosomes. C: Spermatocyte I (early diplotene) with 11 autosomal bivalents and two positively heteropycnotic sex chromosome univalents arranged in parallel (11II+X1X2). D: Spermatocyte I (metaphase I) with 11 autosomal bivalents and two sex chromosome univalents (11II+X1X2). E, H: Mitotic spermatogonial metaphase, 2n♂ = 22 telocentric chromosomes. F, I, K: Mitotic oogonial metaphases, 2n♀ = 24 telocentric chromosomes. G, J, L: Spermatocyte I, metaphase I (G, J) and early diplotene (L) consisting of 10 autosomal bivalents and two sex chromosome univalents (10II+X1X2). Sex chromosomes are positively heteropycnotic at J and L. While they are associated at G, they pair in parallel in J. Mode of pairing is unclear at L. M: Mitotic spermatogonial metaphase, 2n♂ = 30. N: Spermatocyte I, metaphase I. Note 14 autosomal bivalents and two sex chromosome univalents (14II+X1X2). O: Metaphase II, n = 14. P: Metaphase II, n = 16 (14+X1X2). Sex chromosomes are positively heteropycnotic. Arrowhead = bivalent with two chiasmata. Scale bars = 5 µm. page 5 of 9Zoological Studies 62:42 (2023) © 2023 Academia Sinica, Taiwan two sex chromosome univalents (X1X2) in pachytene, diplotene, and metaphase I (Fig. 4E–F), with one chiasma per bivalent, except for two or three bivalents with two chiasmata in late prophase I (Fig. 4F). We found two types of metaphases II, namely with n = 13 and n = 15 chromosomes (13+X1X2) (Fig. 4G–H). The sex chromosomes were positively heteropyknotic in pachytenes, diplotenes, and metaphases II. Based on these data, karyotype was determined as 2n♂ = 28 (26+X1X20). Zodarioidea Zodariidae The oogonial metaphases of Epicratinus sp. were composed of 44 chromosomes (Fig. 4I). All chromosomes were telocentric, with the exception of the second largest pair, which was subtelocentric and corresponded to the X1 chromosomes. These elements show the same morphology at male prophase I (Fig. 4I–J). Diplotene of spermatocytes I consisted of 20 autosomal bivalents and two sex chromosome univalents (X1X2). Bivalents exhibited one chiasma, except for two bivalents with two chiasmata. Subtelocentric X1 is larger than X2 (Fig. 4J). We found two types of metaphases II in spermatocytes II, one with n = 20 and another one with n = 22 chromosomes (20+X1X2) (Fig. 4K–L). Based on these data, karyotype was determined as 2n♂ = 42 (40+X1X20) and 2n♀ = 44 (40+X1X1X2X2). DISCUSSION Oonopidae (Synspermiata, Dysderoidea) Members of Neoxyphinus Birabén, 1953, Neotrops Grismado & Ramírez, 2013, Xestaspis Simon, 1884 Fig. 4. Chromosomes of Corinnidae, Trachelidae, and Zodariidae (RTA Clade). Falconina sp. (Corinnidae) (A–E), Orthobula sp. (Trachelidae) (F–H) and Epicratinus sp. (Zodariidae) (I–L): A: Mitotic spermatogonial metaphase, 2n♂ = 28 telocentric chromosomes. B: Pachytene, 13II+X1X2. Sex chromosomes pair in parallel; they are positively heteropycnotic. C: Spermatocyte I, diplotene. Note 13 autosomal bivalents and two sex chromosome univalents (13II+X1X2). Sex chromosomes pair in parallel on the periphery of the plate. D: Metaphase II, n = 13 telocentric chromosomes. E. Metaphase II, n = 15 (13+X1X2), telocentric chromosomes. Sex chromosomes are associated and positively heteropycnotic. F. Mitotic spermatogonial metaphase, 2n♂ = 21. G: Mitotic oogonial metaphase, 2n♀ = 22 telocentric chromosomes. H: Spermatocyte I, diplotene consisting of 10 autosomal bivalents and a sex chromosome univalent (10II+X). Sex chromosome is positively heteropycnotic. I: Mitotic oogonial metaphase, 2n♀ = 44 telocentric chromosomes, except for the subtelocentric X1. J: Spermatocyte I, diplotene composed of 20 autosomal bivalents and two sex chromosome univalents (20II+X1X2). Sex chromosomes are associated in parallel. K: Metaphase II, n = 20 telocentric chromosomes. L. Metaphase II, n = 22 (20+X1X2). Arrowhead = bivalent with two chiasmata. Scale bars = 5 µm. page 6 of 9 Zoological Studies 62:42 (2023) © 2023 Academia Sinica, Taiwan (present study), as well as Oonops Templeton, 1835 and Ischnothyreus Simon, 1893 (Král et al. 2019), have 2n♂ = 7, X0. These genera, with the exception of Xestaspis, had already been included in the phylogeny of De Busschere et al. (2014). Formula 2n♂ = 7, X0 is considered ancestral for Dysderoidea and Oonopidae by Král et al. (2019). Our study supports this hypothesis for oonopids. This pattern was found in two of the three genera analyzed in the present work. A derived karyotype was found in Cinetomorpha simplex (2n♂ = 9, X0). This genus was not included in the phylogeny of De Busschere et al. (2014). The karyotype of this spider contains an additional autosomal pair in comparison to previously analyzed oonopids. Palpimanidae To date, there are no chromosomal studies on the Palpimanoidea clade (Araujo et al. 2022). According to Wood et al. (2012 2013) and Dimitrov et al. (2017), Palpimanoidea is a sister group of Entelegynae. The relatively high chromosome number found in O. birabeni (Palpimanidae) (2n♀ = 36) is consistent with the relatively high diploid number (2n♂ = 42) of the ancestral Entelegynae karyotype proposed by Král et al. (2006). Linyphiidae, Theridiidae and Theridiosomatidae (Entelegynae, Araneoidea) Agyneta sp. (2n♂ = 24, X1X20, telocentric) is the first Neotropical member of Linyphiidae to be karyotyped. However, it shows the same pattern as most karyotyped species of the family (10 of 15 species karyotyped) (Araujo et al. 2022), including the sister genus Helophora Menge, 1866. Species with different karyotype compositions (Araujo et al. 2022) have been found in genera which are not closely related to Agyneta (Arnedo et al. 2009; Wang et al. 2015). The karyotype found in the three species of Theridiidae analyzed (C. floridanum, Thymoites sp.1, Thymoites sp.2, subfamily Theridiinae) (2n♂ = 22, X1X20, telocentric chromosomes) is the most common for the family (23 of 32 karyotyped species) (Araujo et al. 2022), and it is also found in Theridion Walckenaer, 1805, a notoriously problematic and non-monophyletic genus that includes species phylogenetically close to those studied here (Agnarsson 2004; Liu et al. 2016). The Theridiosomatidae family (Entelegynae, Araneoidea) was also chromosomally analyzed for the first time. Its position within the superfamily Araneoidea is controversial (Griswold et al. 1998; Dimitrov et al. 2017; Wheeler et al. 2017; Fernandez et al. 2018). Chromosomal data has been published only for the two families proposed as sister groups, namely Synotaxidae (1 species, 2n♂ = 24, XY) and Araneidae, whose male diploid numbers range from 13 to 50, with 2n♂ = 24 being more frequent (52 species of 66 karyotyped). The diploid number found in Naatlo sp. (2n♂ = 30, X1X20) was not found in the last two families, as well as in any Araneoidea so far (Araujo et al. 2022). Another peculiarity of Naatlo sp. is the exclusively or almost exclusively biarmed karyotype. Among Araneoidea species that have been karyotyped, only seven species of Araneidae show the same pattern. In some cases, this change in morphology was accompanied by a considerable reduction in the diploid number (2n♂ = 14) (Hackman 1948; Suzuki 1951; Amalin et al. 1993), whereas in others, the diploid number 2n♂ = 24 has been retained (Carandang and Barrion 1994) or slightly increased to 2n♂ = 26 (Prakash and Prakash 2014). Trachelidae, Corinnidae, and Zodariidae (Entelegynae, RTA clade) Concerning Trachelidae (Clade RTA, Dionycha part A), Orthobula sp. (2n♂ = 21, X0) differs from Afroceto plana Lyle & Haddad, 2010 and Trachelas japonicus Bösenberg & Strand, 1906 (2n♂ = 22, X1X20) (Suzuki 1952; Šťáhlavský et al. 2020) by sex chromosome system. The number of autosomal pairs in these trachelids is reduced in comparison to Trachelas sp. (2n♂ = 24, X1X20) (Datta and Chatterjee 1983). Telocentric X of Orthobula possibly involves tandem fusion of X1 and X2. The same type of rearrangement is possibly involved in the autosome number reduction from the 2n♂ = 24, X1X20, found in Trachelas sp. (for review, see Araujo et al. 2012). In Corinnidae (Clade RTA, Dionycha part B), the diploid number of the Neotropical genus Falconina sp. (2n♂ = 28) differs from members of Castianeira Keyserling, 1879 (2n♂ = 26) (Bole-Gowda 1958; Mittal 1966), which is a globally distributed genus (World Spider Catalog 2022). Concerning Zodariidae, the karyotype 2n♂ = 42, X1X20 has been found in two taxa, namely Neotropical Epicratinus sp. (Storeninae) and Pax islamita (Simon, 1873) (Zodariinae) (Král et al. 2011; Kumbiçak et al. 2014) from the Near and Middle East (Jocqué 1991; Henrard 2019). This karyotype is considered by Král et al. (2006) as ancestral for Entelegynae. Presence of this karyotype in two zodariid subfamilies occurring in distinct geographical areas suggests that this pattern is also an ancestral karyotype for the family. Karyotype evolution of some zodariid clades was accompanied by the reduction of diploid numbers as found in the genera Mallinella Strand, 1906 (2n♂ = 22) (Datta and page 7 of 9Zoological Studies 62:42 (2023) © 2023 Academia Sinica, Taiwan Chatterjee 1983 1989) and Zodarion Walckenaer, 1826 (2n♂ = 21, 24, 25, 26 and 29) (Pekár and Král 2001; Pekár et al. 2005a b). Epicratinus sp. differentiated from an ancestral pattern found in Pax islamita by the subtelocentric morphology of the X1 chromosome, possibly originated by pericentric inversion. CONCLUSIONS The main findings of the study are as follows: 1) the confirmation of an ancestral karyotype for Zodariidae and Oonopidae; 2) the relatively high chromosome number in Palpimanidae which supports an hypothesis on relatively high diploid number in ancestral Entelegynae; 3) the peculiar karyotype in theridiosomatid representative. Acknowledgments: This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [Finance Code 001] and Universidade Federal de Mato Grosso do sul (UFMS). We would like to thank Editage (www.editage.com) for English language editing and the reviewer J. Král for his valuable comments and corrections. Authors’ contributions: Collection and identification of specimens: DDD, DA, ADB. Dissection of specimens and preparation of slides: DDD. Chromosomal analyses: DDD, DA. Manuscript preparation: DDD, ADB. Competing interests: Authors declare that they have no conflict of interest. Availability of data and materials: Not applicable. Consent for publication: The authors declare no competing interests. Ethics approval consent to participate: Not applicable. REFERENCES Agnarsson I. 2004. Morphological phylogeny of cobweb spiders and their relatives (Araneae, Araneoidea, Theridiidae). Zool J Linn Soc 141:447–626. doi:10.1111/j.1096-3642.2004.00120.x. Amalin DM, Barrion AA, Jayoma M. 1993. Comparative karyomorphology of two Neoscona species (Araneae: Araneidae). Philippine Entomol 9:1–6. Araujo D, Rheims CA, Brescovit AD, Cella DM. 2008. Extreme degree of chromosome number variability in species of the spider genus Scytodes (Araneae, Haplogynae, Scytodidae). J Zool Syst Evol Res 46:89–95. doi:10.1111/j.1439-0469.2007.00457.x. Araujo D, Schneider MC, Paula-Neto E, Cella DM. 2012. Sex chromosomes and meiosis in spiders: a review. In: Swan A (ed) Meiosis – molecular mechanisms and cytogenetic diversity, 1st edn. Intech, Rijeka. Araujo D, Schneider MC, Paula-Neto E, Cella DM. 2022. The spider cytogenetic database, version 11.0. Available at: www. arthropodacytogenetics.bio.br/spiderdatabase. (Accessed 19 Oct. 2022). Arnedo MA, Hormiga G, Scharff N. 2009. Higher‐level phylogenetics of linyphiid spiders (Araneae, Linyphiidae) based on morphological and molecular evidence. Cladistics 25:231–262. doi:10.1111/j.1096-0031.2009.00249.x. Bole-Gowda BN. 1958. A study of the chromosomes during meiosis in twenty-two species of Indian spiders. Proc Zool Soc Bengal 11:69–108. Carandang RB, Barrion AA. 1994. Karyotype of the egg chromosomes of Argiope luzona (Walck.), and orb-weaving spider (Araneae, Araneidae). Philippine Entomol 9:443–447. Coddington JA, Griswold CE, Dávila DS, Peñaranda E, Larcher SF. 1991. Designing and testing sampling protocols to estimate biodiversity in tropical ecosystems. In: The Unity of Evolutionary Biology: Proceedings of the Fourth International Congress of Systematic and Evolutionary Biology. Dioscorides Press., Portland, Oregon, USA, pp. 44–60. Datta SN, Chatterjee K. 1983. Chromosome number and sexdetermining system in fifty-two species of spiders from NorthEast India. Chromosome Inf Serv 35:6–8. Datta SN, Chatterjee K. 1989. Study of meiotic chromosomes of four hunting spider of north eastern India. Persp Cytol Gen 6:414– 424. De Busschere C, Fannes W, Henrard A, Gaublomme E, Jocqué R, Baert L. 2014. Unravelling the goblin spiders puzzle: rDNA phylogeny of the Family Oonopidae (Araneae). Arthropod Systemat Phylogeny 72:177–192. Dimitrov D, Benavides LR, Arnedo MA, Giribet G, Griswold CE, Scharff N et al. 2017. Rounding up the usual suspects: a standard target-gene approach for resolving the interfamilial phylogenetic relationships of ecribellate orb-weaving spiders with a new family-rank classification (Araneae, Araneoidea). Cladistics 33:221–250. doi:10.1111/cla.12165. Fernández R, Kallal RJ, Dimitrov D, Ballesteros JA, Arnedo MA, Giribet G et al. 2018. Phylogenomics, diversification dynamics, and comparative transcriptomics across the spider tree of life. Curr Biol 28:1489–1497. doi:10.1016/j.cub.2018.03.064. Green DM, Sessions SK. 1991. Amphibian cytogenetics and Evolution. In: Appendix I, Nomenclature for chromosomes. Academic Press., USA, pp. 431–432. Griswold CE, Coddington JA, Hormiga G, Scharff N. 1998. Phylogeny of the orb-web building spiders (Araneae, Orbiculariae: Deinopoidea, Araneoidea). Zool J Linn Soc 123:1– 99. doi:10.1111/j.1096-3642.1998.tb01290.x. Hackman W. 1948. Chromosomenstudien an Araneen mit besonderer berücksichtigung der geschlechts chromosomen. Acta Zoologica Fennica 54:1–101. Henrard A. 2019. Systematics and phylogeny of the ant eating spiders (Araneae; Zodariidae): a total evidence analysis. Tese de Doutorado. UCL-Université Catholique de Louvain. Indicatti RP, Villarreal OM. 2016. Pseudonemesia tabiskey, a new species of Pseudonemesia Caporiacco 1955 and new ultramorphological data for the Microstigmatinae (Araneae: Microstigmatidae). J Nat Hist 50:2153–2167. doi:10.1080/00222 933.2016.1196297. Jocqué R. 1991. A generic revision of the spider family Zodariidae (Araneae). Bull Am Mus Nat Hist 201:1–160. page 8 of 9Zoological Studies 62:42 (2023) © 2023 Academia Sinica, Taiwan Král J, Musilová J, Št’áhlavský F, Řezáč M, Akan Z, Edwards RL et al. 2006. Evolution of the karyotype and sex chromosome systems in basal clades of araneomorph spiders (Araneae: Araneomorphae). Chromosome Res 14:859–880. doi:10.1007/ s10577-006-1095-9. Král J, Kořínková T, Forman M, Krkavcová L. 2011. Insights into the meiotic behavior and evolution of multiple sex chromosome systems in spiders. Cytogenet Genome Res 133:43–66. doi:10.1159/000323497. Král J, Forman M, Kořínková T, Lerma ACR, Haddad CR, Musilová J et al. 2019. Insights into the karyotype and genome evolution of haplogyne spiders indicate a polyploid origin of lineage with holokinetic chromosomes. Sci Rep 9:1–14. doi:10.1038/s41598019-39034-3. Kumbiçak Z, Ekiz E, Çiçekli S. 2014. Karyotypes of six spider species belonging to the families Gnaphosidae, Salticidae, Thomisidae, and Zodariidae (Araneae) from Turkey. Comp Cytogenet 8:93–101. doi:10.3897/compcytogen.v8i2.6065. Levan A, Fredga K, Sandberg AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52:201–220. doi:10.1111/j.1601-5223.1964.tb01953.x. Liu J, May-Collado LJ, Pekár S, Agnarsson I. 2016. A revised and dated phylogeny of cobweb spiders (Araneae, Araneoidea, Theridiidae): a predatory Cretaceous lineage diversifying in the era of the ants (Hymenoptera, Formicidae). Mol Phylogenet Evol 94:658–675. doi:10.1016/j.ympev.2015.09.023. Mittal OP. 1966. Karyological studies on the Indian spiders V. Chromosome cycle in three species of the family Clubionidae. Caryologia 19:385–394. doi:10.1080/00087114.1966.10796229. Pekár S, Král J. 2001. A comparative study of the biology and karyotypes of two central European Zodariid spiders (Araneae, Zodariidae). J Arachnol 29:345–353. doi:10.1636/01618202(2001)029[0345:ACSOTB]2.0.CO;2. Pekár S, Král J, Lubin Y. 2005a. Natural history and karyotype of some ant-eating spiders (Araneae, Zodariidae) from Israel. J Arachnol 33:50–62. doi:10.1636/S03-2. Pekár S, Král J, Malten A, Komposch C. 2005b. Comparison of natural histories and karyotypes of two closely related ant-eating spiders, Zodarion hamatum and Z. italicum (Araneae, Zodariidae). J Nat Hist 39:1583–1596. doi:10.1080/00222930400016762. Prakash A, Prakash S. 2014. Cytogenetical investigations on spiders of semi-arid areas. Indian J Arachnol 3:40–54. Rasband WS. 1997–2021. ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA. Available at http://imagej.nih.gov/ij/. Sakamoto Y, Zacaro AA. 2009. Levan, an ImageJ plugin for morphological cytogenetic analysis of mitotic and meiotic chromosomes. Initial version. An open source Java plugin distributed over the Internet from http://rsbweb.nih.gov/ij/, 2009. Št’áhlavský F, Forman M, Just P, Denič F, Haddad CR, Opatova V. 2020. Cytogenetics of entelegyne spiders (Arachnida, Araneae) from Southern Africa. Comp Cytogenet 14:107–138. doi:10.3897/CompCytogen.v14i1.48667. Suzuki S. 1951. Cytological studies in spiders. I. A comparative study of the chromosomes in the family Argiopidae. J Sci Hiroshima Univ Series B Division 1 12:67–98. Suzuki S. 1952. Cytological studies in spiders II. Chromosomal investigation in twenty two species of spiders belonging to the four families, Clubionidae, Sparassidae, Thomisidae and Oxyopidae, which constitute Clubionoidea, with special reference to sex chromosomes. J Sci Hiroshima Univ Series B Division 1 13:1–52. Wang F, Ballesteros JA, Hormiga G, Chesters D, Zhan Y, Sun N et al. 2015. Resolving the phylogeny of a speciose spider group, the family Linyphiidae (Araneae). Mol Phylogenet Evol 91:135– 149. doi:10.1016/j.ympev.2015.05.005. Wheeler WC, Coddington JA, Crowley LM, Dimitrov D, Goloboff PA, Griswold CE et al. 2017. The spider tree of life: phylogeny of Araneae based on target-gene analyses from an extensive taxon sampling. Cladistics 33:574–616. doi:10.1111/cla.12182. Wood HM, Griswold CE, Gillespie R. 2012. Phylogenetic placement of pelican spiders (Archaeidae, Araneae), with insight into evolution of the “neck” and predatory behaviors of the superfamily Palpimanoidea. Cladistics 28:598–626. doi:10.1111/ j.1096-0031.2012.00411.x. Wood HM, Matzke N, Gillespie R, Griswold CE. 2013. Treating fossils as terminal taxa in divergence time estimation reveals ancient vicariance patterns in the palpimanoid spiders. Systemat Biol 62:264–284. doi:10.1093/sysbio/sys092. World Spider Catalog, 2022. World Spider Catalog. Version 23.5 Natural History Museum, Bern. Available at: http://wsc.nmbe.ch. doi:10.24436/2. (Accessed 19 Oct. 2022). page 9 of 9Zoological Studies 62:42 (2023)