Italian Journal of Zoology ISSN: 1125-0003 (Print) 1748-5851 (Online) Journal homepage: https://www.tandfonline.com/loi/tizo20 Chromosomal similarities between Nephilidae and Tetragnathidae indicate unique evolutionary traits among Araneoidea D. Araujo, E. Paula-Neto, A. D. Brescovit, D. M. Cella & M. C. Schneider To cite this article: D. Araujo, E. Paula-Neto, A. D. Brescovit, D. M. Cella & M. C. Schneider (2015) Chromosomal similarities between Nephilidae and Tetragnathidae indicate unique evolutionary traits among Araneoidea, Italian Journal of Zoology, 82:4, 513-520, DOI: 10.1080/11250003.2015.1078418 To link to this article: https://doi.org/10.1080/11250003.2015.1078418 © 2015 Unione Zoologica Italiana Published online: 18 Aug 2015. Submit your article to this journal Article views: 406 View Crossmark data Citing articles: 1 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tizo21 Italian Journal of Zoology, 2015, 513–520 Vol. 82, No. 4, http://dx.doi.org/10.1080/11250003.2015.1078418 Chromosomal similarities between Nephilidae and Tetragnathidae indicate unique evolutionary traits among Araneoidea D. ARAUJO1*, E. PAULA-NETO2, A. D. BRESCOVIT3, D. M. CELLA4, & M. C. SCHNEIDER5 1Universidade Federal de Mato Grosso do Sul, UFMS, Setor de Biologia Geral, Centro de Ciências Biológicas e da Saúde, Cidade Universitária, Bairro Universitário, Campo Grande, Brazil, 2Departamento de Biologia, Universidade Estadual Paulista, UNESP, Instituto de Biociências, Rio Claro, Brazil, 3Instituto Butantan, Laboratório Especial de Coleções Zoológicas, São Paulo, Brazil, 4In memoriam, and 5Universidade Federal de São Paulo, UNIFESP, Departamento de Ciências Biológicas, Diadema, Brazil (Received 3 September 2014; accepted 21 July 2015) Abstract Nephilid systematics has been subject to several changes in the last years, and the use of non-classical characters could be useful for evolutionary considerations. In this study, we analyzed the mitotic chromosomes of two nephilid spiders, Nephila clavipes and Nephila sexpunctata, using standard staining, silver nitrate impregnation and C-banding techniques, aiming to discuss the chromosomal similarities of Nephilidae and Tetragnathidae, and chromosome evolution within Nephila and Nephilingis. The basic karyotype characteristics observed in these two species (2n♂ =22+X1X20 and monoarmed chromosomes) were similar to those registered for most araneoid families, i.e., Araneidae, Linyphiidae, Nephilidae, Nesticidae and Tetragnathidae. However, the occurrence of both prominent secondary constrictions and nucleolar organizer regions (NORs) is a shared characteristic between Nephilidae and Tetragnathidae, considering that these regions were not observed in any other Araneoidea species cytogenetically examined. Furthermore, in the present study we showed that within Nephila and Nephilingis species, change in the number and location of NORs as well as in the quantity and distribution of constitutive heterochromatin were the main events responsible for chromosome evolution, and that these differences can be useful in the cytotaxonomy of this group. Keywords: Chromosomes, nucleolar organizer region, secondary constriction, Orbiculariae, karyotype evolution Introduction nephilids, raising them to familial status – Nephilidae – constituted of (Clitaetra (Herennia Nephilids are araneoid spiders whose composition, (Nephilengys + Nephila))). Álvarez-Padilla et al. taxonomical ranking, internal and external relation- (2009) and Dimitrov et al. (2009) corroborated the ships, and genera monophyly have been intensively monophyly of Nephilidae and their placement out- discussed in the last years (Wunderlich 2004; side Tetragnathidae, as sister group to Araneidae, Kuntner 2006; Kuntner et al. 2008, 2013; Álvarez- with an internal topology similar to that proposed Padilla et al. 2009; Dimitrov & Hormiga 2009; by Hormiga et al. (1995), Griswold et al. (1998), Dimitrov et al. 2009; Hormiga & Griswold 2014). Álvarez-Padilla (2007) and Dimitrov and Hormiga Several hypotheses have considered nephilids as (2009), with the exclusion of Phonognatha. A recent part of Tetragnathidae and composed of phylogenomic analysis, aiming to discuss the orb- (Phonognatha (Clitaetra (Nephila (Herennia + weavers monophyly (Bond et al. 2014), shows a Nephilengys)))) (Hormiga et al. 1995; Griswold cladogram with Nephilidae closer to Araneidae et al. 1998; Álvarez-Padilla 2007; Dimitrov & than to Tetragnathidae. However, except for a brief Hormiga 2009). According to Wunderlich (2004), mention on the position of Theridiidae, the internal nephilids are a sister group to Araneidae. Kuntner Araneoidea phylogeny is not discussed, because, (2006) proposed a new taxonomical ranking for the *Correspondence: D. Araujo, Universidade Federal de Mato Grosso do Sul, UFMS, Setor de Biologia Geral, Centro de Ciências Biológicas e da Saúde, Cidade Universitária, Bairro Universitário, 79070-900, Campo Grande, Mato Grosso do Sul, Brazil. Tel/Fax: +55 67 33457311. Email: [email protected] © 2015 Unione Zoologica Italiana Published online 18 Aug 2015 514 D. Araujo et al. according to these authors, denser taxon sampling is embryos – three males and five females) from Tupã needed for a number of these clades. (21°56ʹ06” S, 50°30ʹ50” W), state of São Paulo, Kuntner et al. (2013) confirmed Nephilidae as a Brazil. Many attempts were made to obtain chromoso- monophyletic group and refused its placement as a mal preparations of male adult specimens, but the lack Tetragnathidae subfamily, considering that the clade of mitotic and meiotic cells indicated that the sperma- seems to be more closely related to “araneids” than togenesis occurs in an early developmental stage in these to tetragnathids. Moreover, Kuntner et al. (2013) species, as suggested by Araujo et al. (2005) for N. rejected the monophyly of Nephilengys, transferring cruentata. The vouchers were deposited in the some species to a new genus, Nephilingis. The mono- Laboratório Especial de Coleções Zoológicas, Instituto phyly of Nephila was also strongly questioned, but in Butantan (IBSP), city of São Paulo, state of São Paulo this case, the author chose not to formally reclassify (SP), Brazil (curator A.D. Brescovit). The chromoso- those species, which may not belong to this genus. mal preparations were obtained from ovaries of adult Thus, Kuntner et al. (2013) denominated those spe- females and from embryos, following the procedure cies as the “Nephila” clade and proposed two alter- described by Araujo et al. (2008): gonads were dissected native topologies: ((Nephilengys + Herennia)(Nephila in physiologic solution for insects (7.5 g Sodium (“Nephila” (Clitaetra + Nephilingis)))) or Chloride (NaCl), 2.38 g Sodium phosphate dibasic ((Nephilengys + Herennia)(Nephila (Nephilingis (Na2HPO4), 2.72 g Potassium dihydrogen phosphate (Clitaetra + “Nephila”)))). (KH2PO4), in 1 L of distilled water), transferred to The World Spider Catalog (2015) lists 61 species colchicine solution (0.16% in physiologic solution for of Nephilidae, now included in five genera: Clitaetra insects) and left for 2 h. A volume of hypotonic solution (six species from Africa/Asia), Herennia (11 (tap water) equal to that of the colchicine solution was Australasian species), Nephila (38 species with a added. After 15 min of hypotonization, the material was worldwide distribution), Nephilengys (two placed in methanol:acetic acid (3:1) for 60 min. Pieces Australasian species) and Nephilingis (four of tissue were dissociated in a drop of 60% acetic acid on Afrotropical/South American species). the surface of microscope slides. The preparation was Cytogenetically, only Nephila clavata L. Koch, driedonametalheatingplate(35–40°C) and stained 1878, from Japan/India, and Nephilingis cruentata with 3% Giemsa solution. (Fabricius, 1775), under Nephilengys cruentata To establish the constitutive heterochromatin and (Fabricius, 1775), from Brazil, were analyzed, both nucleolar organizer region (NOR) distribution pat- presenting 2n♂ = 24 and/or 2n♀ = 26, with the tern, the chromosomes were submitted to C-banding X1X20/X1X1X2X2 sex chromosome system, and (Sumner 1972) and silver nitrate impregnation telo-acrocentric chromosomes (Suzuki 1950, 1951; (Howell & Black 1980), respectively. The images of Datta & Chatterjee 1988; Araujo et al. 2005). It is the chromosomes were captured using an Olympus worthy to note that Nephila clavata as well as the BX51 microscope coupled to an Olympus DP71 South American Nephila clavipes (Linnaeus, 1767) digital camera with the DP Controller software. and Nephila sexpunctata (Giebel, 1867) might be The chromosomes were morphologically classified formally reclassified soon, and are provisionally trea- following Levan et al. (1964). ted as part of the “Nephila” clade by Kuntner et al. (2013). Results The aim of this paper is to chromosomally char- acterize the two other nephilid species that occur in Mitotic metaphase cells of N. clavipes and N. sex- Brazil, both belonging to the genus Nephila: N. cla- punctata showed 2n = 26 in female adult individuals vipes and N. sexpunctata, to determine the diploid and female embryos, and 2n = 24 in male embryos number, type of sex chromosome system, chromo- (Figure 1). The chromosomes presented telo-acro- somal morphology, distribution of C-banding and centric morphology and gradually decreased in size. nucleolar organizer regions (NORs), in order to dis- The sex chromosomes were not identified in cuss