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Sociobiology 61(1): 21-27 (March, 2014) DOI: 10.13102/Sociobiology.V61i1.21-27

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Sociobiology 61(1): 21-27 (March, 2014) DOI: 10.13102/Sociobiology.V61i1.21-27 Sociobiology 61(1): 21-27 (March, 2014) DOI: 10.13102/sociobiology.v61i1.21-27 Sociobiology An international journal on social insects RESEARCH ARTICLE - ANTS The geneic Characterizaion of Myrmelachista Roger Assemblages (Hymenoptera: Formicidae: Formicinae) in the Atlanic Forest of Southeastern Brazil MA Nakano1, VFO Miranda2, RM Feitosa3, MSC Morini1 1 - Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil. 2 - Universidade Estadual Paulista, Jaboicabal, SP, Brazil. 3 - Universidade Federal do Paraná, Curiiba, PR, Brazil. Aricle History Abstract Edited by: Arboreal ants of the genus Myrmelachista, which have ecologically important relaion- Gilberto M M Santos, UEFS, Brazil ships with diferent vegetable species, are found exclusively in the Neotropical region. Received 27 September 2013 Iniial acceptance 12 November 2013 These ant species are diicult to idenify, and their taxonomy remains controversial; Final acceptance 14 December 2013 moreover, litle is known regarding their biology. The objecive of the present work is to assess the geneic similariies and dissimilariies between and within Myrmelachista spe- Keywords cies, with the goal of expanding knowledge of the relaionships among the taxa of this twigs, ISSR molecular markers, genus. Sample collecion in selected regions of the dense ombrophile forest of south- Formicinae, UPGMA, similarity eastern Brazil yielded 256 nests, which were found in vegetaion or among scatered Corresponding author twigs in the leaf liter; eight species were recorded. A total of 180 specimens were ana- Maria Sanina de Castro Morini lyzed, producing 123 polymorphic fragments. Data analyses revealed similarity relaion- UMC – Núcleo de Ciências Ambientais ships that allowed the examined species to be classiied into the following groups: (1) Laboratório de Mirmecologia Myrmelachista sp. 4, M. nodigera, M. ruszkii and M. gallicola; (2) M. catharinae and M. Av. Dr. Cândido Xavier de Almeida arthuri; (3) M. reiculata; and (4) Myrmelachista sp. 7. The study results also revealed the Souza, 200 M. catharinae M. arthuri Mogi das Cruzes - SP - Brazil existence of two morphological variants of ; was more closely 08780-911 related to one of these M. catharinae variants than to the other variant. The present E-mail: [email protected] work provides important informaion regarding geneic variaion among Myrmelachista species that may contribute to interpreing the complex morphology of this genus. Introduction (Brown, 2000); however, it is known that these species gene- rally feed on extraloral nectaries (Haber et al., 1981) and on Myrmelachista Roger is a genus of the Formicinae animal-derived proteins (Torres, 1984; Amalin et al., 2001; subfamily. The geographical distribution of this genus is re- McNett et al., 2010). stricted to the Neotropical region, and 41% of the species in At present, 69 Myrmelachista species have been described, this genus can be found in Brazil (Kempf, 1972; Fernández & with a few recognized subspecies (Bolton et al., 2006; Bolton, Sendoya, 2004). The species in this genus are arboreal (Long- 2013); the diversity of this genus has most likely been underesti- ino, 2006) and engage in the specialized practice of nesting in mated (Longino, 2006) due to the limited taxonomic knowledge trunk cavities and among twigs (Stout, 1979; Brown, 2000; available regarding Myrmelachista (Snelling & Hunt, 1975). Pre- Longino, 2006; Edwards et al., 2009; Nakano et al., 2012, viously published reports, such as studies by Wheeler (1934) 2013). These ant species may also form complex mutual as- and Longino (2006), are important sources of taxonomic sociations with certain myrmecophytes (Renner & Ricklefs, knowledge regarding Myrmelachista; in particular, the report 1998; Frederickson, 2005; Edwards et al., 2009) or with Coc- by Longino (2006) is the most recent taxonomic review of the cidae and Pseudococcidae species (Kusnezov, 1951; Stout, genus, although this review was restricted to Myrmelachista 1979; Ketterl et al., 2003; Longino, 2006). Little information species found in Costa Rica. In the most recent molecular data- is available regarding the biology of Myrmelachista species based phylogenetic proposals for ants (Brady et al., 2006; Moreau Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 22 MA Nakano et al. – Geneic characterizaion of Myrmelachista assemblages et al., 2006), Myrmelachista is a sister group of Brachymyrmex, and these groups constitute the most basal and closely related formicine groups. Longino (2006) emphasized the dificulty of separating Myrmelachista species based on morphological characters but suggested that the combined use of worker, queen and male characters could improve the accuracy of species distinctions. Because the collection of alate individuals is more dificult than the collection of workers (Nakano et al., 2013), molecular techniques may be utilized to help establish the systematics of the Figure 1. The locations of the Atlantic Forest areas in which Myrmelachista genus. These techniques have been applied for Myrmelachista species were collected. (PNMFAM: Francisco similar analyses of other insect groups (Reineke et al., 1998). Affonso de Mello Municipal Natural Park; PN: Ponte Nova Dam; In particular, ISSRs (inter-simple sequence repeats) may serve PENT: Tietê Springs State Park). as a very important tool for these analyses. ISSR-based tech- niques enable the development of co-dominant microsatellite of 25 individuals and 5 nests per specie), except for M. markers that may be used in population studies (Gupta et al., reticulata (5 individuals and 1 nest) and Myrmelachista sp.7 1994; Bornet & Branchard, 2001; Wolfe, 2005). These tech- (20 individuals and 4 nests). Alate queens (n = 5) were found niques have allowed genetic distinctions to be drawn among only in Myrmelachista sp.7 nests; these queens were also Hymenoptera groups (Borba et al., 2005; León & Jones, 2005; examined by molecular analyses. Al-Otaibi, 2008) and among other Insecta groups (Souza et Species (or morphospecies) were identiied by al., 2008; Luque et al., 2002; Dusinsky et al., 2006). However, comparing the examined specimens with specimens deposited at present, no known published studies in the literature have in the reference collection of the Museum of Zoology of the applied these techniques exclusively for the examination of University of São Paulo. Vouchers were deposited in the ants. Thus, the present work sought to identify relationships myrmecofauna collection of the Alto Tietê Myrmecology and levels of genetic similarity (and dissimilarity) between Laboratory of the University of Mogi das Cruzes and in the and within Myrmelachista species found in the Atlantic Forest Museum of Zoology of the University of São Paulo. of southeastern Brazil, with the goal of providing additional knowledge regarding the relationships among different taxa Genomic DNA extraction of this genus. Genomic DNA from ants preserved at -80ºC was ex- Material and methods tracted using the protocol described by Sambrook et al. (1989). DNA extraction was performed independently for each exam- Samples for molecular analysis ined specimen. Sample integrity and purity were analyzed by 0.8% agarose gel electrophoresis, and spectrophotometric ob- The collection of Myrmelachista nests occurred in three servations were used to assess DNA quantity and purity. different regions of dense ombrophile forest in southeastern Brazil (Fig 1). A total of 256 nests were collected; these nests Ampliication of ISSRs were located either in the forest vegetation or among scattered twigs in the leaf litter (for details about biological material Twenty primers (UBC kit, University of British Co- collecting see Nakano et al., 2012, 2013). The nests housed lumbia) were tested; among these primers, the 10 primers that eight Myrmelachista species: M. arthuri Forel, 1903; M. generated the most distinguishable bands were selected, and catharinae Mayr, 1887; M. gallicola Mayr, 1887; M. nodigera optimal annealing temperatures were determined for each Mayr, 1887; M. reticulata Borgmeier, 1928; M. ruszkii Forel, of these 10 primers (Table 1). Each ampliication reaction 1903; Myrmelachista sp.4; and Myrmelachista sp.7. was performed in a tube containing a total volume of 20 µL. Whole nests containing living workers were stored at The reaction mixture included the following reagents: buffer -80°C. To minimize the degradation of genomic DNA, the (1.6X), deoxynucleotide triphosphates (dNTPs) (0.2 mM), screening and identiication of individuals were performed primer (0.8 µM), MgCl2 (1.5 mM), DNA (8 ng), Taq DNA after specimens were frozen in ice-immersed Petri dishes. polymerase (1 U) (GoTaq® Flexi DNA Polymerase, Promega) Workers from each nest were also collected in the ield and and autoclaved Milli-Q H2O. The ampliications were per- stored in 70% ethanol for subsequent morphological identi- formed in a thermal cycler (Peltier Thermal Cycler PTC-200, ication. MJ Research). The following temperature conditions were A total of 180 specimens were assessed. For most of utilized for the ampliication reactions: an initial denaturation the examined species, ive individuals per nest were selected at 94°C for 3 min; 40 cycles of denaturation at 92°C for 1 min, from ive nests (randomly) for molecular identiication (total annealing (at an optimized temperature for each primer) for 2 Sociobiology 61(1): 21-27 (March, 2014) 23 min and extension at 72°C for 2 min; and a inal extension at graphical representations
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