DOI: 10.5061/dryad.547d7wm86
title: Phylogenomic reconstruction reveals new insights into the evolution and biogeography of Atta leaf-cutting ants (Hymenoptera: Formicidae)

description: Atta Fabricius is an ecologically dominant leaf-cutting ant genus, the
 major herbivore of the Neotropics, and an agricultural pest of great
 economic importance. Phylogenetic relationships within Atta have until now
 remained uncertain, and the delimitation and identification of a subset of
 Atta species are problematic. To address these phylogenetic uncertainties,
 we reconstruct the most comprehensive phylogenetic estimate to date of
 Atta by employing ultraconserved elements (UCEs). We recovered 2340 UCE
 loci from 224 Atta specimens, which include 14 out of the 15 identifiable
 species from across their geographic distributions, and 49 outgroup
 specimens. Our results strongly support the monophyly of Atta and of the
 four clades that coincide with the previously recognized subgenera
 Archeatta Gonçalves, Atta s.s. Emery, Epiatta Borgmeier, and Neoatta
 Gonçalves. The Archeatta clade contains three species occurring in North
 and Central America and the Caribbean and is the sister group of the
 remainder of all other Atta species. The Atta s.s. clade is composed of
 two species occupying North, Central, and South America. The Epiatta clade
 contains seven entirely South American species and the two species of the
 Neoatta clade occur in Central and South America. Divergence-dating
 analyses identify a series of major events in the Miocene, such as the
 divergence of Acromyrmex Mayr and Atta 16.7 million years ago (Ma) and the
 crown-group origin of Atta around 8.5 Ma. Extant Atta species evolved very
 recently, originating in the early Pleistocene, approximately 1.8 to 0.3
 Ma (crown-group ages). We provide the first evidence that Atta goiana
 Gonçalves belongs to the Epiatta clade and that Atta robusta Borgmeier is
 the species with the youngest crown-group age of 0.3 Ma. The very young
 ages of Atta and its component species indicate a recent, rapid radiation.
 Biogeographic analyses suggest that the range of the most recent common
 ancestor of Atta consisted of the combined North/Central America and NW
 South America bioregions and that one daughter lineage subsequently
 dispersed into South America, rapidly diversifying in the newly formed
 Cerrado biome and Chaco, and further dispersing into the Atlantic Forest,
 Caatinga, and Pampas bioregions.
description: We extracted
 DNA non-destructively from specimens using Qiagen DNeasy Blood and Tissue
 kit (Qiagen Inc., Valencia, CA). We quantified each DNA extraction with a
 Qubit 3.0 Fluorometer employing the High Sensitivity Kit
 (Thermo Fisher Scientific, Inc.).
 Sheared the DNA target size to an average fragment of 300-600 bp using a
 Qsonica Q800R3 Sonicator (Qsonica LLC, Newton, CT,
 U.S.A.). The fragmented DNA was used as
 input for libraries preparation using the Kapa Hyper Prep
 Library Kit (Kapa Biosystems, Inc., Wilmington, MA, U.S.A.) as described
 in Faircloth <i>et al.</i> (2015) with iTru Adapters protocol.
 We implemented all magnetic bead clean-up steps (Fisher <i>et
 al.</i>, 2011) using a SPRI substitute (Rohland & Reich,
 2012) as described in Faircloth <i>et al.</i> (2015). We used
 dual-indexing TruSeq adapters (Faircloth & Glenn, 2012; Glenn
 <i>et al.</i>, 2019) for ligation. We amplified 15 µL of the
 library by PCR using the KAPA HiFi ReadyMix (Kapa Biosystems, Inc.,
 Wilmington, MA, U.S.A.) with 25 µL of HiFi mix, 2.5 µL of each of Illumina
 TruSeq i5 and i7 primers, and 5 µL nuclease-free
 ddH<sub>2</sub>O. We employed the thermal cycler program: 98
 °C for 45 s; 14 cycles of 98 °C for 15 s, 60 °C for 30 s, 72 °C for 60 s;
 and final extension at 72 °C for 5 m. We purified DNA products using 1.2x
 speedbeads and rehydrated the purified product in 22 µL of Elution Buffer
 (EB, pH= 8). We quantified individual libraries using a Qubit 3.0
 Fluorometer using the Broad Range Kit. We pooled eight to 12 post-PCR
 libraries at equimolar concentrations into 22 pools. We adjusted pool
 concentrations to 147 ng/µL by drying samples in a vacuum centrifuge for
 45-60 m at 60 °C, and then re-suspending them in nuclease-free water. We
 quantified each pool concentration in a Qubit 3.0 Fluorometer with the
 Broad Range Kit. We enriched each pool using 9446 baits (myBaits®; Arbor
 BioSciences) targeting 2524 conserved loci in Hymenoptera (Branstetter
 <i>et al.</i>, 2017b) for 24 h at an incubation temperature of
 65 °C. Enrichment, bead-cleaning, and PCR reaction procedures followed
 Borowiec (2019). The resulting reactions were purified using 1.0X
 speedbeads and the enriched pools were then rehydrated in 22 µL EB. We
 quantified each enriched pool using the Qubit 3.0 Fluorometer and the
 Broad Range Kit. We verified the enriched
 pools DNA concentration with qPCR on an Applied Biosystems
 7500 Real-Time PCR System (Applied Biosystems™, Thermo Fisher Scientific,
 Inc.) using the KAPA Library Quantification Kit (Kapa Biosystems, Inc.).
 We then used the resulting concentration estimates to pool the libraries
 at equimolar concentrations in a single final pool. We sent two final
 enriched pools to the University of Utah High Throughput Genomics Core
 Facility for quality control and sequencing of two full lanes of a HiSeq
 2500 (125 Cycle Paired-End Sequencing v4) run.