Origin of specimens and taxonomic history

Ants and their associates were collected at several tropical sites (see Additional file 1: Table S1). For one site, La Selva Biological Station (LSBS) in Costa Rica (N10 25.847 W84 00.404, altitude 67 m), the only location where the new species was found, we provide comprehensive community data that allowed us to assess the specificity of host-myrmecophile interactions in great detail (for more information on collection methods see [30]). At LSBS we collected myrmecophiles during host colony emigrations from all of the six local Eciton species: E. burchellii foreli Mayr, 1886 (12 colonies), E. dulcium crassinode Borgmeier, 1955 (11 colonies), E. hamatum Fabricius, 1781 (10 colonies), E. lucanoides conquistador Weber, 1949 (2 colonies), E. mexicanum s. str. Roger, 1863 (10 colonies) and E. vagans angustatum Roger, 1863 (8 colonies). For ant identifications we used the keys and species distribution maps of Watkins [40, 41] and Longino [42]. The sampling took place from February to April 2013 and March to April 2014.

Morphological protocol

The species description follows the conventional terminology as used in current histerid taxonomic literature (e.g., [39, 43–47]). Body parts were measured using a Zeiss Stemi SV 11 stereomicroscope with an ocular micrometer. Measurements represent the range (minimum and maximum), are grouped at the beginning of the description to facilitate comparisons, and are abbreviated as follows: L (mm – dorsal length along midline from anterior pronotal margin to posterior elytral margin); W (mm – width at widest point); E/PnL (ratio – elytral length/pronotal length); E/PnW (ratio – elytral width/pronotal width); Pn W/L (ratio – pronotum width/length); EL/W (ratio – elytral length/width); Pr/Py (ratio – propygidium length/pygidium length); Thoracic sterna – pro-, meso-, metasternum (mm – lengths along midline); Tibiae – pro, meso, meta (mm - straight line length from base to apex, ignoring curvature). Primary type label information is listed verbatim, with the different labels separated by forward slashes.

We realized during collections that the new species was found primarily attached to workers of a certain size. To quantify this, we exclusively collected ant workers with the new beetle species attached for two different colonies. The head widths of those ant workers were then compared to a sample of workers from the same colonies that covered the entire body size range. Note that the ants used to assess body size were not random samples; rather they were obtained to gain an estimate of the range of body sizes present within a colony. A Leica Z16 APO stereomicroscope equipped with a Leica DFC450 camera and the processing software Leica Application Suite (version 4) were used to take frontal images of ant heads. A Nikon stage micrometer was used to calibrate the scale bars. Head width was defined as the straight line between the most distant points of the head, which was a line through the middle of the eyes. It was measured using the software ImageJ (version 1.47).

Molecular protocol

Morphological analysis was supplemented with genetic analysis for three reasons. First, we applied a broad-scale DNA barcoding approach to the entire Eciton-associated myrmecophile fauna (unpublished data and [30, 48]), which includes species from different taxonomic groups including millipedes, mites, silverfish, phorid flies, beetles, and wasps. DNA barcoding helped us to sort the specimens into pre-defined units, which were then carefully inspected by taxonomic experts. This streamlines the identification of species. Second, DNA barcoding allowed us to screen for possible cryptic diversity [30, 48]. Third, COI barcoding supported the generic placement of the new species, which was originally achieved by comparing morphological genus level synapomorphies [46]. While DNA barcoding is less effective in resolving taxonomic units above the species level [49, 50], it can still predict the taxon affiliation of an undescribed species at lower taxonomic scales, such as the genus, quite well [51].

We amplified the animal barcoding gene cytochrome oxidase I (COI) [52]. Forty-four specimens of the new species from eight different E. mexicanum colonies were genetically analyzed (range: 1–14 specimens per colony, mean = 5.5 ± 3.74 specimens per colony). In total, we performed genetic analyses on 58 histerid specimens including taxa expected to be closely and distantly related to our candidate genus for the new species, i.e. Nymphister. These taxa were chosen based on phylogenetic analyses of morphological and genetic characters of the subfamily Haeteriinae [34, 46, 53], as well as on specimen availability.

DNA was extracted from whole specimens using the QIAGEN® DNeasy® Tissue Kit for 96-well plates. We followed the standard protocol except for a shortened digestion step of 2–3 h, which allowed us to keep all samples as voucher specimens in absolute ethanol. The mitochondrial COI barcode region (658 base pairs (bp)) was amplified in standard polymerase chain reactions (PCRs). For N. kronaueri specimens (for species identifications see result section) we used the primers LCO1490 and HCO2198 [54]. These primers did not amplify consistently for other taxa, and therefore we used a combination of newly designed primers that are located a few base pairs inwards of the COI barcode fragment: Ecc1F (5’AACYTTATAYTTTATCTTTGGNGCWT3’; forward primer), Ecc2F (5’GCAGGAATAGTAGGAACATCTCTTAG3’; forward primer) and Ecc1R (5’AAWAGRTGTTGRTATARAATAGGGTC3’, reverse primer). PCRs were set up as described previously [30]. Purification and sequencing of PCR products were outsourced to Macrogen USA. All PCR products were sequenced in both directions.

Analysis of molecular data

Workflows, including information about collection, DNA extraction, PCR and DNA sequencing, were tracked with the laboratory management software Geneious (version R10.0.5) (http://www.geneious.com, [55]) with the plugin ‘biocode’ (version 3.0.1) [56]. Sequences were trimmed and aligned in Geneious. A neighbor-joining tree based on Tamura-Nei distances with bootstrap support (1000 replicates) was created using the Geneious Tree Builder. To support the generic placement of the new species, we were seeking for the most similar sequence in a set of potential candidates using a distance-based tree method. Note that our approach did not intend to fully resolve the phylogenetic relationships of the species studied here.

Data/specimen storage and research permits

In total, 67 voucher images from 19 specimens including dorsal, ventral, lateral views, as well as frontal images of the head were uploaded together with collection records and DNA sequences to the Barcode of Life database (http://www.boldsystems.org/; Additional file 1: Table S1). GenBank accession numbers for COI sequences can be found in Additional file 1: Table S1. Images were taken with the Leica Z16 APO stereomicroscope described above and processed using the software Helicon Focus (v. 5.3). Specimens were deposited at the following institutions: the Louisiana State Arthropod Museum, Baton Rouge, USA (LSAM), the National Museum of Costa Rica, San Jose, Costa Rica (MNCR), the Field Museum of Natural History, Chicago, USA (FMNH), the Connecticut State Museum of Natural History, USA (CSMNH), and the private collection of A. Allen, Boise, ID, USA (AA). Ant workers of all colonies are vouchered in CvB’s personal collection. Research and specimen export permits for Costa Rica were issued by the ‘Ministry of the Environment, Energy and Technology’ (MINAET; permit numbers: 192-2012-SINAC and R-009-2014-OT-CONAGEBIO).

Nomenclatural act

The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new species name contained herein is available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank Life Science Identifiers (LSID) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/“. The LSID for this publication is: urn:lsid:zoobank.org:pub:0D0FD20C-D73E-4326-B574-F3523EBD96F4.