Research Article |
Corresponding author: Christopher M. Wilson ( christopher.wilson2@unt.edu ) Academic editor: Michael Ohl
© 2016 Christopher M. Wilson, Autumn Smith-Herron, Jerry L. Cook.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wilson CM, Smith-Herron A, Cook JL (2016) Morphology of the male genitalia of Brachymyrmex and their implications in the Formicinae phylogeny. Journal of Hymenoptera Research 50: 81-95. https://doi.org/10.3897/JHR.50.8697
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The male genitalia of Brachymyrmex are examined and terminology clarified. We document two conformation groups based on the lateral carina, conformation 1 has a complete lateral carina that reaches the apex of the valviceps and in conformation 2 the lateral carina is broken with the apical end projected dorsally. Previously documented species are evaluated in the context of this new information. We offer support for the movement of Brachymyrmex into the resurrected tribe Myrmelachistini based on the morphology of the penisvalvae, and offer definitive methods of assigning unknown specimens to this genus. This study provides histological methodologies for the preparation, differentiation, and permanent storage of minute ant genitalia structures and the associated musculature.
Brachymyrmex , genitalia, morphology, penisvalvae, phylogeny
The ant genus Brachymyrmex Mayr, 1868 exists in a state of taxonomic confusion. The small workers are notably devoid of good species delineating characters, and their distributions are not well represented in collections. This “miserable little genus” (Creighton 1950) has been largely ignored in systematic myrmecological works. Currently there are 60 valid species, of which only 21 contain male descriptions, and five of these have genitalia illustrations. The male genitalia offer species level characters that are desperately needed for this taxonomically challenging group (
Brachymyrmex was described by Mayr (1868) from the type species Brachymyrmex patagonicus.
Santschi (1923) provided the last revision of the group, designating many new species and subspecies. His descriptions, however, are of little value, as they are short and include few comparative characters. In his revision Santschi erected the subgenus Bryscha, which he defined by having dimorphic workers, short and flared sepals of the proventriculus, and erect hairs on the scape and legs. Bryscha was provisionally synonymized under Brachymyrmex by
The genus has a wide diversity of life histories. Most species are generalist nesters that nest in leaf litter, under stones, or under bark; at least two species have a distinct replete caste; while others are arboreal nesting under epiphyte mats or in small plant cavities. By contrast, the closely-related Myrmelachista are obligate arboreal nesting ants (
As our understanding of ant evolution progresses, the need for additional taxonomic and systematic information is increasing. The primary source for morphological characters in ants is worker morphology. This trend is easy to understand as workers are ubiquitous in the environment, often being the only caste collected and certainly the most prevalent. Queens and males on the other hand are less-frequently encountered, as they are usually within nests or produced only for brief periods. The reproductive casts have thus been largely neglected. Males are largely ignored, and therefore represent an untapped source of morphological characters for taxonomic and phylogenetic use. Moreover, when collected, males may be difficult or even impossible to associate with their workers. However, males may be associated through careful collecting and molecular phylogenies. A recent movement among ant researchers has been to pay more attention to males, both when collecting in the field and unassociated males (
The most useful character for males are arguably the genitalia, and males, like workers, range in size from the Dorylus “sausage flies” to remarkably small Brachymyrmex. Viewing and manipulating the male genitalia of larger species is often simple enough; most genitalia are even large enough to point mount for permanent storage. Smaller species, however, are neither simple to manipulate nor store. This has led some authors to not fully dissect the genitalia from small males obtaining images in situ either through compound microcopy or scanning electron microscopy (SEM) (
Here we present methods for dissection and permanent storage via slide mounting of small ant genitalia, with an additional option for muscle fixture and the ability to clear structures for SEM. Slide mounting genitalia allows examination at much higher magnifications than under stereoscopic microscopy, without the need for sputter coating or environmental SEM prep. Additionally the remainder of the specimen remains intact, losing, at most, only the last three segments. Notes on the genitalia of Brachymyrmex and their implications on the Formicinae phylogeny are presented using the described dissection methodologies. It is our hope that this paper provides a greater understanding of the male genitalia characters for Brachymyrmex, and encourages renewed interest in this “miserable little genus”.
For this study we sampled 16 species/morphospecies of Brachymyrmex, specimens were taken from the first author’s personal collection (CMWC), the Sam Houston State University Entomology Collection (SHSUE), the John T. Longino collection (JTLC), or accessed via AntWeb.org. Specimens from the collections were either in 95% EtOH or dried. Once dissected, bodies without genitalia were returned to the collections dried and point mounted, with the genitalia deposited in the CMWC on slides. The genitalia where dissected under an Olympus SZX12 dissecting microscope with a maximum 115× magnification mounted with a Olympus DP72 camera. Examined under an Olympus BX53 compound microscope with 10×, 40×, and 60× planapochromatic objectives with differential interference contrast prism mounted with a Olympus DP72 camera, and an Olympus B-Max 41 compound microscope with 10×, 40×, and 60×, with universal and planapochromatic objectives with either bright-field or phase-contrast condenser mounted with a Olympus DP-12 camera. Additional specimens were prepared for SEM using a Vega Scan high vacuum micrograph. Photos taken with the aforementioned cameras were stacked by hand using Adobe Photoshop CS6.
The procedures for the unstained whole mounts are as follows. The terminal segments of male reproductive organs were removed with a surgical scalpel and size 000 insect pins. Tissues were transferred and presoaked in a 20% sodium hydroxide (NaOH) solution for ca. 30 minutes to dissolve muscle and soft tissues. The remaining sclerites were neutralized with a 20% solution of Acetic acid (C2H4O2) for 5 minutes. Dehydration of tissues was initiated in 70% ethanol and continued at 20 min intervals through a graded series of ethanol washes, ending in two final washes with 100% ethanol. Tissues were cleared to near transparency with an initial introduction to xylene via a 50:50 solution of xylene:ethanol, and two 20 minutes washes in histological grade xylene. Prepared specimens were permanently mounted with dammar balsam mounting media. Alternatively, specimens can be directly taken form 100% ethanol and mounted in euparal. Slides are placed on a pre-warmed slide warmer and allowed to cure overnight.
The procedures for stained whole mounts are as follows. The terminal segments of male reproductive organs were removed with a surgical scalpel and size 000 insect pins. In order to examine the insertion and attachment sites for all muscles, excised tissues were not dissolved. Hydration of reproductive tissues were initiated in 70% ethanol and regressed to a final wash in deionized water. Prepared tissues were stained for 20 minutes in Harris Hematoxylin (without counterstain), and washed in an acid alcohol de-stain for 5–10 seconds. Stained tissues were then dehydrated with ethanol through an ascending graded series, cleared in xylene and permanently mounted with Damar Balsam mounting media. Examination of prepared tissue mounts follows the description above. The figures presented here are grouped by similar morphology and grouping statements accompany the respective figures.
The terminology herein follows
The paired penisvalvae of Brachymyrmex show tremendous variation in general shape, ranging from scimitar, to sickle-shaped, and quadrate. All species in which the males have been examined show dentition on the ventral ridge of the penisvalvae, which is conspicuously absent in Myrmelachista (though it should be noted that dentition is present throughout many of the formicids) (Fig.
As the base of the valvura is produced laterally, it may be referred to as the “lateral apodeme” (Fig.
Two main conformation patterns can be seen in the penisvalval morphology among the Brachymyrmex we sampled. In conformation 1 the lateral carina is complete, running from the lateral apodeme to the apex of the valviceps. In this conformation group the lateral carina forms a sharp curve dorsally near the carina’s base (Fig.
External genitalia of Brachymyrmex patagonicus, compound light microphotograph of the penisvalva in ectal view A lateral view of the genital capsule with the telomere pulled back to reveal the volsella and penisvalva BSEM of the penisvalva in ectal view C magnified ectal view of the penisvalva showing the separation of the valvura, the proximal end of the lateral carina, and severed muscular tissue form muscles k, and iD dorsal view of the genital capsule E apical view of the genital capsule F Colors: Blue penisvalvae; Green volsella; Red telomere; Purple baismere.
Paramere, volsella, and penisvalva of Brachymyrmex obscurior A–C Brachymyrmex coredmoyi (basimere not present) AntWeb.org, CASENT0740911 Photograped by Veronica M. Sinotte D–F Grouping: lateral carinae complete; digitus elongate and bifurcate apicaly; valvura parallel to the long axis of the valviceps.
The lateral carina of conformation 2 is incomplete being broken apically of the lateral apodeme into an anterior portion, which is continuous with the lateral apodeme, and a posterior portion that is raised dorsally above the blade of the valviceps (Fig.
Paramere, volsella, and penisvalva of Brachymyrmex CMW-011 (basivolsella fragmented) A–C Brachymyrmex CMW-012 (basivolsella fragmented) D–F Brachymyrmex cavernicola G–I Brachymyrmex heeri J–L Grouping: lateral carinae broken; digitus elongate, apex curving ventrad and not bifurcate apically; valvura oblique to the long axis of the valviceps.
Paramere, volsella, and penisvalva of Brachymyrmex CMW-007 (basivolsella fragmented) A–C Brachymyrmex CMW-009 (basivolsella fragmented) D–F Brachymyrmex JTL-004 (basivolsella fragmented) G–I Brachymyrmex JTL-005 (basivolsella fragmented) J–L Brachymyrmex depilis (basivolsella fragmented) M–O Grouping: lateral carinae broken; digitus lobate; valvura perpendicular to the long axis of the valviceps.
The volsella of Brachymyrmex follows the pattern of some Myrmelachista where the cuspis is reduced to a small triangular protrusion affixed to the wall of the digitus (B. obscurior, (Fig.
The modification to the lateral carina of the valviceps seen in both conformation groups seem to be related to the method in which the volsella rests against the valviceps in situ. As a result the dorsal surface of the volsella in conformation 1 is unbroken, while the dorsal surface of conformation 2 has a distinct concavity medially (Fig.
The telomeres are separated from the basimere by a membrane. Of the structures associated with the genital capsule, the telomeres of Brachymyrmex show the least variation in terms of overall shape and size. They are generally short and broad with numerous elongate flexuous setae. Of the taxa we examined, the telomeres of B. CMW-007, and B. CMW-009 (Fig.
Our findings demonstrate that males of this genus may offer more taxonomic information for understating the evolution within the genus, and for species delineations than their worker counterparts. The morphology of the penisvalva lateral carina displayed in the two conformation groups seen in Brachymyrmex likely represent two distinct clades within the genus, and when compared on an intraspecific level there seems to be little variation in genital morphology. Of course additional sampling is needed to confirm this. In regard to the tribal position of Brachymyrmex in light of the conclusive phylogeny of
Within Brachymyrmex
Future researchers collecting Brachymyrmex are encouraged to exhaustively, and carefully hunt for males along with associated workers. Those running Malaise and UV light traps should also be alert for male specimens. To that regard Brachymyrmex holds a number of invasive ant species, B. patagonicus for example is a widespread and has reached pest status in the United States (
The authors would like to thank Brendon Boudinot for reviewing earlier versions of this manuscript, his comments substantially improved this work. We would also like to thank Jack Longino for contributing much-needed specimens without which much of this genus would still be a mystery.
Specimen locality data
Data type: Specimen locality data
Explanation note: Locality data for dissected specimens.