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Investigating the Functional Morphology ofGenitalia During Copulation in the GrasshopperMelanoplus rotundipennis (Scudder, 1878) viaCorrelative Microscopy

Derek A. Woller* and Hojun Song

Department of Entomology, Texas A&M University, College Station, Texas

ABSTRACT We investigated probable functions of theinteracting genitalic components of a male and afemale of the flightless grasshopper species Melanoplusrotundipennis (Scudder, 1878) (frozen rapidly duringcopulation) via correlative microscopy; in this case, bysynergizing micro-computed tomography (micro-CT)with digital single lens reflex camera photography withfocal stacking, and scanning electron microscopy. Toassign probable functions, we combined imaging resultswith observations of live and museum specimens, andfunction hypotheses from previous studies, the majorityof which focused on museum specimens with few inves-tigating hypotheses in a physical framework of copula-tion. For both sexes, detailed descriptions are given foreach of the observed genitalic and other reproductivesystem components, the majority of which are involvedin copulation, and we assigned probable functions tothese latter components. The correlative microscopyapproach is effective for examining functional morphol-ogy in grasshoppers, so we suggest its use for otheranimals as well, especially when investigating bodyregions or events that are difficult to access and under-stand otherwise, as shown here with genitalia and cop-ulation. J. Morphol. 278:334–359, 2017. VC 2017 Wiley

Periodicals, Inc.

KEY WORDS: anatomy; insect; Acrididae; sexual selec-tion; speciation

INTRODUCTION

The evolution of animal genitalia is an activearea of research in evolutionary biology and it isgenerally accepted that sexual selection is themajor driving force that explains the observeddivergence of genitalic components often foundamong closely related species (especially in malesand particularly in insects), with these divergen-ces playing a potentially significant role in specia-tion (Eberhard, 1985; Arnqvist et al., 2000;Arnqvist and Rowe, 2002; Hosken and Stockley,2004; Ritchie, 2007; Hotzy et al., 2012; Richmondet al., 2012; Simmons, 2013). Insights into theunderlying mechanisms of this evolutionary pro-cess, however, are difficult to gain for a number ofreasons. For one, insect genitalia are extremelydiverse and often relatively complex, especially in

males, in terms of the number of componentsinvolved in copulation and reproduction (Eberhard,1985; Arnqvist, 1997; Eberhard, 2010). This rela-tive complexity has often masked our ability tounderstand functional genitalic morphology, partic-ularly during the copulation process. This processis difficult to examine intensely due to its often-hidden nature (internal components shielded fromview by external components) and because it isoften easily interrupted by active observation, butsome studies investigating function have manipu-lated genitalia as a way around such issues(Brice~no and Eberhard, 2009; Grieshop and Polak,2012; Dougherty et al., 2015). Adding further com-plexity to understanding function, several studieshave demonstrated that individual genitalic compo-nents may play different roles during copulationand evolve at different rates (Song and Wenzel,2008; Song and Bucheli, 2010; Rowe and Arnqvist,2011). Furthermore, when studying the evolution ofgenitalia, there has been a continued general biastoward examining the morphology of male genitaliafor various reasons, including the difficulties inexamining female genitalic morphology (Eberhard,1985) (e.g., a common lack of rigidity), assumptionsthat female components do not appear to vary asmuch between conspecifics (which may also be truein some cases) (Eberhard, 1985; Ah-King et al.,

Additional Supporting Information may be found in the onlineversion of this article.

Contract grant sponsors: Orthopterists’ Society Research Grant(2012, to D.A.W.), and NSF Grant IOS-1253493 and Texas A&MUniversity start-up (to H.S.).

*Correspondence to: Derek A. Woller, Minnie Belle Heep Center,Room 412, Campus MS 2475, College Station, TX 77843-2475.E-mail: [email protected]

Received 24 August 2016; Revised 18 November 2016;Accepted 3 December 2016.

Published online 23 January 2017 inWiley Online Library (wileyonlinelibrary.com).DOI 10.1002/jmor.20642

VC 2017 WILEY PERIODICALS, INC.

JOURNAL OF MORPHOLOGY 278:334–359 (2017)

2014), and because differences in male componentsare typically more obvious (Eberhard, 1985; Arnqv-ist, 1997). However, male and female genitalia havepresumably co-evolved, and, therefore, should bestudied together.

We are currently in the midst of an insect (andother invertebrates) morphology renaissance eversince a pioneering study in by H€ornschemeyeret al. (2002) used micro-computed tomography(micro-CT) to examine beetle morphology. Thispowerful imaging technology is non-invasive andnon-destructive and has been used by many toinvestigate the morphology of insect morphologyin novel ways (e.g., Jongerius and Lentink, 2010;Wipfler et al., 2011; Wojcieszek et al., 2012;Breeschoten et al., 2013; Faulwetter et al., 2013;Michalik et al., 2013; Beutel et al., 2014; Grecoet al., 2014; Simonsen and Kitching, 2014). One ofthe primary benefits of micro-CT is the ability tobuild three-dimensional (3D) reconstructions ofany anatomical component included in the high-resolution scanning process (Fig. 1A–D). For thisstudy, we couple this technology synergisticallywith two other imaging methods, digital singlelens reflex (DSLR) camera photography with focal

stacking and scanning electron microscopy (SEM),a method known as correlative microscopy (Caplanet al., 2011), to investigate the copulation processin the context: functional morphology. Compre-hending the function of morphology, principallyhow the male and female parts interact, is a keyto better understanding genitalia evolution (Kellyand Moore, 2016). The copulation process ininsects has previously been difficult to study infine detail because of the cryptic nature of interac-tions between male and female genitalia and theinvasive techniques that were necessary for exami-nation. This frontier is ripe for exploration withrelatively few studies focusing on functional geni-talic morphology in insects (e.g., Eberhard andRamirez, 2004; Brice~no and Eberhard, 2009; Grie-shop and Polak, 2012; Brice~no et al., 2016; Rheber-gen et al., 2016), with even fewer utilizing micro-CT to do so (e.g., Dougherty et al., 2015; Holwellet al., 2015; Mattei et al., 2015; Schmitt and Uhl,2015). In terms of the order Orthoptera, only oneother study of a similar nature has yet beenundertaken (with Metrioptera roeselii (Hagenbach,1822) (Ensifera: Tettigoniidae) in Wulff et al.(2015), but, to our knowledge, our study is the first

Fig. 1. Melanoplus rotundipennis, abdomina of original male and female specimens frozen incopula compared to their 3D-reconstructions using micro-CT scanning (for all, female is aboveand male is below): A. left lateral view; B. right lateral view; C. left lateral view of 3D-reconstructions; D. right lateral view of 3D-reconstructions.

335FUNCTIONAL MORPHOLOGY DURING COPULATION

Journal of Morphology

to use micro-CT to examine functional morphologyduring the copulation process within the suborderCaelifera (true grasshoppers). Although this studyis focused specifically on elucidating the functionalrole of genitalic morphology during the copulationof grasshoppers, we think that our findings andmethodology have wider implications for other ani-mals, especially arthropods.

Our study subject, Melanoplus rotundipennis(Scudder, 1878) (Orthoptera: Caelifera: Acrididae:Melanoplinae) (Fig. 2A–C) is a member of the PuerGroup, a group of 24 small, flightless grasshoppersthat reside in xeric habitats (e.g., scrub, pine flat-woods, and sandhills). Additionally, they appear tobe strongly associated with scrubby oaks, are dis-tributed across the southeastern United States

(here defined as Alabama, North and South Caroli-na, Georgia, and Florida), and many are endemic torelatively small regions. Externally, all speciesresemble one another to a strong degree, but it isthe male genitalia, primarily the aedeagus, thatseparate them due to their highly divergent forms(Hubbell, 1932). These collective factors make thePuer Group an excellent system in which to exam-ine the evolution of genitalia. Of all group members,M. rotundipennis was chosen for four reasons, thefirst being that it is the most widely distributed (inboth Florida and southern Georgia), meaning thatlocating populations, normally quite abundant, waseasy and integral to encouraging specimens to matein the lab within habitat boxes (Fig. 2C). The secondis that intraspecific variation in male genitalia,especially in the aedeagi, is negligible (Squitieret al., 1998), http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1097-4687/homepage/ForAuthors.html, which may also be true for female genitalia.Third, the species is one of the largest in the PuerGroup with an average length of 15 mm in malesand 20 mm in females and the aedeagus is males inalso one of the most prominent, making this speciesone of the easiest to work with overall. Finally, the58 named genitalic and other reproductive systemcomponents across both sexes (33 male, 25 female)of M. rotundipennis can arguably be labeled as themost complex in the Puer Group in terms of numberand structural shape based on comparative observa-tions, making this species interesting to study.

The general functions of grasshopper genitaliaduring copulation have been described and specu-lated on by numerous sources (Federov, 1927; Bol-dyrev, 1929; Snodgrass, 1935; Kyl, 1938; Roberts,1941; Dirsh, 1956; Randell, 1963; Gregory, 1965;Otte, 1970; Pickford and Gillott, 1971; Whitmanand Loher, 1984; Snodgrass, 1993; Eades, 2000;Chapman, 2012; Song and Mari~no-P�erez, 2013),with the majority focused on museum specimensand many biased toward male anatomy. Only fiveof those studies (Boldyrev, 1929; Kyl, 1938; Grego-ry, 1965; Pickford and Gillott, 1971; Whitman andLoher, 1984) investigated hypotheses in a physicalframework of copulation to better examine thelargely cryptic interactions of the external andinternal components of both sexes. For furtherperspective, there are 12,219 species of Caeliferacurrently known worldwide (Cigliano et al., 2016)and each of the five previous framework studiesfocused on four different species and one subspe-cies across two families of grasshoppers (Acrididaeand Romaleidae). This means that our collectiveunderstanding of functional genitalic morphologyin grasshoppers is still quite poor, hence our deci-sion to also utilize a physical framework of copula-tion to investigate previous findings and positedhypotheses by answering two broad questions: 1)what role(s) do the genitalic and other reproduc-tive system components of males play during

Fig. 2. Melanoplus rotundipennis, live photos of: A. male; B.female; C. in copula pair.

336 D.A. WOLLER AND H. SONG


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copulation? Additionally, as noted previously,female morphology is frequently ignored, so femalegenitalia was intentionally made a focus of thisstudy, both on their own and how they interactwith male genitalic morphology, leading us to ask:2) what role(s) do the genitalic and other reproduc-tive system components of females play duringcopulation? Furthermore, the majority of previousstudies relied on standard dissections, drawings,and, occasionally, photographs, which were uti-lized well to gather evidence to support observa-tions. Imaging technology, although, is continuallyadvancing, so we harnessed the combined power ofthree of these technologies (DSLR with focal stack-ing, SEM, and micro-CT) and our own observa-tions of live and museum specimens for twoprimary reasons: to investigate the general valueof correlative microscopy and to decide if such anapproach is more effective for examining genitalicmorphology and function for at least grasshoppers.

MATERIALS AND METHODS

Taxon Sampling

The present study is based on M. rotundipennis (Scudder,1878) (Orthoptera: Caelifera: Acrididae: Melanoplinae). Utilizedspecimens (see Supporting Information for full locality informa-tion) mainly came from Florida with two from southern Georgiaand included: museum, recently collected, and laboratory-observed within habitat boxes. Specimens in the latter categorywere collected from different locations in Florida, with each boxcontaining up to 10 individuals of mixed sex ratios, mainly toobserve mating behavior. Specimen numbers and sexes used foreach imaging technique are as follows: 1) DSLR camera:10#,11$; 2) SEM: 5#, 1$; 3) micro-computed tomography(micro-CT): 1#, 1$.

Dissections

The internal genitalia and parts of the reproductive systemsof male and female specimens were dissected from both freshlycollected and museum specimens (rehydrated by being dippedbriefly into boiling water) and removed from the body usingstandard procedures for male grasshoppers (Hubbell, 1932) andfemale grasshoppers (Slifer, 1939) and the assistance of a LeicaMZ16 microscope system. Intact dissected components were putin 0.65 ml vials containing a 10% KOH solution and placed intoa boiling water bath for up to an hour to clear away obstructingtissues. The specimens were then removed and further dissect-ed as necessary for examination and imaging. In the case of themale, this meant fully separating the epiphallus from the ecto-phallus and endophallus and, occasionally, the former from thelatter. For the female, this meant removing all exterior abdomi-nal segments to reveal hidden components.

Terminology

Terminology was derived and synthesized (and, in somecases, modified) from a number of sources: Snodgrass, 1935,1993; Slifer, 1939, 1940a, 1940b, 1943; Roberts, 1941; Dirsh,1956, 1965, 1973; Eades, 1961; Randell, 1963; Gregory, 1965;Uvarov, 1966; Ander, 1970; Am�ed�egnato, 1976; Whitman andLoher, 1984; Key, 1989; Stauffer and Whitman, 1997; Eades,2000; Gordh and Headrick, 2001; Chapman, 2012; and Songand Mari~no-P�erez, 2013. The reproductive system is definedhere as any anatomical components used by both sexes for theproduction of offspring, with genitalia defined as a subset of

those components used specifically for copulation between amale and female. To aid in comprehension, the orientation ofalmost all of the DSLR and SEM images and 3D-reconstructions was made uniform for both sexes (head facingleft) and reflects the natural positions of all anatomy. Finally,the 3D-reconstructions of male components were colored inshades of green, purple, and blue while female componentswere colored in shades of yellow, orange, pink, and red.

Imaging and 3D-Reconstructions

Digital single lens reflex. Digital images of anatomicalcomponents of freshly collected and museum specimens were tak-en in the Song Laboratory of Insect Systematics and Evolutionusing a Visionary Digital imaging system equipped with a CanonEOS 6D DSLR camera combined with a 100 mm/65 mm lens (thelatter often coupled with a 23 magnifier) to take multiple imagesat different focal lengths. The resulting files were converted fromRAW to TIFF format using Adobe Lightroom (v.4.4), stacked intoa single composite image using Zerene Stacker (v.1.04), and thenAdobe Photoshop CS6 Extended was used to add a scale bar andadjust light levels, background coloration, and sharpness. Photo-graphs of live specimens were taken in the same lab in a simulat-ed habitat using a Canon EOS 6D Digital SLR Camera equippedwith a 100 mm lens and a Canon MT-24EX Macro Twin LiteFlash paired with two multi-directional halogen lights for addi-tional lighting. Photographs of the specimens used for micro-CTscanning were taken by a colleague in Germany using a KEY-ENCE VHX-2000 series digital microscope.

Scanning electron microscopy. Genitalia of both sexescame from specimens that had been cleared in KOH asdescribed above. All images were taken with a JEOL JSM-6480 after genitalia were attached to standard metal stubswith carbon glue and coated with 30 nm of gold palladium.

Micro-CT. Scanning was performed on a copulating pair ofmale and female specimens that were brought to the lab fromthe field, kept within a habitat box with other specimens ofboth sexes, fed Romaine lettuce daily, observed until copulating,carefully isolated into a smaller container, and then frozen rap-idly in a 2808C freezer within 30 min of the male penetratingthe female. Several other copulating pairs were also frozen thisway and the set that looked the most intact was chosen to bethe micro-CT subject. This specific pair was then prepared forthe critical point drying process by removing the sample fromthe freezer and cutting through the entirety of their abdomensat or around segment 6. This smaller sample was then fullydehydrated over 24 hours within a 1.65 ml vial containing100% ethanol. The sample was transferred next to a specialmicrovial punctured with a syringe for fluid/gas exchange andrun through a Tousimis Samdri-790 Semi-Automatic CriticalPoint Drying Apparatus. Then, the final dried sample wasscanned using an X-radia 400 (Carl Zeiss X-ray Microscopy,Pleasanton) with absorption contrast and a spatial resolution of2.6 mm. The resulting scans were mirrors of the sample.

3D-reconstructions. The micro-CT scanning processresulted in a stack of 1,943 TIFF images that was thenimported into the program Amira (FEI, v. 6.0.1) and examinedin three two-dimensional (2D) planes (X-Y, X-Z, and Y-Z) tolocate all desired anatomical structures, which were then trans-formed into three-dimensional (3D) reconstructions using theSegmentation Editor. The file sizes of the four halves of the fullexterior terminalia of the male and female seen only in theinteractive reconstruction were found too large to work with,and thus, were made smaller with Resample module. Once cre-ated, each reconstruction was then isolated from the rest usingthe Surface View module and all were then exported as stereoli-thography (STL) files. The program Blender (v.2.76) was thenused to apply smoothing algorithms to the individual STL filesas follows: for almost all anatomical components, the SmoothVertex option was applied with “1” as the Smoothing value and“30” as the Repeat value, but, for the four halves of the termi-nalia, “0.5” was the Smoothing value and “5” was the Repeatvalue. Smoothed reconstructions were then exported as updated



STL-files and imported back into Amira for color assignmentsusing the Surface View module. Note that the smoothing pro-cess reduces general roughness, which can disconnect objectsformerly tightly connected (e.g., see Fig. 4B,C: the base of thespermathecal duct no longer touches the apex of the bursacopulatrix). Blender was also used to mirror the STL-filesaround the y-axis to match the original sample. All figureswere assembled in Adobe Photoshop CS6 Extended and allreconstructions had their original backgrounds replaced witha uniform color. Photos of the original sample were taken inGermany once the micro-CT scanning process was completedin China. During the journey to Germany, the specimensshifted slightly, which is why they do not exactly match thereconstructions (Fig. 1).

Special features. The included video (Supporting Infor-mation Fig. S1) is intended to be an animated, visual overviewof the anatomical components that were reconstructed in 3Dand was initially created using Amira’s Animation Director.Next, the video was exported as a series of image files andimported into Windows Live Movie Maker (v. 2011) where itwas mirrored around the y-axis to match the original sample,transitions and titles were added, and it was then exported asan MP4 file.

The included interactive 3D-figure (Fig. 14) requires a 3DPDF-enabled viewer such as Adobe Reader and is intended tosimulate what we can see and do in Amira and to also assistthe reader in better understanding how the anatomical compo-nents of both sexes interact because 2D-images of such

Fig. 3. Melanoplus rotundipennis, 3D-reconstructions (in copula positions) of male and female components of genitalia and otherparts of the reproductive system (male components are colored in shades of green, purple, and blue; female components are coloredin shades of yellow, orange, pink, and red): A. dorsal view, posterior to right; B. left lateral view, posterior to right; C. right lateralview, posterior to left; D. posterior view; E. ventral view, posterior to right.



http://onlinelibrary.wiley.com/doi/10.1002/jmor20642/suppinfo

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components can be difficult to mentally place spatially in rela-tion to each other. The program PDF3D ReportGen (v. 2.13.0,Build 8317 364) was used to accomplish this by combining allSTL files generated in Amira and a text file (of the type IV)containing associated titles for each component that was modi-fied from a template included with PDF3D. The color of eachSTL file was matched as closely as possible to the color of its2D counterpart in the figures and video using PDF3D’s “pickscreen color” option in the Visual Effects tab and clicking onthe colors in the figures. Additionally, to reduce file size andoptimize movement speed in the 3D-PDF, the following simplifi-cation options within PDF3D (Advanced tab) were employed,all with a value of 150,000: threshold triangles count, thresholdline segments count, and threshold points count. Locations forplacement of associated component titles were found usingAmira by attaching the Line Probe module to each desired STLfile, resulting in exact x, y, z coordinates.

High-resolution versions of the static figures (TIFF), interac-tive 3D figure (PDF), video (MP4), 3D-reconstructions (STL –uncolored), and the micro-CT data used to build the 3D-reconstructions (TIFF) are available for download at Morpho-Bank (O’Leary and Kaufman, 2012) under Project P2517:http://morphobank.org/permalink/?P2517.

RESULTS

All terminology used for the genitalic and otherreproductive system components are included inthis section with occasional modifications fromoriginal sources to aid in uniformity and ease ofunderstanding. Additionally, definitions are givenfor those components that are either not obviouslydelimited in referenced figures or are potentiallyunique to this species and/or the Puer Group. Dis-coveries that are possibly novel are further elabo-rated on later. There are no intended statementsof homology regarding terminology and given defi-nitions, and they may only apply to this speciesand/or the Puer Group. The genitalia and otherreproductive system components of M. rotundipen-nis examined in this study are arguably the mostcomplex compared to other Puer Group species,consisting of 58 named components (many paired)for both sexes combined, 33 for males and 25 forfemales. To aid in comprehension, after a briefoverview of mating behavior for this species is giv-en, a detailed overview follows of the observedcomponents of external and internal genitalia andother parts of the reproductive system with orien-tation descriptions based on relative repose posi-tions (i.e., when copulation is not occurring). Themajority of these components were transformedinto 3D-reconstructions (e.g., Figs. 3, 4) and anoverview is provided separately for each sex, aswell as for what components are touching while incopula (Figs. 3, 4C) solely based on the 3D-reconstructions. Additionally, further comprehen-sion can be gained by watching the video of the3D-reconstructions (Supporting Information Fig. S1)and exploring the interactive 3D-reconstructions(Fig. 14).

Mating Behavior

Habitat boxes often contained up to 10 M. rotun-dipennis individuals of mixed sex ratios, but matingdid not seem to occur until at least two males werein the same habitat box with at least one female and

Fig. 4. Melanoplus rotundipennis, left lateral, cut-away viewsof 3D-reconstructions (in copula positions) of male and femalecomponents of genitalia and other parts of the reproductive sys-tem (in all, posterior to right): A. male external and internal; B.female external and internal (muscle tissue of spermathecal com-plex removed); C. close-up view of combined male and female(muscle tissue of spermathecal complex removed).



http://morphobank.org/permalink/?

then, occasionally, one would attempt copulation in amanner similar to what Bland (1987) observed in M.childsi Otte, 2012 (2011) (formerly belonging to M.tequestae Hubbell, 1932) and what Otte (1970)observed in melanoplines in general. The male beganhis copulation attempt by moving slowly behind afemale and then suddenly jumping on her back. Thefemale would then often kick at him wildly with herhind tibiae and sometimes jump around the cage withthe male clinging tight. Usually, the female wouldeventually calm down, allowing the male to positionhis abdomen under hers, invert his supra-anal plateto expose his phallic complex, and attempt to matewith her. The general in copula positions for grass-hoppers (e.g., Figs. 3, 4) are as follows: the male gainsaccess to the female’s internal genitalia by pullingdown her subgenital plate with his epiphallus andthen rotates his phallic complex anterodorsallyaround 908 (or more), inserting it through her vulva(e.g., Chapman, 2012). After successful mounting,defined here as a female allowing a male to insert hisadeagus into her for an uninterrupted period, (Figs.2C) the M. rotundipennis pair were carefully

transferred to a private container for periodic obser-vations during which the pair would stay still or wan-der around slowly. We also observed the femora ofboth sexes vibrating intermittently along with period-ic pulsations of both abdomens. Despite a femaleseeming to be receptive, some males were unsuccess-ful for unknown reasons, in either attempting to pulldown the female’s subgenital plate or inserting theiradeagus. These rejected males would then dismountand wander away, sometimes not trying to mateagain for hours or days, if at all.

Male: External and Internal Genitalia andOther Parts of the Reproductive System

External (Fig. 5): Seven anatomical componentsare present, three of which are paired. The furculae(Figs. 4A, 5A–E) are prominent and extend from theposterior margin of tergite 10 (Fig. 5C–E) andslightly across the base of the supra-anal plate (orepiproct; Figs. 4A, 5A–E) that also emerges fromtergite 10. The cerci (Figs. 4A, 5A–E) extend poste-riorly toward the cone-like pallium (Fig. 5C–E), theapex of the subgenital plate (Fig. 5D,E). The

Fig. 5. Melanoplus rotundipennis, 3D-reconstructions (in copula positions) and DSLR images ofmale external genitalic components: A. 3D, left lateral view, posterior to right; B. 3D, dorsalview, posterior downwards; C. DSLR, dorsal view; D. DSLR, left lateral view; E. DSLR, left later-al view, made translucent with KOH.



paraprocts (Figs. 4A, 5A–E) are in-between thesupra-anal plate and the cerci, also extending later-ally and posteriorly, and attach, via a membranealong the middle of their dorsal edge, to the base ofthe cerci. The subgenital plate resembles the gener-al form found in many Acrididae species, almostappearing to comprise two segments due to themedial transverse groove that can be stretchedapart to a degree due to membranes in-between.

Internal (Fig. 6A): Two main anatomical com-ponents are present: the genital chamber (cavityenclosed by the supra-anal plate (Figs. 4A, 5A–E)dorsally and subgenital plate plus pallium (Fig.5C–E), laterally, posteriorly, and ventrally) and thephallic complex (Fig. 6A), which comprises threesubcomponents: epiphallus (Figs. 6B,C, 7F,G), ecto-phallus (Figs. 6D,E, 8A), and endophallus (Figs.6F,G, 8A), each of which are divided further into

Fig. 6. Melanoplus rotundipennis, 3D-reconstructions (based on in copula positions, but rotated into repose positions) and a DSLRimage of male internal genitalia and greater reproductive system components (posterior to right unless otherwise noted with oneexception - spermatophore only present during copulation, so this component always points anteriorly): A. all components, quasi-dorsal view; B. epiphallus, dorsal view, posterior downwards; C. epiphallus, left lateral view; D. ectophallus, dorsal view; E. ectophal-lus, left lateral view; F. endophallus, dorsal view; G. endophallus, left lateral view; H. DSLR, discarded spermatophore, left lateralview; I. greater reproductive system, left lateral view; J. greater reproductive system, dorsal view.



more subcomponents. The epiphallus (Figs. 6B,C,7F,G) consists of six subcomponents (five of whichare paired) and is the most dorsal part of the phal-lic complex, resting above the ectophallus (Figs.6D,E, 8A) and endophallus (Figs. 6F,G, 8A), con-nected to these two components via muscles andthick membranes. The ancorae (Figs. 4A, 6B,C,7F,G) curve ventrally at a steep angle and the ante-rior projections (Figs. 6B, 7F,G) curve inwardsslightly while the posterior projections (Figs. 6B,C,

7F,G) do not. Additionally, the anterior projectionshave what appear to be multiple sensory receptors(not shown here) scattered about dorsally withmore found in the median sclerotized cleft formedbetween the anterior projections and the ancorae.The bridge (Figs. 6B, 7F,G) is thick and bendsstrongly inwards in the middle when viewed anteri-orly while the lateral plates (Fig. 6B,C, 7F) are alsothick, but unremarkable. The lophi (Figs. 4A, 6B,C,7F,G) are covered with minute ridges that resemble

Fig. 7. Melanoplus rotundipennis, DSLR images of male internal genitalia and greater reproductive system components (posteriorto right unless otherwise noted): A. all components, left lateral view; B. phallic complex, left lateral view; C. phallic complex, dorsalview; D. phallic complex, ventral view; E. phallic complex, posterior view; F. epiphallus, dorsal view, posterior downwards; G. epi-phallus, anterior view, dorsal downwards.



fish scales that are oriented toward the anterior ofthe epiphallus (Fig. 9G).

The ectophallus (Figs. 6D,E, 8A) is the middlephallic complex component that is firmly connectedto, and largely surrounds, the endophallus (Figs.6F,G, 8A) via thin membranes and consists of oneprimary component, the cingulum [that wouldresemble the letter “H” if flattened (Roberts, 1941)]with four subcomponents (only two of which aretypically paired). The apodemes of cingulum (Figs.4A, 6D,E, 7B,C,E, 9A) are of varying sizes and cur-vature while the zygoma (Figs. 4A, 6D,E, 7B,C, 9A)can be described as double-layered and complete,although atypical (because it slightly extends poste-riorly over the valves of aedeagus (Figs. 4A, 7B,9B), but just ventral to it is an almost-identical sec-ond region that contains a medial gap. The rami(Figs. 4A, 6D,E, 7B,D, 9A) are well-developed,extend a little ways ventrally beyond the aedeagus,curve inwards slightly, and have what appear to besensory receptors (not shown here) at the apex ofthe posterior region. The sheath of aedeagus (Figs.4A, 6D,E, 7B–D, 9A) is a somewhat misleadingterm for what is observed in this species because itis only vaguely sheath-like. In fact, it would be bet-ter described as two individual lobes that extend alittle ways beyond the apices of the rami and arelightly attached via membranes to the basal portionof the dorsal valves of aedeagus, but for ease ofunderstanding, will be continued to be referred toas a single component. Additionally, the sheath ofaedeagus curves upwards, almost reaching the topof the dorsal valves of aedeagus in some specimens(Fig. 7B), curve slightly inwards, and are also cov-ered with tiny spines arranged in overlapping rowsthat point anteriorly (Fig. 9F).

The endophallus (Figs. 6F,G, 8A) is the most ven-tral part of the phallic complex and the primary por-tion that contains components of a species-specificnature, and consists of nine subcomponents (six ofwhich are paired), with six more closely connectedthat belong to the greater reproductive system (Fig.7A). The apodemes of endophallus (Figs. 4A, 6F,G,7B–E, 8B,D, 9A) are typical for acridids as is itswhitish, opaque connective tissue (Figs. 4A, 6F,G,7C,E, 8D), presumably, and, more than likely, so arethe greater reproductive system components: ejacu-latory duct (Figs. 4A, 6I,J, 7A), ejaculatory sac(Figs. 4A, 6I,J, 7A), gonopore (not pictured becauseit is a hole connecting the sacs), and spermatophoresac (Fig. 6 I,J). The duct and sacs can still be verydifficult to see after dissection, being often obscuredby vestiges of undissolved musculature (the whitishcoloration of the components blends in well with thewhitish musculature) and by their ethereal natureand relative plasticity of their shapes; therefore, werelied on the 3D-reconstructions to guide us to thegeneral shapes. The spermatophore (Fig. 4A, 6H–J)is a temporary creation of the greater reproductivesystem during copulation and, thus, its relative

orientation is the opposite of other male internalcomponents because the phallic complex is rotatedaround 908 (or more) during copulation (e.g., Fig.4A,C). The spermatophore comprises two compo-nents (anterior to posterior): 1) tube region (thebody, which is lightly sclerotized; Fig. 4A, 6H–J)and 2) reservoir (bulbous head; Fig. 6H–J). Addi-tionally, flexures (Figs. 6G, 7B, 8B, 9A, 10E) arepresent and have obvious articulations (not labeled,but see Figs. 6G, 7B, 8B, 10E) between them and

Fig. 8. Melanoplus rotundipennis, DSLR images and 3D-reconstructions (based on in copula positions, but rotated intorepose positions) of male internal genitalic components (in all,posterior to right): A. DSLR, separated ectophallus and endo-phallus, left lateral view; B. DSLR, partially separated endo-phallus, left lateral view; C. 3D, ventral valve of aedeagus, leftlateral view; D. DSLR, partially separated endophallus, dorsalview; E. 3D, ventral valves of aedeagus, dorsal view.



the aedeagus while the gonopore processes (Figs.6G, 7B,D, 8B, 10E) are near the apical end of theapodemes of endophallus and slope dorsally towardthe flexures.

The aedeagus, the intromittent organ, is highlyspecies-specific and, here, both its dorsal valves(Figs. 4A, 6F,G, 7B–E, 8B,D, 9A,B, 10E) and ven-tral valves (Figs. 4A, 6F,G, 7B-E, 8B–E, 9B, 10E)

are quite long and gently curve upwards whenviewed laterally. For this species, both sets ofvalves can be imagined as nested structures inwhich the concave dorsal valves almost fullyencompass the tubular ventral valves. The dorsalvalves are open along the entirety of their ventralside, fused for more than 3=4 of their length with athin, median dorsal cleft appearing for the

Fig. 9. Melanoplus rotundipennis, SEM images of male internal genitalic components: A. ectophallus and endophallus, left lateralview, posterior to right; B. dorsal and ventral valves of aedeagus, posterior view; C. close-up, lateral view of left ventral valve ofaedeagus; D. close-up, left lateral view of dorsal valves of aedeagus; E. further magnified close-up, left lateral view of dorsal valves ofaedeagus; F. sheath of aedeagus, quasi-left lateral view, posterior to right; G. right lophus of epiphallus, quasi-left lateral view.



remainder, and their entire outer surface is cov-ered with microscopic leaf-like projections that areanteriorly projected (Fig. 9A,D,E). The ventralvalves are subcylindrical, apically taper to points,and are enclosed by the dorsal valves on all sidesexcept ventrally. However, the ventral valves cansometimes extend a bit beyond the start of thedorsal valves, and can also often be seen throughthe walls of the dorsal valves, especially laterally,when sclerotization is light or a specimen hasbeen fully cleared in KOH (e.g., Fig. 7B). Often,these valves may appear to be attached for somelength basally, but are actually only attached vialight membranes. In fact, the valves can be fullyseparated into two nearly identical halves, eachappearing to be smooth, but, on closer inspection,are actually quite rugose (Fig. 9C). Three lengthvariations have been observed for the ventralvalves: they do not extend beyond the apex of thedorsal valves (Figs 7B 9A), they extend a littleways beyond the apex (Fig. 8A), and they extendquite a bit beyond the apex (Figs. 7A, 9B). Due tothe unique shape of both valve sets, the phallot-reme (not labeled, but see Figs. 7B, 8A,B), thechannel formed by the inner space of these valvesthrough which the spermatophore (Fig. 4A, 6H–J)travels, is, thus, unique to this species. The archof aedeagus (Figs. 6F,G, 7B, 8B,D, 10C) rises fromthe median, dorsobasal region of the dorsal valvesof aedeagus.

Female: External and Internal Genitalia, andOther Parts of the Reproductive System

External (Fig. 11): Nine anatomical compo-nents are present (six of which are paired) andone has two subcomponents (one of which ispaired). The supra-anal plate (or epiproct) (Figs.11A,B) is attached to tergite 10 (Fig. 11A,B) at itsbase with the cerci (Fig. 11A,B) extending posteri-orly. The paraprocts (Figs. 11A,B) are similar tothose in males, although larger and with a morerounded shape. Dorsal (Figs. 11A–C, 12B), inner(Fig. 11B), and ventral valves of ovipositor (Figs.11A–D, 12A,B), and lateral basivalvular sclerites(Figs. 11B,C, 12A,B) are unremarkable and resem-ble those of other melanoplines, if not the majorityof acridids that lay eggs in soil (Stauffer and Whit-man, 1997). Similarly, the subgenital plate (Figs.4B, 11B–H), which often has species-specific char-acters, such as the shape of its posterior margin,resembles that of all Puer Group species with themargin possessing an ephemeral triangular projec-tion that is attached firmly to the underside of theegg guide, extending about 1=4 to 1=2 of its length(partially seen in Fig. 10A,B). The subgenital platecontains two subcomponents: the lophi receptacles(Fig. 11E–H) and egg guide (Figs. 4B, 11D–H),both of which also resemble those of all PuerGroup species. Additionally, the apical third of thesubgenital plate that is bent downwards into the

Fig. 10. Melanoplus rotundipennis, 3D-reconstructions (A,B are in copula positions, C,D,E rotated into repose positions) of variousgenitalic components: A. complete male and female external, posterior view; B. partial male external and internal, plus female exter-nal, posterior view; C. partial male phallic complex, quasi-right lateral view, posterior to left; D. partial male phallic complex, quasi-ventral view, posterior downwards; E. endophallus, ventral view, posterior to right.



male’s genital chamber during copulation (Fig. 4B)and contains the lophi receptacles is more heavilysclerotized than the rest (Fig. 11G,H).

Internal (Fig. 13A): Eight main anatomicalcomponents are present (three of which are paired)and two have subcomponents. The genital chamberis the cavity containing the internal genitalia andparts of the reproductive system, but can be difficultto delimit. Here, it seems to be delimited dorsally

by the apodemes of ovipositor (Fig. 12A,B) and ven-trally by the subgenital plate (Figs. 4B, 11B–H),with the spermathecal complex (Fig. 13H,I) as themost anterodorsal component, median oviduct (notpictured) as the most ventral, and vulval sclerite(Figs. 4B, 12A–E,G,H, 13B–E) as the most posterior.The apodemes of ovipositor and anterior basivalvu-lar sclerites (Fig. 12A,B) are unremarkable andresemble those of other melanoplines.

Fig. 11. Melanoplus rotundipennis, DSLR images and 3D-reconstructions (in copula positions) of female external genitalia andgreater reproductive system components (posterior to right unless otherwise noted): A. DSLR, dorsal view; B. DSLR, left lateralview; C. DSLR, ventral view; D. DSLR, ventral view, made translucent with KOH; E. 3D, subgenital plate, posterior view; F. 3D,subgenital plate, quasi-left lateral view, posterior to right; G. DSLR, subgenital plate, dorsal view; H. DSLR, subgenital plate, ventralview.



The spermathecal complex (Fig. 13H,I), too, is essen-tially similar to other members of the Puer Group andcontains four subcomponents with one of these dividedfurther into two more subcomponents. The entire com-plex is covered in what Snodgrass (1993) called a“muscular sheath” while Whitman and Loher (1984)referred to it as “glandular tissue” in Taeniopoda eques(Burmeister, 1838) (Romaleidae) and which a more

detailed histological study by Ahmed and Gillott(1982) called a “small discrete bundle of muscle fibers.”This is further supported by Lay et al. (1999) in whichit is noted that “longitudinal and transverse musclesoverlay” the entire complex. Here, we follow Gos�alvezet al. (2010) in calling it the more general “muscletissue” (partially seen in Figs. 12A, 13H). The sperma-thecae comprises two subcomponents: preapical and

Fig. 12. Melanoplus rotundipennis, DSLR and SEM images of female external and internal genitalia, and greater reproductive sys-tem components (posterior to right unless otherwise noted): A. DSLR, ventral view; B. DSLR, ventral view, posterior downwards; C.DSLR, bursa complex, dorsal view; D. DSLR, bursa complex, ventral view; E. DSLR, close-up of vulval sclerite, posterior view, dorsalupwards; F. DSLR, partially separated bursa complex, quasi-dorsal view; G. SEM, bursa complex, ventral view; H. SEM, close-up ofposterior region of bursa complex, ventral view.



apical diverticula (Figs. 4B, 12A, 13H,I), with the for-mer very obvious, comparatively long, and slightly bul-bous, and the latter only occasionally slightly bulbousat its apex, but often resembling the spermathecalduct (Fig. 4B, 12A, 13H,I), for which the spermathecaeform the apex. The duct itself appears to vary in lengthto a degree (Fig. 12A, 13H,I) and is typically com-pressed into loosely tangled coils (Fig. 13H,I). Unlikein Melanoplus sanguinipes (Fabricius, 1798), M. rotun-dipennis and all other examined Puer Group speciesdo not have a U-shaped bend in the duct toward thebasal end (Pickford and Gillott, 1971). Furthermore,for M. rotundipennis and other Puer Group members,

the spermathecae and spermathecal duct, althoughsometimes long, were not observed to extend beyondthe basal arms of the subgenital plate (Fig. 3A–C,E),with spermathecal ducts typically compressed intoloosely tangled coils. The delimitation of the sperma-thecal aperture seems clear here and is the slit con-necting the spermathecal duct to the bursa copulatrix(not labeled, but see Fig. 4B).

The bursa complex (Fig. 13A,E) comprises twosubcomponents: the armor of bursa copulatrix(Figs. 4B, 12B–D,F,G, 13E–G) and the bursa copu-latrix (Figs. 4B, 12B–D,F,G, 13E–G), and has theoverall appearance of a scorpion’s tail, with,

Fig. 13. Melanoplus rotundipennis, 3D-reconstructions (in copula positions) and a DSLR image of female internal genitalia andgreater reproductive system components (posterior to right unless otherwise noted): A. all components, left lateral view; B. vulvalsclerite, dorsal view; C. DSLR, vulval sclerite, posterior view, dorsal upwards; D. vulval sclerite, ventral view; E. bursa complex andvulval sclerite, left lateral, cut-away view; F. bursa complex, left lateral view; G. bursa complex, dorsal view; H. spermathecal com-plex, quasi-dorsal view; I. spermathecal complex, ventral view (muscle tissue of spermathecal complex removed).



approximately, its apical fourth being curved backon itself ventrally except in the 3D-reconstructions(discussed later). Interestingly, this curvature wasobserved to point left or right (Fig. 12B) dependingon the individual (and even within the same popu-lation) with no apparent pattern yet known. Thearmor of bursa copulatrix (Figs. 4B, 12B–D,F,G,13E–G) is an outer covering that surrounds themajority of the inner bursa copulatrix, has aribbed appearance (Fig. 12D,G) with flexiblemembrane-like portions in-between. Furthermore,

it is tough to the touch (of forceps), despite it notappearing to be heavily sclerotized as in othercomponents, and it also does not possess the yel-lowish coloration often associated with sclerotiza-tion (Fig. 12F). The bursa copulatrix (Figs. 4B,12B–D,F,G, 13E–G), conversely, does have the yel-lowish coloration (Fig. 12F), but is much weakerto the touch and only appears to be lightly sclero-tized, and to varying degrees among specimens.Overall, the bursa copulatrix is broadly tubular,but a portion just before its apex constricts into anarrower tube with its actual apex being quitebulbous (to varying degrees) and extending beyondthe armor, sometimes quite extensively (Fig. 12B).

The vulval sclerite (Figs. 4B, 12A–E,G,H, 13B-E) is found ventrally at the entrance to the bursacomplex, also known as the vulva (not labeled, butsee Fig. 12A–H), and often extending slightlybeyond. Sclerotization levels for this componentvary between specimens (Figs. 12D,E,G,H, 13B)and some may even be divided in two (Fig. 13C),but, in general, it is often plate-like, curves gentlydorsally (lightly concave from an anterior view),has two lateral arms that extend posteriorly, andsometimes has slight anterodorsal protrusions(Fig. 13C). Finally, the Comstock-Kellog glands(not pictured) do not appear to be as obvious as inother Melanoplus species (Slifer and King, 1936;Slifer, 1940b), but are present in M. rotundipennis,are of a typical shape, and almost translucent(made even fainter with KOH).

In copula. External: During copulation, theapical third of the female’s subgenital plate is bentat an almost 908 angle ventrally, is wedged intothe male’s genital chamber, and is dominantlyresting atop his right paraproct and basal end ofhis right cercus, but is not quite touching hisfolded-in supra-anal plate or furculae (Figs. 3C,10A,B). Additionally, the apical half of the female’segg guide is bent upwards and to the left (Fig.10A,B), most likely a result of encountering themuscles that surround the male’s phallic complex.Finally, the male’s cerci are pressing slightly onthe female’s abdomen toward the base of her later-al basivalvular sclerites (Fig. 1A–D).

Internal: During copulation, the right lophus ofthe male’s epiphallus is inserted for some distanceinto the female’s corresponding right lophus recep-tacle (Fig. 10B) while the majority of his aedeagusis inserted into her bursa complex, specificallycoming into direct contact with her bursa copula-trix for some distance (Fig. 4C). The dorsal, con-cave surface of the female’s vulval sclerite isentirely touching part of the basal, dorsal regionof the male’s dorsal valves of aedeagus (Fig. 4C).Finally, the male’s spermatophore is emergingfrom between the apices of his dorsal and ventralvalves of aedeagus, so that the rest of its tuberegion is extended through the remainder of thefemale’s bursa copulatrix. Here, the apex of the

Fig. 14. Melanoplus rotundipennis, interactive 3D-viewingwindow containing 3D-reconstructions and associated labels ofmale and female components of genitalia and other parts of thereproductive system of in copula specimens (male componentsare colored in maroon (Exterior) and shades of green, purple,and blue; female components are colored in white (Exterior) andshades of yellow, orange, pink, and red). The 3D-PDF interfaceis filled with useful abilities and we welcome exploration, buthere is a short guide to getting started quickly (the functionalityof this guide depends on your version of Adobe Reader): at thetop of the viewing window is a toolbar with shortcuts, of whichthe most useful is the one that resembles a phylogenetic treecalled “Model Tree.” Clicking on this icon opens the Model Treepane to the left of the viewing window and, from here, you cancheck/uncheck boxes (clicking on “1” signs reveals full tree)associated with each component (name first, then body region,and then sex) as well as their associated labels (“LABELS-Ana-tomical_Components,” naming scheme identical to components).These abilities are also present in the viewing window by right-clicking, choosing “Part Options,” and then “Hide,” and this func-tion can be applied to both components and labels. There arealso 15 pre-set views (Left Lateral View is Home) that we thinkyou may find interesting and these can be accessed from the“View” dropdown menu of the viewing window toolbar, in a win-dow below the model tree pane, or by right-clicking and choosing“Views.” You may also change lighting schemes by clicking onthe lamp icon (CAD Optimized Lights is default) in the viewingwindow toolbar. Finally, basic movements within the viewingwindow are as follows: hold down left mouse button to rotate inany direction, scroll middle mouse button to zoom in and out,hold down right mouse button and move mouse forward andback to more quickly zoom in and out (or hold Shift 1 left mousebutton), and hold Ctrl 1 left mouse button to pan around. Moremovement abilities can be accessed from the far left arrowedicon of the viewing window toolbar or by right-clicking and look-ing in “Tools.”



tube region has swollen to completely fill theinterior of the bulbous apex of the bursa copula-trix, presumably because it is close to rupturing(Fig. 4C).

DISCUSSION

Functional Morphology

Unsurprisingly, but rarely demonstrated inaction, external and internal anatomical compo-nents of the male and female reproductive system,mainly genitalic, in M. rotundipennis are involvedin copulation, often appearing to possess multiplefunctions. Herein, the probable functions of 45 ofthe 58 named components will be discussed inmore detail as well as other items of interest, likenovel discoveries, starting, in general, with themale (32 male components) and then moving tothe female (13 female components; summarized inTable 1). Twelve female components were notobserved to be involved in copulation: tergite 10(Fig. 11A,B), supra-anal plate (Fig. 11A,B), cerci(Fig. 11A,B), paraprocts (Fig. 11A,B), median ovi-duct (not pictured), dorsal, inner, and ventralvalves of ovipositor (Figs. 11A–D, 12A,B), apo-demes of ovipositor (Fig. 12A,B), lateral basivalvu-lar sclerites (Figs. 11B,C, 12A,B), anteriorbasivalvular sclerites (Fig. 12A,B), and Comstock-Kellog glands (not pictured). Of these, the majorityare most likely involved in laying eggs whileothers may be involved with structural support ofthe abdomen, assist in waste excretion, and/or sen-sory reception (possibly even during copulation)(Snodgrass, 1935; Eisner et al., 1966; Uvarov,1966; Snodgrass, 1993; Stauffer and Whitman,1997). For the males, the lone exception was thefurculae (Figs. 4A, 5A–E), which were initiallythought to be involved in copulation, but may notbe. Observations were based on correlative micros-copy combining DSLR and SEM images withmicro-CT-based 3D-reconstructions, as well asobservations of museum specimens and live speci-mens, and inferences based on past publicationson other species of Acrididae, some within thesame genus.

Male: External and Internal Genitalia, andOther Parts of the Reproductive System

External: All but one of the seven named exter-nal components that we observed appear to beinvolved in copulation. The primary probable func-tion of the supra-anal plate, although, as in allother Acridoidea with eversible phallic complexes,is to act as the protective dorsal surface (a “roof”)for the genital chamber and its contents. Whenswung downwards, however, it may also serve asan effective wall to ensure against the possibilityof the female’s subgenital plate (Figs. 4B, 11B–H),or even debris, making its way further anterior

into the more delicate areas of the male’s repro-ductive and digestive systems.

During observations of live copulation attemptsthe cerci (Figs. 4A, 5A–E) were able to be flippedinwards and outwards, most likely due to muscu-lar action assuming the four muscles mentionedby Snodgrass (1935) are present that attach thecerci to both tergite 10 (Fig. 5C–E) and the supra-anal plate. Although not observed directly, a possi-ble reason for cerci inversion is to assist in scoop-ing the phallic complex up and out of the genitalchamber, with the possible assistance of the supra-anal plate and paraprocts (Figs. 4A, 5A–E). In thein copula 3D-reconstructions (Fig. 3A–E), the cerciare not inverted and are back in their originalpositions, which must occur at some point duringthe process of copulation, because the cerci havebeen observed to push against the female’s abdo-men, possibly for support during copulation and/orto further prevent the female from leaving. Uvarov(1966) and Chapman (2012) mention somethingsimilar occurring across Acrididae, but use theterm “grip” instead, although it is unclear if thissuggests the cerci are pushing against the female(as we observed) or are actually pulling her towardthe male. Kyl (1938) too, says the cerci “grasp theabdomen of the female” in Melanoplus differentia-lis (Thomas, 1865), but then goes further bydescribing that the effect of this action pulls downthe egg guide, opening the genital chamber of thefemale slightly. We know now that, more thanlikely, the epiphallus is the component responsiblefor this in all grasshoppers, but Snodgrass (1935)seems to clarify the possible gripping role of thecerci by noting that they “grasp the base of thesubgenital plate of the female.” Boldyrev (1929)goes further and actually calls cerci “organs ofattachment,” specifically in reference to Locustamigratoria (Linnaeus, 1758), but also notes thattheir primary purpose is as “organs of orientation.”Furthermore, it appears there might be a mildinvagination toward the basal end of the female’slateral basivalvular sclerites (Figs. 11B,C, 12A,B)that the apices of the cerci might be able to pushinto slightly (Fig. 1A–D), but further investigationis needed to clarify this issue.

Moreover, cerci in numerous insects have beenobserved to be sensory structures, sensitive tosounds, movement of air, and touch, primarily dueto the abundance of sensilla on them (Snodgrass,1935; Uvarov, 1966; Snodgrass, 1993; Gordh andHeadrick, 2001; Chapman, 2012). So far, for thePuer Group, the presence of such sensilla has yetto be confirmed, but the abundance of setae on thecerci of all species suggests it. Thus, it isquite possible that if such sensilla are present,they might be used as contact receptors duringcopulation and/or may even have functions notinvolved in copulation. Moreover, grasshopper cer-ci, which are often species-specific (particularly in



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Melanoplus) may be important for appropriatesensory stimulation and recognition of conspecificmales by the female, a hypothesis in need ofexperimental investigation (Eberhard, 1985).Finally, the cerci (at least the one on the right inthis case), in conjuction with the paraprocts,appear to assist in supporting the posterior portionof the apical third of the female’s subgenital platewhen it is bent downwards into the male’s genitalchamber (Figs. 3C, 10A).

The paraprocts (Figs. 4A, 5A–E) may have atleast two probable functions. The first one mightbe to assist in flipping the cerci down into the gen-ital chamber due to their membranous connection(Fig. 5A,B). When the supra-anal plate is swungdown into the genital chamber, it pushes the para-procts downwards as well and brings the cerciwith them. This proposed function, supported byexperimentation with freshly killed specimens inthe lab using fine-tipped forceps, is less likely, ifmuscles turn out to be present at the base of thecerci. When the supra-anal plate was pushedinwards with forceps, the paraprocts were as welland pulled the cerci with them. Evidence con-tained in the 3D-reconstructions (Figs. 3C, 10A)suggests the other possible function of the para-procts (at least the one on the right in this case) isthe same as the cerci: support for the posteriorportion of the apical third of the female’s subgeni-tal plate when it is bent downwards into themale’s genital chamber.

The obvious extension of the pallium (Fig. 5C–E) seems clear: because the aedeagus is one of thelongest in the Puer Group and is strongly curvedupwards, it needs an extended cover for protec-tion. When preparing for copulation, the rotatorsof phallus and retractors of pallium muscle, specif-ically the posterior portion (muscle 266, specifi-cally 266B in Eades, 2000) pulls the palliumdownwards, exposing the aedeagus’ apex. Theprobable function of the subgenital plate (Fig.5D,E) is not unique to this species and also seemsclear: it is able to stretch ventroposteriorly tosome degree via its medial membranous connec-tion, which, most likely, assists in moving thephallic complex somewhat toward the posterior ofthe genital chamber, so the supra-anal plate, para-procts, and cerci are able to swing inwards.

As mentioned, the function of the furculae (Figs.4A, 5A–E) during copulation is still unknown atthis time, although it is quite possible that they donot play a role. If they do, although, one sugges-tion is that they might possess sensory receptors(as seen on other male components) that mightassist in assessing the location of a female’s abdo-men during copulation. However, preliminarySEM observations of the furculae of different PuerGroup species did not reveal their presence.Regardless, it can be unquestionably stated thatthe furculae in M. rotundipennis stay rigid and

keep their original positions when the supra-analplate (Figs. 4A, 5A–E) is swung downwards intothe genital chamber (presumably via muscularaction along tergite 10; Fig. 3C). Numerous mem-bers of the Puer Group possess species-specific fur-culae, but others lack them entirely, and the sameobservations can be applied to other Acrididae spe-cies, further suggesting that they may not play arole in copulation.

Internal: All the named internal componentsthat we observed appear to be involved in copula-tion. The genital chamber encloses the phalliccomplex (Fig 6A) until the male is ready toattempt copulation. Then, the external genitaliccomponents are pushed out of the way (presum-ably via muscular action), so the phallic complexcan be partially everted dorsally by muscularaction and the inflation of surrounding mem-branes. The mechanism behind such inflation iscurrently unknown, but may be due to an increasein hemolymph-related hydrostatic pressure causedby muscular action (Whitman and Loher, 1984;Lawry, 2006; Chapman, 2012). Following that, theepiphallus (Figs. 6B,C, 7F,G) is pushed higherthan the other phallic complex componentsbecause it is the most dorsal and not directly con-nected to the rest. The ancorae (Figs. 4A, 6B,C,7F,G) of the epiphallus are then used as hooks(maybe individually or together) to catch the pos-terior margin of the female’s subgenital plate(Figs. 4B, 11B–H). This action pulls the subgenitalplate down into the anterior of the male’s genitalchamber, thus opening access to the female’s vul-va. More than likely, the sensory receptors on theanterior projections (Figs. 6B, 7F,G) and near theancorae (mentioned earlier and possibly seen herefor the first time in acridids) play roles in thisaction. In the 3D-reconstructions, also note thatthe epiphallus is not symmetrically aligned withthe female’s subgenital plate, but, rather, skewedslightly toward the left (Fig. 10B). This is mostlikely due to the fact that even though a male ismounted on top of a female, he must lower hisabdomen below and to either side of a female’sabdomen, resulting in asymmetrical copulation(Fig. 2C), which, in the case of the male used forthe 3D-reconstructions, was from the left side ofthe female (Figs. 1A–D, 14).

After the apical third of the female’s subgenitalplate is pulled down into the anterior of the male’sgenital chamber, the entire phallic complex isswung dorsally in a quick motion, continuing in acounterclockwise arc anteriorly with the majorityof the aedeagus (Figs. 4A, 7B,C, 9A) entering thefemale’s bursa complex through the vulva (notlabeled, but see Fig. 12A–H). Such a motion meansthat all phallic complex components (previouslydescribed in a state of repose; e.g., Fig. 6A) haverotated around 908 (or more), resulting in orienta-tions that can now be described as ventral/



anteriorly projecting (e.g., Fig. 4A). In these newin copula positions (Fig. 3A–E, 4A,C, 5A,B, 10A,B)is how descriptions of the probable function(s) ofthe remaining components of the phallic complexwill be described herein; referenced figures shouldbe carefully examined to maximize comprehension.Once rotation of the phallic complex has occurred,and (presumably) the aedeagus enters the female’sbursa complex, at least one lophus on the male’sepiphallus is able to be inserted into a female’scorresponding lophus receptacle of the subgenitalplate (Fig. 10B). There is little doubt that this pin-ning action ensures that the female’s subgenitalplate stays down to allow for continued access bythe male to the female’s internal genitalia. Thescales observed on the lophi (Fig. 9G) all but con-firm this hypothesis, especially in light of the factthat said scales are directed anteriorly (posteriorlyduring copulation) and would, thus, act as minia-ture friction anchors as a lophus is pushed into areceptacle and pulled down and back. If a femalewere found to possess similar structures that facein the opposite direction the friction effect shouldbe even greater. The other three subcomponents ofthe epiphallus: anterior projections, posterior pro-jections, bridge, and lateral plates (Figs. 6B,C,7F,G) appear to have no specialized function (apossible exception are the scattered sensory recep-tors on the anterior projections) other than 1) topotentially act as structural support during copu-lation, in some cases being able to flex to a degreeas evident by the shape of the bridge, and 2) topossibly also serve as attachment sites for muscles(e.g. lateral plates: Eades, 2000).

As mentioned previously, a male asymmetricallycopulates with a female, so, like the epiphallus,the ectophallus (Figs. 6D,E, 8A) and endophallus(Figs. 6F,G, 8A) are also asymmetrically alignedwith the female’s internal genitalia. The particularmale used for the 3D-reconstructions entered thefemale from her left side (Figs. 1A–D, 14), so thephallic complex components are therefore skewedslightly at an angle to the right meaning that theaedeagus also enters the female’s bursa complexat a slight angle (Fig. 3D). The fact that the phal-lic complex is able to be twisted a fair range man-ually with forceps supports the idea thatasymmetrical entry is possible, but we do notthink that it has been noted or demonstratedbefore. The probable functional role of the ecto-phallus overall is not fully clear compared to theother two phallic complex components. However,the apodemes of cingulum (Figs. 4A, 6D,E, 7B,C,E,9A) have a muscle attached to them called the pro-tractors of cingulum (only found in Melanoplus)that, when combined with the retractors of cingu-lum muscle (attached to the zygoma: Figs. 4A,6D,E, 7B,C, 9A; muscles 267XC and 278, respec-tively, in Eades, 2000) would most likely assist, inconjunction with other related muscles, in rotating

the phallic complex for copulation and back againto its resting state (Eades, 2000). Additionally, theectophallus acts as general structural support forthe entire complex and most likely as a leveragepoint for the aedeagus, particularly during itsinsertion into the female’s bursa complex (andextraction) and throughout spermatophore trans-fer during copulation. This leverage suggestionstems from the fact that the arch of aedeagus isinserted into the gap in the lower region of thezygoma (Figs. 7B, 10C) and it is quite possiblethat the upper region portion is extended to pro-vide limited articulation and structural support(acting as a plate to push against) for the aedea-gus during the copulation process.

For Melanoplinae, Eades (2000) made no men-tion of any muscles being attached directly to therami (Figs. 4A, 6D,E, 7B,D, 9A) and there do notseem to be any in this species (Fig. 7A), so it isstrongly possible there are none meaning that therami may just be supports and attachment pointsfor the sheath of aedeagus (Figs. 4A, 6D,E, 7B–D,9A). Note, although, that the rami appear to belaterally compressed inwards during copulation ascaptured in the 3D-reconstructions (Fig. 10C,D);such action may tighten the sheath around thedorsal valves of aedeagus for an unknown reasonor may just be a by-product of muscle movementelsewhere. Additionally, it is currently unknownwhat role is played by the sensory receptorsobserved on the posterior apices of the rami. Theprobable function of the minute spines seen on thesheath of aedeagus (Fig. 9F) that are potentiallynew to science is also unknown at this time, but,given their placement in relation to the female’sanatomy (Fig. 4C), it is feasible that they eitherplay a role in stimulating the region surroundingthe female’s vulva (Eberhard, 1985) or act as addi-tional grippers (possibly for gripping the ventralside of her abdomen), further securing the male’saedeagus to the female, but they do not appear tobe sensory receptors for the male’s use.

The subcomponents of the endophallus (Figs.6F,G, 8A–E, 10E), and the associated subcompo-nents of the greater reproductive system (Figs.6H–J, 7A), serve numerous probable functionsduring copulation. The apodemes of endophallus(Figs. 4A, 6F,G, 7B–E, 8B,D, 9A) provide attach-ment points for a number of muscles with manyprobable functions related to spermatophore pro-duction and insemination (Snodgrass, 1935; Rob-erts, 1941; Gregory, 1965; Hartmann, 1970;Pickford and Gillott, 1971; Whitman and Loher,1984; Snodgrass, 1993; Eades, 2000), but themajority of these muscles will not be discussedhere as it is outside of this study’s scope. Insemi-nation results from sperm contained within thesemen contained in a male’s spermatophore (Figs.4A, 6H–J) and the process of its creation can differfrom species to species, so it will only be briefly



touched on here, based on synthesized evidencefrom studies of other grasshoppers, including someMelanoplus species (Boldyrev, 1929; Snodgrass,1935; Kyl, 1938; Roberts, 1941; Gregory, 1965;Uvarov, 1966; Hartmann, 1970; Pickford and Gil-lott, 1971; Whitman and Loher, 1984; Snodgrass,1993; Eades, 2000; Chapman, 2012). The first stepin the process usually begins shortly after a malebegins copulating with a female, which signals thetransfer of accessory gland secretions to move intothe ejaculatory duct (Figs. 4A, 6I,J, 7A). Thesesecretions are used to construct the spermato-phore’s reservoir (Fig. 6H–J), followed by its tuberegion (Figs. 4A, 6H–J), and then, it begins to fillwith semen. Next, the reservoir and tube regionmove to the ejaculatory sac (Figs. 4A, 6I,J, 7A)where they are enlarged and continue to fill withsemen. Then, the adductor of endophallic apo-demes (muscle 283 in Eades (2000), extendingbetween the two apodemes of endophallus) iscontracted, laterally separating the gonopore pro-cesses (Figs. 6G, 7B,D, 8B, 10E) and openingthe gonopore (not pictured). This transfers thespermatophore from the ejaculatory sac to thespermatophore sac (Fig. 6I,J) in which the sperma-tophore’s reservoir will remain to continually addsemen to the spermatophore’s tube region duringcopulation until empty. Finally, via rhythmic con-tractions of the muscles surrounding the sperma-tophore sac, the semen-filled tube region ispumped through the phallotreme (which furthershapes the spermatophore into its final tubularform), out of the aedeagus (Fig. 4A), and into thefemale (Fig. 4C), followed by the eventual ejectionof the spermatophore from the body of both sexeson the completion of sperm transferal. The exactsources of these muscular contractions are stillbeing debated, but it has been suggested that oneor more of the following muscles are responsible:adductor of endophallic apodemes, outer compres-sors of ejaculatory sac (muscle 2813 in Eades,2000), and/or inner compressors of ejaculatory sac(muscle 284 in Eades, 2000).

The probable function of the connective tissue ofapodemes of endophallus (Figs. 4A, 6F,G, 7C,E,8D), seemingly not previously identified, named,or described (composition is reminiscent of thefemale’s egg guide: Figs. 4B, 11D–H) by otherstudies, is not fully clear at this time. More thanlikely, although, based on examination with for-ceps and consideration, it serves to snap the apo-demes back into their starting positions after eachcontraction by the adductor muscles (muscle 283in Eades, 2000) that cover most of it, so that fur-ther musculature is not needed to expand the apo-demes again; in this way, the connective tissuewould act in a similar manner as a spring-loadedbellows. The flexures (Figs. 6G, 7B, 8B, 9A, 10E),the membranes connecting them to the valves ofaedeagus (Fig. 8A,B), and the articulations

between these subcomponents (not labeled, but seeFigs. 6G, 7B, 8B, 10E) possibly function, as otherphallic complex components appear to, as supportand leverage points for the dorsal and ventralvalves, allowing the aedeagus greater movementand positioning ability (Whitman and Loher,1984).

Based on the 3D-reconstructions, the aedeaguscan be inserted into the bursa complex almostentirely, its apex reaching just to the bend in thebursa complex, with essentially only the portiontouching the sheath of aedeagus left outside thefemale (Fig. 4C). The mid-basal region of the dor-sal valves of aedeagus appear to rest, and mostlikely push downwards, on the female’s vulvalsclerite (Fig. 4C). Such support may assist themale in guiding his aedeagus into the bursa com-plex and additionally offer purchase during inser-tion and extraction as well as spermatophoretransfer. The tiny, leaf-like posterior-oriented pro-jections observed all over the exterior of the dorsalvalves (Fig. 9A,D,E) were seemingly observed foronly the second time in at least Acridoidea andthe first in Acrididae, despite Eades (2000) com-menting that it was yet unknown if such micro-structures were confined to the species they wereoriginally found on (T. eques in Whitman andLoher, 1984) or if they might be found in otherspecies of Acridomorpha. The probable function ofthese projections is not entirely clear here, but twopossible explanations are as follows: 1) as stimula-tors for the interior of the female’s bursa copula-trix (Figs. 4B, 12B–D,F,G, 13E–G) as suggestedbroadly by Eberhard (1985) and/or 2) as friction-structures for improved gripping while insertedinto the bursa copulatrix as suggested for T. equesby Whitman and Loher (1984).

The only functions of the ventral valves (Figs. 4A,6F,G, 7B–E, 8B–E, 9B, 10E) hypothesized at thistime for this species are assisting in the shapingand guiding of the spermatophore (Figs. 4A, 6H–J).As the semi-gelatinous spermatophore is exudedthrough the phallotreme during copulation itappears to mostly rest between the ventral valves,eventually passing further through them dorsallynearer to their apex, emerging beyond both sets ofvalves, and then into the apical remainder of thebursa copulatrix (Fig. 4C). The ventral valves ofaedeagus, it should be noted, are quite soft and flex-ible compared to the much more rigid, and seeming-ly inflexible, dorsal valves, so it would make sense ifthe primary, if not only, probable functions of theventral valves for this species are spermatophoreshaping and guidance. Interestingly, in T. eques, thedorsal valves only possess transverse ridges while itis the ventral valves that have their exterior cov-ered in microstructures that are described asbackward-pointing spines. These spines serve mul-tiple functions during copulation in T. eques, includ-ing anchoring the aedeagus to the basal region of



the female’s spermathecal duct (a bursa copulatrixis not present) and assisting in the removal of emp-ty spermatophores, so that more can be transferredduring a single copulation session (Whitman andLoher, 1984). In contrast, in M. rotundipennis, it isthe dorsal valves that contain the microstructures,which are far from being spines (we have describedthem as “leaf-like projections”) and are not asdensely packed. Furthermore, it is doubtful thatthese microstructures could assist in spermato-phore removal because the dorsal valves are fusedfor almost their entire length as opposed to theindependently articulating structures in T. eques.Finally, the ventral valves appear to lack any sort ofridges and only seem to be rugose (Fig. 9C), but, sofar, only the apical third have been examined usingSEM.

As a whole, the phallic complex is consistent insize and shape across the geographic range of thespecies. Slight differences do exist, although, par-ticularly in the lophi of epiphallus (Figs. 4A, 6B,C,7F,G) and apodemes of cingulum (Figs. 4A, 6D,E,7B,C,E, 9A; Squitier et al., 1998). Such differencesin grasshoppers may be unique to an individual,population-specific [e.g., in Schistocerca lineataScudder, 1899 (Acrididae) (Song and Wenzel,2008)], or even simply developmental in nature[e.g., in S. americana (Drury, 1770) (Song, 2004)].

Female: External and Internal Genitalia, andOther Parts of the Reproductive System

External: The only named external componentsthat we observed that appear to be involved in copu-lation are the subgenital plate (Figs. 4B, 11B–H),its lophi receptacles (Fig. 11E–H), and, possibly, theattached egg guide [Figs. 4B, 11D–H; its primaryfunction appearing to be to guide an emerging eggupwards into the intervalvular space (Snodgrass,1935)]. The subgenital plate and its egg guide, intheir normal state of repose, block entry to the vul-va, which is centered just beneath the base of theventral valves of ovipositor (Figs. 11A–D, 12A,B).The egg guide, at rest, passes dorsally between theventral valves (Fig. 11D). When the male’s ancorae(Figs. 4A, 6B,C, 7F,G) pull the apical third of thefemale’s subgenital plate and egg guide downwardsinto his genital chamber, her vulva is exposedthrough which the male is able to insert his aedea-gus into the female’s bursa complex (Fig. 4C). Thelophi receptacles then receive at least one lophusfrom the male’s epiphallus per copulation event (inthis case, the right lophus: Fig. 10B), which mostlikely acts as a friction anchor to keep the subgeni-tal plate and egg guide from being retracted and,thus, close the vulva. Note again that the heaviersclerotization of the apical third of the subgenitalplate (Fig. 11G,H) is also the portion that is bentdownwards at an approximately 908 angle (Figs.4B, 11E,F). The fact that a bend takes place where

the sclerotization level lessens is structurally logi-cal, but also because the entire subgenital plate isquite long and would not fit into the male’s genitalchamber anyway.

In the 3D-reconstructions, the egg guide, which isapparently highly flexible, can be seen being bentupwards and to the left near its middle (Fig.10A,B). However, what the egg guide is pushingagainst is not shown and was not clearly observed,but, based on its position, just anterior to the male’sapodemes of cingulum (Fig. 4C), it can be inferredto be the muscles that surround the ectophallus andendophallus of the male’s phallic complex. The posi-tion of the egg guide gives rise to three possibilities:1) that it is simply random chance, 2) the egg guideprovides some level of support for the male to pushagainst because the subgenital plate is essentiallyhollow, or 3) a mixture of both meaning that theposition can change, but the egg guide could still beutilized by the male for support.

Internal: The only named internal componentsthat we observed that appear to be involved incopulation are the genital chamber, bursa complex(Fig. 13A,E), and vulval sclerite (Figs. 4B, 12A–E,G,H, 13B–E). The genital chamber, other thancontaining the internal genitalia and other partsof the greater reproductive system, seeminglyplays only a single, but important, role in copula-tion: without the subgenital plate being pulleddownwards by the male, thereby essentially wid-ening the genital chamber dorsoventrally, hewould not be able to access the vulva and beginthe copulation process. Collectively, the bursa com-plex receives the male’s aedeagus (Figs. 4A, 7B,C,9A) and spermatophore (Fig. 4A, 6H–J), althoughneither directly contact the armor of bursa copula-trix (Figs. 4B, 12B–D,F,G, 13E–G). The ribbing ofthe armor (Fig. 12D,G) probably serve one to twofunctions, the first being reinforcement tostrengthen the bursa copulatrix (Figs. 4B, 12B–D,F,G, 13E–G) from either strong movements ofthe aedeagus during insertion and extraction aswell as spermatophore transfer, or contact withthe leaf-like projections that cover the male’s dor-sal valves of aedeagus (Fig. 9A,D,E). The secondpossible function could be expansion in that theribs may allow for the flexible membrane-like por-tions between them to stretch, akin to an accordi-on, in order for the bursa copulatrix to betteraccommodate aedeagi, perhaps of varying sizes,and their movements (and possibly even those ofthe spermatophore).

The bursa copulatrix (Figs. 4B, 12B–D,F,G,13E–G) appears to function as the primary recep-tacle for the male’s aedeagus (Figs. 4A, 7B,C, 9A)and may be lined with nerve endings for the possi-ble purpose of stimulation (Eberhard, 1985) by theleaf-like projections on the aedeagus (Fig.9A,D,E). Another possibility is that the bursa cop-ulatrix is lined with rough material, possibly the



afore-mentioned light sclerotization (Fig. 12F),that the male is able to grip onto via friction. Thebulbous apex appears to be able to shift in size(Fig. 12B) and may do so to accommodate theincrease in size of the apex of the male’s sperma-tophore (Figs. 4A,B, 6I,J) as semen is continuallyadded from the spermatophore’s reservoir into itstube region (Figs. 4A, 6H–J). Presumably, theapex of the bursa copulatrix continues to expanduntil the spermatophore’s apex ruptures (Fig.6H), most likely sending semen into the sperma-thecal duct (Figs. 4B, 12A, 13H,I) through thespermathecal aperture where the sperm wouldmake their way toward the spermathecae (preapi-cal and apical diverticula) (Figs. 4B, 12A, 13H,I).In addition to the observations of the 3D-reconstructions, this hypothesis is based on evi-dence from previous studies on other species, twoalso belonging to Melanoplus, in which a sperma-tophore (and in the non-Melanoplus species, theaedeagus) was introduced directly into thefemale’s spermathecal duct where it rupturedeither further in or at the first narrow U-shapedbend, a structurally limiting situation similar towhat we have observed in M. rotundipennis (Kyl,1938; Gregory, 1965; Pickford and Gillott, 1971;Whitman and Loher, 1984; Lay et al., 1999). Themuscle tissue of spermathecal complex (partiallyseen in Figs. 12A, 13H) is presumably responsiblefor moving the sperm back toward the eggs forthe purpose of fertilization (Snodgrass, 1993). Thevulval sclerite (Figs. 4B, 12A–E,G,H, 13B–E) maybe an uncommon component in grasshopper geni-talia because it has only seemingly been identi-fied once previously in a distantly related genus,Praxibulus Bol�ıvar, 1906 (“dorsal sclerite of bursacopulatrix” in Key, 1989), and is only found in afew other Puer Group species. In addition toseemingly serving as reinforcement to theentrance to the bursa copulatrix and as the con-nector between the latter and the vulva, thissclerite possibly also functions as a support platefor the male’s aedeagus to slide along and pushagainst during insertion and extraction as well asspermatophore transfer.

On the whole, the 3D-reconstructions of all com-ponents of both sexes reflect the results gainedusing DSLR and SEM imaging methods with onedifference for an internal female component: the“tail” of the bursa complex (so-called here becauseof its overall resemblance to a scorpion’s tail asnoted earlier) is bent ventrally at rest (e.g., Fig.12B), but is bent dorsally when the aedeagus hasbeen inserted and the spermatophore is beingtransferred (e.g., Fig. 4C). We know the entire bur-sa complex has not been rotated for two reasons:1) the vulval sclerite (e.g., Figs. 12B, 13E) is ori-ented correctly and 2) the membranes anchoringthe base of the bursa complex to the body (Fig.12A) would not allow for full rotation, anyway.

Before the 3D-reconstructions were completed, itseemed apparent that the length and curvature ofthe aedeagus had evolved to fit better into the sim-ilarly long and curved bursa complex (the fact thatit naturally bends left or right may not be a factoras it appears to be flexible: Fig. 12B). In fact, simi-larly matched structures between the sexes alsoappear to exist in many of the Puer Group speciesdissected and examined, but all of these situationsrequire much further investigation. The final 3D-reconstructions of M. rotundipennis, although,while not necessarily disputing the idea of corre-sponding genitalic structures, do complicate mat-ters. This is because it appears that even if the“tail” of the bursa complex was bent in the restingventral position the aedeagus would barely curveinto it anyway since almost the entirety of itslength has already been inserted (Fig. 4C). Thespermatophore, in the 3D-reconstructions, is thestructure that moves the rest of the way throughthe bursa complex and into the apex of the bursacopulatrix (Fig. 4C); it seems possible the sperma-tophore would do this regardless of “tail” orienta-tion as it is essentially a tube being pushedthrough a slightly larger tube. Therefore, a reasonfor the ventroposteriorly curving nature of thecomplex is still unclear, but because at leastpopulation-level variation is known to exist (Squit-ier et al., 1998), it is plausible that variation inthe length of aedeagi also exists. Another possiblereason for the length of the bursa complex is tosimply give the spermatophore more room to enteras it does not seem that the spermatophore wouldbe able to move beyond the spermathecal apertureonce its apex becomes engorged with semen.

As for why the 3D-reconstructions and the otherimaging methods differ, we offer two plausibleexplanations: 1) the freezing method used to cap-ture copulation also caused the “tail” and sperma-tophore to shift. We know this occurred with thefemale’s valves of ovipositor (Fig. 11B) because thedorsoventral widening observed here (Fig. 1A–D)occurs on all female specimens frozen at 2808C.Normally, based on multiple observations of labpopulations, the valves are kept in a “closed” stateduring copulation and such forced widening mayalso have affected musculature. This may alsohave shifted other linked components, althoughnothing other than the “tail” was noticed. 2) Pres-sure from the inserted aedeagus and/or theextruding spermatophore may have shifted the“tail’s” position, meaning that the movement ofthis component is entirely natural. Clearly, aswith many other discussed items, this situationwarrants further investigation.

ACKNOWLEDGMENTS

Thirty-one individuals and groups across the U.S.and the world made this study possible and we



wish to thank them for their contributions: first andforemost, for facilitating micro-CT scanning, photo-graphing the micro-CT sample, assisting withAmira comprehension, and suggesting manuscriptimprovements: B. Wipfler, Jena Institut f€ur Spe-zielle Zoologie und Evolutionsbiologie mit Phyleti-schem Museum, Friedrich-Schiller University,Germany; micro-CT scanning assistance: S. Ge,Institute of Zoology, Chinese Academy of Sciences,Beijing; critical point drying: S.M. Fullerton (1940–2014) and S.L. Kelly (who also helped collect speci-mens), Department of Biology, University of CentralFlorida (UCF); SEM use, training, and scheduling:Y. Sohn, K. Scammon, and K. Glidewell, MaterialsCharacterization Facility, AMPAC, UCF; specimenloans: M.F. O’Brien, Collections Manager, UMMZ’sInsect Division and P. Skelley, FSCA EntomologySection Administrator; field assistants (UCF under-graduate/graduate students, friends, and familymembers) who collected specimens via FloridaDepartment of Environmental Protection, Divisionof Recreation and Parks, Florida Park ServiceResearch/Collecting Permits #10031210 and05281410 (chronological order): S. Gotham, C. Gale,E. Kosnicki, E. Kerr-Woller (and for continuedencouragement), P.H. and K.L. Woller, I.J. Stout,and A.B. Orfinger; guides to the micro-CT popula-tion: B. Gochnour and H. Stephens; micro-CT dataassistance and software training: T. Porri, ResearchAssociate with the Cornell Biotechnology ResourceCenter Imaging Facility; Amira Customer SupportSpecialists, especially K. Tinoco; PDF3D’s technicalsupport team; 3D PDF beta tester: M.L. Ferro;manuscript improvements: D.C. Eades, J.G. Hill, M.Schmitt, and two anonymous reviewers.

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