Proteomic diversification of spermatostyles among six species of whirligig beetles.

Seminal fluid protein composition is complex and commonly assumed to be rapidly divergent due to functional interactions with both sperm and the female reproductive tract (FRT), both of which evolve rapidly. In addition to sperm, seminal fluid may contain structures, such as mating plugs and spermatophores. Here, we investigate the evolutionary diversification of a lesser-known ejaculate structure: the spermatostyle, which has independently arisen in several families of beetles and true bugs. We characterized the spermatostyle proteome, in addition to spermatostyle and FRT morphology, in six species of whirligig beetles (family Gyrinidae). Spermatostyles were enriched for proteolytic enzymes, and assays confirmed they possess proteolytic activity. Sperm-leucylaminopeptidases (S-LAPs) were particularly abundant, and their localization to spermatostyles was confirmed by immunohistochemistry. Although there was evidence for functional conservation of spermatostyle proteomes across species, phylogenetic regressions suggest evolutionary covariation between protein composition and the morphology of both spermatostyles and FRTs. We postulate that S-LAPs (and other proteases) have evolved a novel structural role in spermatostyles and discuss spermatostyles as adaptations for delivering male-derived materials to females.

Morphology of male and female reproductive systems in the ground beetle Apotomus and the peculiar sperm ultrastructure of A. rufus (P. Rossi, 1790) (Coleoptera, Carabidae).

Relatively few studies have focused on evolutionary losses of sexually selected male traits. We use light and electron microscopy to study the male and female reproductive anatomy of Apotomus ground beetles (Coleoptera, Carabidae), a lineage that we reconstruct as likely having lost sperm conjugation, a putative sexually selected trait. We pay particular attention to the structure of the testes and spermatheca. Both of these organs share a strikingly similar shape-consisting of long blind canals arranged into several concentric overlapping rings measuring approximately 18 mm and 19.5 mm in total length, respectively. The similarity of these structures suggests a positive evolutionary correlation between female and male genital organs. Males are characterized by unifollicular testes with numerous germ cysts, which contain 64 sperm cells each, and we record a novel occurrence of sperm cyst "looping", a spermatogenic innovation previously only known from some fruit fly and Tenebrionid beetle sperm. The sperm are very long (about 2.7 mm) and include an extraordinarily long helicoidal acrosome, a short nucleus, and a long flagellum. These findings confirm the structural peculiarity of sperm, testis, and female reproductive tract (FRT) of Apotomus species relative to other ground beetles, which could possibly be the result of shifts in sexual selection.

Novelty and emergent patterns in sperm: Morphological diversity and evolution of spermatozoa and sperm conjugation in ground beetles (Coleoptera: Carabidae).

The beetle family Carabidae, with about 40,000 species, exhibits enough diversity in sperm structure and behavior to be an excellent model system for studying patterns and processes of evolution. We explore their potential, documenting sperm form in 177 species of ground beetles using light microscopy and collecting data on one qualitative and seven quantitative phenotypic traits. Our sampling captures 61% of the tribal-level diversity of ground beetles. These data highlight the notable morphological diversity of sperm in ground beetles and suggest that sperm in the group have dynamic evolutionary histories with much morphological innovation and convergence. Sperm vary among species in total length (48-3,400 µm), head length (0.5-270 µm), and head width (0.2-6.3 µm). Most ground beetles make sperm with heads that are indistinct from the flagella at the gross morphological level. However, some or all Omophron, Trachypachus, and Dyschiriini make broad-headed sperm that show morphological differences between species. Most ground beetles package their sperm into groups of sperm, termed conjugates, and ground beetles show variation in conjugate form and in the number and arrangement of sperm in a conjugate. Most ground beetles make sperm conjugates by embedding their sperm in a hyaline rod or spermatostyle. The spermatostyle is remarkably variable among species and varies in length from 17 to 41,000 µm. Several unrelated groups of ground beetles make only singleton sperm, including Nebriinae, Cicindelinae, many Trechinae, and the tribe Paussini. In order to study patterns in sperm evolution, we combine these data with a low-resolution phylogeny of ground beetles. Results from modern comparative analyses suggest the following: (a) sperm differ from conjugates in some aspect of their underlying evolutionary process, (b) sperm have influenced conjugate evolution and vice versa, and (c) conjugation with a spermatostyle likely evolved early within the history of Carabidae and it has been lost independently at least three times.

Phylogenetic placement of the Pacific Northwest subterranean endemic diving beetle Stygoporus oregonensis Larson & LaBonte (Dytiscidae, Hydroporinae).

Stygoporus oregonensis Larson & LaBonte is a little-known subterranean diving beetle, which, until recently, had not been collected since the type series was taken from a shallow well in western Oregon, USA, in 1984. Here we report the discovery of additional specimens collected from a nearby well in the Willamette Valley. Sequence data from four mitochondrial genes, wingless, and histone III place Stygoporus Larson & LaBonte in the predominantly Mediterranean subtribe Siettitiina of the Hydroporini. Morphological support for these results is discussed, and details of the collecting circumstances of the new specimens are presented. We argue that the biogeographic patterns of Nearctic Siettitiina highlight the likelihood of additional undiscovered subterranean dytiscids in North America.

Up high and down low: Molecular systematics and insight into the diversification of the ground beetle genus Rhadine LeConte.

Rhadine LeConte is a Nearctic genus of flightless ground beetles that is poorly studied despite its relevance to evolutionary studies of subterranean fauna. Adults are notable for their slender and leggy habitus and the wide variety of habitat preferences among species, with several known only from mountaintops while others are restricted to caves or more general subterranean habitats. In central Texas, USA there are several cave endemics relevant to conservation. Here we present the first phylogenetic hypothesis for the overall structure of the genus with an emphasis on the troglobites in central Texas. We infer the phylogeny of Rhadine from ∼2.4-kb of aligned nucleotide sites from the nuclear genes, 28S rDNA and CAD, and the mitochondrial gene COI. These data were obtained for 30 species of Rhadine as well as from members of their putative sister group, Tanystoma Motschulsky. Results reveal that Rhadine is polyphyletic, and morphological characters that have been traditionally used to classify the genus into species groups are shown to be convergent in many cases. Rhadine aside from two species of uncertain placement is composed of two major clades, Clades I and II that both include epigean and subterranean species in very unequal proportions. Clade I is primarily composed of subterranean species, and Clade II includes many epigean species and high altitude montane endemics. A clade of troglobitic, cave-restricted species in Texas includes several species of large-eyed cave Rhadine. The slender habitus typical of some species [e.g., R. exilis (Barr and Lawrence), R. subterranea (Van Dyke), R. austinica Barr] evolved independently at least three times. Major biogeographic and evolutionary patterns based on these results include: troglobitic species north of the Colorado River in Texas (that also lack lateral pronotal setae) are found to comprise a monophyletic group, beetles in caves south of the Colorado River likely form another monophyletic group, and the "species pairs" of troglobitic Rhadine known to occur in the same caves are not resolved as each other's sister group, suggesting that these caves were colonized independently by more than one lineage of Rhadine. Our divergence time estimates support a Miocene age for the split between Clade I and II Rhadine and indicate that all subterranean Clade I Rhadine began diversifying in the late Miocene-early Pliocene, contemporary with cave formation in the Balcones Escarpment.

Prionohydrus, a New Genus of Noterini Thomson (Coleoptera: Noteridae) From South America With Three New Species and Its Phylogenetic Considerations.

A new genus of Noterini, Prionohydrus Gómez and Miller gen. n., and three new species are described from South America: Prionohydrus matogrossensis Gómez and Miller sp. n. from Brazil and Prionohydrus marc Gómez and Miller sp. n. and Prionohydrus ubercornis Gómez and Miller sp. n. from Venezuela. The new genus was scored for characters presented in a recent phylogenetic analysis of the family, and key morphological characters are illustrated, including male and female genitalia. A parsimony analysis of these data resulted in a single, most parsimonious solution, with members of the new genus resolved as sister to Mesonoterus Sharp with moderate support. Prionohydrus is similar to Mesonoterus and can be distinguished from this and other Noterini based on a combination of the following: 1) distal attachment of protarsus to protibia; 2) posterior protibial spine present; 3) single, weakly curved, apical protibial spur present; 4) last abdominal tergum with large medial spine flanked by numerous setae; 5) posterior mesotibial spur strongly serrate; 6) anterior metatibial spur strongly serrate; 7) prosternal process narrow, rounded apically, and widest near the middle; 8) prosternum longitudinally broad and glabrous; 9) weak, angular setae along posterodistal margin of metafemur present; 10) pronotal bead moderately broad; 11) cluster of setae at apex of medial metacoxal lobe present; and 12) antenna of males expanded, from slightly incrassate to strongly incrassate.

A phylogenetic review of the North American band-winged grasshopper genus, Encoptolophus Scudder with description of Nebulatettix gen.n. (Orthoptera: Acrididae: Oedipodinae).

A taxonomic review of the North American band-winged grasshopper genus Encoptolophus Scudder (Orthoptera: Acrididae: Oedipodinae) was conducted. This genus is hypothesized to be non-monophyletic following a cladistic analysis of the genera in the Chortophaga genus group. We examined all species currently classified in this genus group for morphological characters and one behavioral character. The phenotypic character data were combined with three mitochondrial genes: cytochrome c oxidase subunit II, 16S rRNA and 12S rRNA. A parsimony analysis was performed on the combined data resulting in two equally parsimonious trees. Encoptolophus, as historically defined, is resolved in three separate clades. The results support erection of a new genus, Nebulatettix Gómez, Lightfoot & Miller gen.n. to comprise one of the groups historically classified in Encoptolophus. In addition, we transfer the species Encoptolophus californicus Bruner to Chimarocephala Scudder, comb.n., a combination used historically. The evolution of certain characters in the Chortophaga group is discussed, and a key to the genera is provided.