A second locality for the Namib darkling beetle Onymacris brainei (Tenebrionidae, Coleoptera) and first report on its molecular phylogenetic placement

Abstract Onymacris brainei Penrith, 1984 – the most recent species of Onymacris to be described – is known only from its type locality in the Namib Desert, adjacent to the Cunene River in northern Namibia. No additional specimens are known to have been collected beyond the type series. Herein, we report on eight specimens discovered at a second site near the original locality. DNA from four beetles was used to determine the phylogenetic placement of O. brainei among congeners, based on sequence data from one nuclear (histone III) and two mitochondrial (cox1, cox2) genes. Maximum likelihood analysis identifies O. brainei as a member of the ‘white’ Onymacris clade, in which it forms a strongly supported subclade with O. bicolor and O. marginipennis. More broadly, its phylogenetic placement augments previous molecular results that revealed a sister taxon relationship between the ‘white’ Onymacris and a second genus, Physadesmia. The paraphyly of Onymacris with respect to Physadesmia highlights a need for nomenclatural change, but revision should await acquisition of genetic data for the few species outstanding in both genera.


Introduction
The darkling beetle genus Onymacris is a diverse assemblage of fast-running diurnal species endemic to Africa's Namib Desert and adjacent southwestern edges of the Kalahari. As substrate specialists, these beetles are restricted to loose sand that characterizes hummocks, dry riverbeds, and dune fields, where they occur in abundance and assume key ecological roles as detritivores (Louw 1983;Hanrahan and Seely 1990). The genus belongs to the flightless tribe Adesmiini, which includes 240+ species and is distributed largely within southwest Africa-a geographic center where adesmiines exhibit their greatest ecological breath and morphological diversity (Koch 1962;Penrith 1979). Regionally, Onymacris represents one of the tribe's more species-rich genera, with 26 currently recognized taxa (14 species and 12 subspecies) that include distinctive 'white' species, whose elytral color ranges from pure white to yellow or tan (Fig. 1). White elytral coloration, an unusual characteristic among beetles in general and darkling beetles in particular, is one of many remarkable evolutionary features observed among Namib tenebrionids that are unknown in beetles from other deserts (Hamilton and Seely 1976;Endrödy-Younga 1978;Roberts et al. 1991).
'White' Onymacris are restricted to the northern Namib, where they are patchily distributed, often with limited geographic ranges. Onymacris brainei-the most recent member of the genus to be described (Penrith 1984)-represents this case in the extreme: it is known only from the type locality in northern Namibia, just south of the Cunene River along the Angolan border (Fig. 2). Steven Braine collected the first specimens (3 males, 2 females) on 24 February 1983 and brought them to the attention of Mary-Louise Penrith, who at that time was actively revising the southern African Adesmiini (Penrith 1975(Penrith , 1979(Penrith , 1984(Penrith , 1986. Early in the following year (12-15 February 1984), Penrith and Ruth Müller collected 16 additional specimens at the original locality, which provided sufficient material for describing the new species, named in Braine's honor (Penrith 1984). Onymacris brainei is distinguished from other 'white' species by the presence of three broad, pale yellow to tan stripes on otherwise white elytra (Fig. 3).
To our knowledge, no other specimens or localities for O. brainei have been documented since its description. In 2015, some 30 years after Penrith and Müller's expedition, we searched the general vicinity of the type locality in an attempt to update the status of O. brainei. Herein, we report on eight additional specimens taken from a second site. Importantly, these beetles provided a source of fresh tissue for DNA extraction, gene sequencing, and phylogenetic analysis. Hence we also offer the first report on the molecular phylogenetic placement of O. brainei among its congeners. Penrith (1984) reported the type locality as "Kunene R. east of dunes at 17.12S, 12.10E," where beetles were collected "on dune hummocks." Working from this geographic ap-  (Fig. 2). Several beetles were observed, of which eight specimens were captured, euthanized (ethanol injection), and carded.

Molecular phylogenetic analysis
Rear legs from four of the eight beetles were preserved in RNAlater for subsequent DNA isolation using Qiagen's DNeasy kit. The mitochondrial genes cytochrome oxidase I (cox1) and cytochrome oxidase II (cox2) and a nuclear gene (histone III, H3) were amplified using the primers and PCR conditions listed in Table 1. Amplicons were cleaned using exoSAP-IT (USB Corp.) and sequenced on an Applied Biosystems 3130 capillary sequencer. Sequences were edited and assembled in Sequencher 4.9 (GeneCodes, Ann Arbor, MI) and aligned using ClustalX ver. 2.0 (Larkin et al. 2007), after which sequences were translated to ensure a correct reading frame. Sequences are available through GenBank (Table 2).
DNA sequences for O. brainei were combined with sequence data previously generated for Onymacris (Table 2) to yield a concatenated dataset-cox1 (1212 bp), cox2 (688 bp), and H3 (317 bp)-representing 18 of the 26 currently recognized species/ We also incorporated species sequences representing three additional adesmiine genera: Physadesmia (represented by P. globosa), shown to be the sister taxon to the white Onymacris clade (Lamb and Bond 2013) as well as Eustolopus octoseriatus and Adesmia cribripes, which served as outgroups.
We used maximum likelihood (ML) to analyze the concatenated gene dataset. The ML analysis, executed in RAxML ver. 7.2.8 (Stamatakis 2006), comprised 1,000 random sequence addition replicates (RAS) using the commands "-# 1000" and "-m GTRGAMMA." Bootstrap support values were calculated using the same search parameters with 1,000 replicates, and bootstrap results were applied to the best tree recovered in the RAS search.

New locality for Onymacris brainei
The second locality for Onymacris brainei was discovered on 22 May 2015. Based on the general geographic information provided in Penrith (1984), this new site is estimated to lie ~ 15-20 km SSW of the type locality (Fig. 2). The second site closely resembles the original locality's physical and ecological description, characterized by vegetated dune hummocks on which nara (Acanthosicyos horridus), an iconic Namib endemic, is the prevalent floristic component. Beetles were observed under and, in some cases, on hummock vegetation.

Elytral color variation
As noted, Onymacris brainei is diagnosed by the presence of three broad yellow to tan stripes on white elytra. Specifically, this patterning involves a prominent dorsal stripe that is bisected by the elytral suture and flanked by a slightly narrower lateral stripe on either side. All three stripes bear diffuse edges that coalesce anteriorly near the pronotum, taper posteriorly, and terminate at (or just before) the apical declivity. Table 2. GenBank accession numbers for adesmiine sequences used in the ML analysis.

Species
GenBank White elytral coloration is not due to any pigment product but rather a function of reflectivity involving microscopic "bubbles" within the cuticle (Kühnelt 1957). Thus, the stripes represent pigment expression within an otherwise colorless elytral matrix. Penrith (1984) noted that both stripe width and degree of pigment suffusion between stripes varied considerably across the type series. Our eight specimens of O. brainei exhibit comparable levels of dorsal color variation (Fig. 3).

Genetic variation
DNA sequences were invariant for the nuclear gene H3 but did exhibit variation for both mitochondrial genes (two haplotypes for each gene); mean sequence divergence for the cox1 and cox2 was 1.49 % and 0.05%, respectively.

Molecular phylogenetic placement of Onymacris brainei
ML analysis of the concatenated dataset identified O. brainei as sister to O. marginipennis + O. bicolor in a highly supported clade (BS = 100%) that is sister to a second 'white' clade comprising the three subspecies of O. langi represented in our dataset (Fig. 4). Overall, the ML topology is essentially identical to ML and Bayesian phylogenies previously derived from a larger multilocus dataset (Lamb and Bond 2013), which not only identified two distinct, well supported clades -one containing all 'white' species, the other, exclusively black species -but also revealed that Onymacris is paraphyletic. All three phylogenies [i.e., this report; Lamb and Bond (2013)] depict Physadesmia globosa as the sister taxon to the 'white" Onymacris lineage in a highly supported clade (herein, BS = 99%).

Discussion
In her paper originally describing Onymacris brainei, Penrith (1984) also reported the first cladistic analysis for the genus Onymacris, based on 23 morphological characters.
To her credit, she examined several additional characters but rejected them "owing to suspected parallelism" or because "the direction of development could not be ascertained." Her analysis recovered two major clades: an all-black clade comprising six species ( Our ML phylogeny corroborates bicolor-brainei-marginipennis monophyly but differs by depicting O. bicolor and O. marginipennis as sister species. To this end, we note a preliminary aspect of the molecular results-our somewhat limited geographic representation for O. bicolor and O. marginipennis. Relative to the other 'white' taxa, both these species have extended ranges and were recognized historically as being polytypic (Péringuey 1885;Koch 1952). Indeed, O. bicolor was for some time treated as two separate species (Koch 1952;Penrith 1975 Onymacris not only offers incremental support for the 'white' clade but, more broadly, augments a diphyletic Onymacris relative to Physadesmia (Lamb and Bond 2013). Penrith (1979) described the genus Physadesmia for three species [Physadesmia globosa (Haag), P. bullata (Péringuey), and P. aculeata (Péringuey)] formerly in Physosterna. (Of note, Physosterna was subsequently reduced to a subgenus of Adesmia (Penrith 1986)). She also observed that "Physadesmia and Onymacris are evidently very closely related, being separated only by the hypertrophy of the spurs and claws and the shortening of the tarsi in Onymacris." Support for her observation was provided in the first cladistic analysis of adesmiine genera, which recovered a clade comprising Onymacris, Physadesmia, and a third genus, Eustolopus (Penrith 1986). A refined phylogenetic view of Onymacris-Physadesmia, revealed herein and earlier (Lamb and Bond 2013), identifies a need for nomenclatural changes that will reflect the new found relationship between white Onymacris and Physadesmia. However, molecular genetic data are still missing for key taxa: two white Onymacris (O. langi langi and O. candidipennis, the latter being the type species of the genus) as well as the remaining two species of Physadesmia (P. bullata and P. aculeata). Though recognizing the necessity for taxonomic change (i.e., either subsuming Physadesmia or assigning the black species of Onymacris to a new genus), we consider this move to be premature at present and refrain from such effort until relationships for the remaining few species of Onymacris and Physadesmia have been thoroughly explored.
"Rediscovery" is a beguiling catchword, conveying equal parts accomplishment and optimism upon finding species thought to be rare or possibly extinct. We were indeed relieved to locate new specimens of O. brainei-a species gone unreported for 33 years. However, a claim of rediscovery might be overstated: the hiatus is attributable in large degree to the northern Namib's remote setting and limited accessibility. A more telling discovery may be the genetic divergence (1.49%, cox1) observed among individuals at the new locality, which could possibly indicate a historically larger geographic distribution. It is worth noting that O. candidipennis, once thought to be restricted to the Namib's northern terminus in Angola, has been reported from Namibia at the Cunene River, near the type locality for O. brainei (Penrith 1984). Moreover, O. bicolor and O. marginipennis, the two species most closely related to O. brainei, occur on both sides of the Cunene. Thus, future assessment on the status of O. brainei (regarding genetic variation as well as range delimitation) should involve surveys of suitable habitat from the type locality west to the Cunene mouth, in Angola as well as Namibia. Close proximity of both type and new localities to the contiguous Skeleton Coast (Namibia) and Iona (Angola) national parks offers promise that additional populations of O. brainei might be discovered within park boundaries, where they would be afforded full protection.