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Research Article
A second locality for the Namib darkling beetle Onymacris brainei (Tenebrionidae, Coleoptera) and first report on its molecular phylogenetic placement
expand article infoTrip Lamb, Eugene Marais§, Jason Bond|
‡ East Carolina University, Greenville, United States of America
§ National Museum of Namibia, Windhoek, Namibia
| Auburn University, Alabama, United States of America
Open Access

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.

Keywords

Adesmiini, Namib Desert, Onymacris, Tenebrionidae

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).

Figure 1. 

Color variation among members of the ‘white’ Onymacris clade, as represented by: (top row, left to right) Onymacris bicolor, O. marginipennis, O. candidipennis, and (bottom row, left to right) O. langi visseri, O. langi cornelii, and O. langi meridionalis.

‘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, 1979, 1984, 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).

Figure 2. 

Map illustrating the type locality for Onymacris brainei (star), surveyed sites with appropriate habitat (white circles), and the second locality for O. brainei (red circle).

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.

Methods

Field survey for Onymacris brainei

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 approximation, we searched a series of appropriate sites (i.e., vegetated hummocks) across the region on 21–22 May 2015. Three of these sites yielded other white Onymacris (O. bicolor, O. langi cornelii), and at a fourth, final site (17°17.87'S; 12°06.20'E), we succeeded in locating O. brainei (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).

PCR primers and amplification conditions.

Gene Primer Annealing Cycles Reference
cox1 TY-J-1460 50°C 35 Simon et al. (1994)
TL2-N-3014
C1-J-2183 sequencing only
cox2 TL2-J-3037 50°C 35
TK-N-3785
H3 Hex AF 61.5°C 45 Odgen and Whiting (2003)
Hex AR

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/subspecies. Those taxa unavailable to us for sequencing included O. candidipennis and O. langi langi, both ‘white’ beetles from Angola, as well as the ‘black’ beetles O. plana debilis and O. paiva conjuncta (though our dataset contains their nominate subspecies). 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.

GenBank accession numbers for adesmiine sequences used in the ML analysis.

Species GenBank GenBank GenBank
cox1 cox2 H3
Onymacris brainei MF459686 MF459688 MF459690
Onymacris brainei MF459687 MF459689
O. bicolor JX448896 JX448934 JX448972
O. marginipennis JX448907 JX448945 JX448983
O. langi cornelii JX448900 JX448938 JX448976
O. langi meridionalis JX448909 JX448947 JX448985
O. langi visseri JX448921 JX448959 JX448997
O. boschimana JX448897 JX448935 JX448973
O. multistriata JX448912 JX448950 JX448988
O. hottentota JX448901 JX448939 JX448977
O. plana JX448915 JX448953 JX448991
O. lobicollis JX448906 JX448944 JX448982
O paiva JX448913 JX448951 JX448989
O. rugatipennis JX448917 JX448955 JX448993
O. laeviceps JX448904 JX448942 JX448980
O. u. unguicularis JX448919 JX448957 JX448995
O. u. schulzeae JX448920 JX448958 JX448996
Physadesmia globosa JX448887 JX448925 JX448963
Eustolopus octoseriatus JX448886 JX448924 JX448962
Adesmia cribripes JX448889 JX448927 JX448965

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.

Results

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. 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).

Figure 3. 

Specimens of Onymacris brainei from the second locality, illustrating variation in degree of elytral striping.

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%).

Figure 4. 

ML consensus topology of Onymacris, with bootstrap support indicated by black (> 95%), gray (> 90%), and white (> 70%) nodes. Inset at lower left is a ML tree showing branch lengths.

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 (O. boschimana, O. laeviceps, O. lobicollis, O. multistriata, O. paiva, O. rugatipennis), and a second clade composed of three additional black species (O. hottentota, O. plana, O. unguicularis) and the ‘white’ species group. Regarding relationships within Penrith’s ‘white’ group, O. brainei was placed with O. bicolor and O. marginipennis, united by the loss of pseudopleural crests along the elytral margins. Furthermore, Penrith’s cladogram depicts O. brainei and O. marginipennis as sister species on the basis of one synapomorphy–the epistome bearing a deep median emargination.

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). Thus, while the precise sister status of O. brainei remains equivocal (pending further geographic sampling of O. bicolor and O. marginipennis, particularly Angolan populations), the strongly-supported monophyly of O. bicolor + O. brainei + O. marginipennis is unlikely to change.

The molecular phylogenetic placement of O. brainei with other ‘white’ 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.

Acknowledgements

We thank Patrice Bouchard, Edie Jeffreys, and Mike Wooten for their assistance in the field. Michael Brewer graciously provided access to his laboratory’s composite imaging system. Specimens were processed under Research/Collecting Permit # 2015/2015 provided by Namibia’s Ministry of Environment and Tourism. Funding for this project was provided by Research and Exploration grant # 9582-14 from the National Geographic Society.

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