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LAB PROTOCOL
From museum drawer to tree: Historical DNA
phylogenomics clarifies the systematics of
rare dung beetles (Coleoptera: Scarabaeinae)
from museum collections
Fernando LopesID
1
*, Nicole Gunter
2
, Conrad P. D. T. GillettID
1
, Giulio Montanaro
1
,
Michele Rossini
1,3
, Federica LosaccoID
1
, Gimo M. Daniel
4,5
, Nicolas StraubeID
6
,
Sergei Tarasov
1
1Finnish Museum of Natural History, University of Helsinki, Helsinki, Uusima, Finland, 2Biodiversity and
Geosciences Program, Queensland Museum Kurilpa, Brisbane, Queensland, Australia, 3Department of
Agronomy, Food, Natural Resources, Animals and Environment (DAFNAE), University of Padova, Veneto
Region, Padua, Italy, 4Department of Terrestrial Invertebrates, National Museum Bloemfontein,
Bloemfontein, Free State province, South Africa, 5Department of Biological and Environmental Sciences,
Walter Sisulu University, Mthatha, South Africa, 6Department of Natural History, University Museum of
Bergen, Vestland, Norway
*fernando.vieiralopes@helsinki.fi
Abstract
Although several methods exist for extracting and sequencing historical DNA originating
from dry-preserved insect specimens deposited in natural history museums, no consensus
exists as to what is the optimal approach. We demonstrate that a customized, low-cost
archival DNA extraction protocol (*€10 per sample), in combination with Ultraconserved
Elements (UCEs), is an effective tool for insect phylogenomic studies. We successfully
tested our approach by sequencing DNA from scarab dung beetles preserved in both wet
and dry collections, including unique primary type and rare historical specimens from inter-
nationally important natural history museums in London, Paris and Helsinki. The focal speci-
mens comprised of enigmatic dung beetle genera (Nesosisyphus,Onychothecus and
Helictopleurus) and varied in age and preservation. The oldest specimen, the holotype of
the now possibly extinct Mauritian endemic Nesosisyphus rotundatus, was collected in
1944. We obtained high-quality DNA from all studied specimens to enable the generation of
a UCE-based dataset that revealed an insightful and well-supported phylogenetic tree of
dung beetles. The resulting phylogeny propounded the reclassification of Onychothecus
(previously incertae sedis) within the tribe Coprini. Our approach demonstrates the feasibil-
ity and effectiveness of combining DNA data from historic and recent museum specimens to
provide novel insights. The proposed archival DNA protocol is available at DOI 10.17504/
protocols.io.81wgbybqyvpk/v3.
PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0309596 December 31, 2024 1 / 18
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OPEN ACCESS
Citation: Lopes F, Gunter N, Gillett CPDT,
Montanaro G, Rossini M, Losacco F, et al. (2024)
From museum drawer to tree: Historical DNA
phylogenomics clarifies the systematics of rare
dung beetles (Coleoptera: Scarabaeinae) from
museum collections. PLoS ONE 19(12): e0309596.
https://doi.org/10.1371/journal.pone.0309596
Editor: Pankaj Bhardwaj, Central University of
Punjab, INDIA
Received: May 27, 2024
Accepted: August 15, 2024
Published: December 31, 2024
Copyright: ©2024 Lopes et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: “***PA AT ACCEPT:
Please follow up with authors for data in NCBI and
whether it has been uploaded***“Yes - all data are
fully available without restriction; The data
presented in this study can be accessed at Open
Science Framework and Protocols.io via the two
following hyperlinks: dx.doi.org/10.17504/
protocols.io.81wgbybqyvpk/v2 (DOI 10.17504/
protocols.io.81wgbybqyvpk/v2); https://osf.io/
mxwj7/ (DOI 10.17605/osf.io/mxwj7). Raw
Introduction
Museomics, a term encompassing procedures allowing access to and analysis of the historical
genomic data preserved in biological specimens deposited in natural history museums, is pro-
viding unprecedented opportunities to investigate evolutionary histories [1,2]. Together with
concomitant advances in high-throughput sequencing technologies and bioinformatics,
museomics has paved the way for the exploitation of an ever-broader diversity of taxonomic
and temporal sampling [3,4]. Importantly, by enabling access to genomes already preserved in
existing museum specimens, museomics can circumvent the need for costly, laborious and
unpredictable bespoke fieldwork, to achieve taxon sampling objectives [2,3]. Museomics is
also compatible with physically preserving the morphological integrity of specimens when
non-destructive DNA extraction methods are employed. This is of paramount importance to
natural history museums and the scientific community because it ensures that intact voucher
specimens will remain available for study by future generations [5,6]. Indeed, the importance
of museomics can only heighten as the necessity for inclusion of recently extinct species within
phylogenies becomes increasingly inevitable [7]. Progress in insect museomics has already
greatly contributed to the study of insects—Earth’s most diverse organisms [5,8]. Although
notable recent achievements in DNA extraction mean that the recovery of DNA from dry-
pinned museum specimens is no longer remarkable [9], challenges still remain [2]. Specifically,
the DNA in many dry-preserved museum specimens is fragmented and prone to contamina-
tion, whilst the comparatively small amount of tissue present in small insects can further limit
the success of DNA extractions [10].
In recent years, a variety of molecular methods have been developed to obtain historical
DNA data at a genome-wide scale [3,4], including approaches exploiting both whole-genome
(shotgun) and reduced representation sequencing [7,11,12]. Many widely used methods rely
on standard DNA extractions using commercial DNA kits, followed by the construction of
DNA libraries based on hybridization capture approaches that combine restriction enzyme
fragmentation and RNA probe capture. For instance, hyRAD uses a double enzymatic restric-
tion of DNA extracts from fresh samples (containing well-preserved DNA) to produce RNA
probes that serve as baits for capturing homologous fragments from historical (more
degraded) DNA libraries [13,14]. However, standard DNA extraction, typically undertaken
with commercially available kits, is optimized for high molecular weight DNA, only inef-
fectively capturing lower-weight short fragments, which are precisely those expected from
degraded historical samples. Furthermore, reduced representation approaches exploiting
restriction enzymes require a comparatively large initial amount of source DNA, not easily
obtained from small insect specimens [15]. Moreover, those methods tend to be costly when
extensively sampling a wide range of insect taxa. They are also labor-intensive because they
require the creation of custom RNA probes for each taxon being studied. Crucially, such meth-
ods are susceptible to the drawbacks associated with restriction enzymes. These include the
potential for enzyme mismatch, either due to point mutations because target taxa are too dis-
tantly related or due to DNA fragmentation at restriction sites (especially in poorly preserved
samples); both processes that can lead to missing data [16].
Within museomics studies, more cost-effective genome reduction methods, such as Ultra
Conserved Elements (UCEs) and Anchored Hybrid Enrichment (AHE) are rising in popular-
ity due to their ability to target specific informative loci within a focal group [17–20]. While
standard extraction from dry-preserved specimens may yield adequate DNA for UCE and
AHE sequencing [20,21], its success varies based upon specimen preservation. Therefore,
exploring the effectiveness of more sensitive DNA extraction methods is essential, especially
since their application in entomological collections remains poorly investigated [18].
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sequencing data can be found at NCBI BioProject
PRJNA1031114.
Funding: This research received funding from the
Research Council of Finland (grant #331631) and a
3-year grant (grant #79783104) from the
University of Helsinki to Sergei Tarasov.
Networking was supported by a Finnish Museum
of Natural History Pentti Tuomikoski Fund award to
Conrad P. D. T. Gillett. Nicole Gunter was
supported by the National Science Foundation
(USA, grant DEB-1942193). The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
In this study, we aim to bridge this gap by assessing a cost-effective (*€10 per sample)
archival DNA extraction protocol [22] specifically tailored to historical insect specimens and
downstream UCE sequencing. We applied this protocol, in combination with standard DNA
extraction from fresh specimens, in addition to compiling relevant sequences deposited in
GenBank, to explore the phylogenetic relationships of eleven species and subspecies of dung
beetles (Coleoptera: Scarabaeinae) represented by historical specimens from three museums:
The Natural History Museum, London (NHMUK); the Muse
´um National d’Histoire Natur-
elle, Paris (MNHN); and the Finnish Museum of Natural History, Helsinki (MZHF). We
selected to focus our study on scarab dung beetles because they are of considerable biological
interest, for providing important ecosystem services including nutrient cycling and secondary
seed dispersal [23], in addition to having proven to be a dependable ‘proxy’ bioindicator taxon
indicative of wider biodiversity patterns [24–27]. Hence, robustly infering their systematics is
fundamental to accurate interpretation of their wider ecological significance.
The selected specimens are of diverse ages and represent enigmatic species of questionable
phylogenetic assignment. The oldest specimen, the holotype of Nesosisyphus rotundatus Vin-
son, 1946 collected in 1944, and deposited in NHMUK, is a potentially extinct species from
Mauritius, not previously included in molecular phylogenies (e.g., Tarasov & Dimitrov (2016)
[28]). The extremely rare (i.e. apparently infrequently collected and represented only by very
few museum specimens) Oriental genus Onychothecus Boucomont, of uncertain taxonomic
affinity [28] and hitherto lacking DNA data, was represented by a specimen collected in 1985
that is held in MNHN. Finally, nine poorly-known taxa belonging to the endemic Madagascan
genus Helictopleurus D’Orbigny were represented by specimens collected between 2003–2010
and deposited in MZHF.
Our archival DNA extraction protocol yielded DNA of sufficiently high quality for success-
ful UCE sequencing using the recently designed probe set for scarab beetles [29]. To elucidate
the phylogenetic position of the selected enigmatic species, we expanded our taxon sampling
to include additional dung beetle species represented by alcohol-preserved specimens,
extracted using a standard commercial DNA extraction kit protocol. In the following sections,
we discuss the phylogenetic position of the focal species based on our results and implement
necessary taxonomic changes. We also explore the broader application of the proposed extrac-
tion protocol to a wide range of historical specimens of insects and other taxa.
Materials and methods
This research did not involve human participants or live animals, so ethical approval was not
required. The study followed the guidelines of the Madagascar Institut pour la Conservation
des Ecosystèmes Tropicaux (MICET), the Mauritian National Parks and Conservation Service
(NPCS), the Finnish Museum of Natural History Research Programme in Systematics and
Evolution, and all other contributing institutions, ensuring adherence to ethical standards in
scientific research.
Taxon sampling
We compiled a dataset of UCE sequences from 96 beetles (S1 Table), encompassing mostly
scarabaeoid beetle lineages from various biogeographical regions. Our dataset combined 70
newly-sequenced specimens for this study with existing data for 26 specimens from a previous
study available on GenBank [29]. The ingroup consisted of 67 samples belonging to 42 genera
or subgenera of true dung beetles of the subfamily Scarabaeinae. The outgroup consisted of 29
samples (of 26 genera) belonging either to scarab beetle families and subfamilies other than
Scarabaeinae, or to non-scarab beetles (two species of Silphidae). Fifty-nine of the newly
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sequenced samples (representing 40 genera) originated from frozen (-20˚C) alcohol-preserved
“wet collection” specimens that were sourced from five natural history museums. A further 11
historical samples belonging to three genera (Nesosisyphus Vinson, 1946, Onychothecus and
Helictopleurus) were selected from the dry collections of three museums, and formed the focal
taxa for our study (Table 1).
DNA extraction and sequencing
We applied an optimized archival DNA extraction protocol to the 11 historical samples. The
protocol described in this peer-reviewed article is published on Protocols.io (DOI 10.17504/
protocols.io.81wgbybqyvpk/v3) and is included for printing purposes as (S1 File). Briefly, the
protocol is a customization of the archival DNA extraction protocol and Guanidine treatment
described by Straube et al. (2021) [22] which was influenced by the studies of Dabney et al.
(2013) [30] and Rohland et al. (2004) [31]. The new approach was first proposed for wet-pre-
served vertebrates and is based on the binding of DNA to a PCR purification silica membrane
in the presence of a chaotropic salt (guanidine hydrochloride) buffer (S2 Table and at
Protocols.io). The method uses an extension reservoir attached to a commercial silica spin col-
umn, able to retain DNA fragments of lengths varying from 70 bp to 4 kbp. This adaptation
allows for a more than tenfold increase in the ratio of binding buffer to sample and enhances
the recovery of short DNA fragments, typically present in historical samples [22]. We further
customized this protocol into a non-destructive extraction using dry-preserved beetle speci-
mens from several museum entomological collections, as specified in step 4 of the protocol. In
short, we optimized how samples were prepared for the lysis step by not physically destroying
body parts. Extractions from the 11 dry-preserved museum specimens (S1 Table) were under-
taken in a dedicated “clean room” for historical samples at MZHF.
The DNA of the 59 wet-preserved museum specimens was extracted using the QIAamp
DNA Micro Kit (QIAGEN), following the manufacturer’s protocol. After DNA extraction,
dual-indexed paired-end Illumina libraries were prepared and enriched using the UCE Scara-
baeinae probe-set Scarab 3Kv1 [29] and sequenced at RAPiD Genomics LLC (Gainesville, FL,
U.S.A.) utilizing their high-throughput workflow with proprietary chemistry. Briefly, the DNA
was sheared to a mean fragment length of 500 bp, followed by end-repair and A-tailing, incor-
poration of unique dual-indexed Illumina adaptors, and PCR enrichment. Samples were
pooled equimolarly and sequenced on an Illumina NovaSeq 6000 S4 flow cell (2x150 bp).
Table 1. Dry-preserved scarab dung beetle specimens from natural history museum collections, used in historical DNA extractions. Natural History Museum, Lon-
don (NHMUK); Muse
´um National d’Histoire Naturelle, Paris (MNHN); and Finnish Museum of Natural History (MZHF).
Sampling Year of Sampling Museum Origin
Helictopleurus fasciolatus fasciolatus (Fairmaire, 1898) 2003 MZHF Madagascar
Helictopleurus fasciolatus obscurus Lebis, 1960 2009 MZHF Madagascar
Helictopleurus fasciolatus pseudofasciolatus Montreuil, 2007 2010 MZHF Madagascar
Helictopleurus neuter (Fairmaire, 1898) 2009 MZHF Madagascar
Helictopleurus nicollei Lebis, 1960 2008 MZHF Madagascar
Helictopleurus perrieri (Fairmaire, 1898) 2006 MZHF Madagascar
Helictopleurus sinuatocornis (Fairmaire, 1898) 2003 MZHF Madagascar
Helictopleurus undatus (Olivier, 1789) 2008 MZHF Madagascar
Helictopleurus near furcicornis Lebis, 1960 2008 MZHF Madagascar
Nesosisyphus rotundatus Vinson, 1946 [holotype] 1944 NHMUK Mauritius
Onychothecus tridentigeris Zelenka, 1992 1985 MNHN Thailand
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Nesosysiphus rotundatus, a monoinsular endemic species from Mauritius that is known
only from six pinned specimens, was represented by its holotype, deposited in NMHUK (Fig
1B). The tiny specimen, one of the smallest scarab dung beetles in the world (*4 mm), was
collected by J. Vinson in 1944 [32]. This specimen was carefully relaxed and disarticulated.
Only the prothorax (with exposed internal tissues) and the attached forelegs (but not the head)
were used during the digestion step of the extraction (Fig 1B), resulting in a total of 17.03 ng of
DNA that generated 1,528 UCE loci after sequencing. Following DNA extraction, the digested
body parts remained well-preserved with no visible external deterioration, and the specimen
was afterward successfully reassembled (Fig 1B). Onychothecus tridentigeris Zelenka, 1992 is a
much larger, very rare species from Thailand, that was represented by a non-type specimen
(*20 mm) deposited in MNHN (Table 1,Fig 2). We removed the entire left middle leg from
the specimen, which was destructively used in the DNA extraction process (Fig 2B and 2C),
resulting in 17.40 ng of DNA that generated 1,692 UCE loci. The remainder of the specimen
survived intact.
Data processing
Demultiplexing and trimming were performed by RAPiD Genomics LLC using Illumina
bcl2fastq2 2.20 [34] with default settings. Our UCE datasets were assembled using the package
Phyluce 1.7.3 [35] following the workflow available on GitHub. Raw demultiplexed reads were
first cleaned using Illumiprocessor 2.0 [36] with default parameters set on Phyluce to remove
residual adapter contamination. Cleaned reads were inspected for quality using FastQC 0.12.0
[37]. After, cleaned pair-end reads were assembled into contigs with Spades 3.15.4 [38] and
default parameters set on Phyluce. The Scarab 3kv1 UCE probes [29] were matched to the
assembled contigs in Phyluce, with a minimum identity of 80% and coverage of 80×to avoid
off-target contaminating sequences [29,39]. UCE loci were then extracted from the sequenced
data. We harvested UCE loci from the available whole beetle genomes on GenBank [29] (S1
Table. Last access previous data harvesting on 23.05.2023) using using faToTwoBit (https://
genome.ucsc.edu/), Phyluce and the Scarab 3Kv1 probe set [29], as described in the Phyluce
tutorial, and combined them with our newly sequenced data. The UCE loci were aligned in
MAFFT 7.475 [40], using the default Phyluce settings and the command -no-trim to provide
internal trimming, as recommended for analysis of divergences over 50 million years old [35].
The resulting alignments were parsed to a parallel wrapper around Gblocks 0.91 [41] to elimi-
nate poorly aligned positions and divergent regions using the settings: b1 = 0.5, b2 = 0.85,
b3 = 8, b4 = 10 [41,42]. Summary statistics for the generated datasets were computed using
the program AMAS [43].
Phylogenomics
For phylogenomic analyses, we constructed data matrices for concatenated species trees in
IQ-Tree 2.0.7 [44] with 50% and 70% complete data, allowing up to 50% and 30% missing taxa
for each locus, respectively [21,45]. Hereafter, the 50% and 70% complete datasets are named
the 50p and 70p datasets. Full and partitioned UCE alignments are available on the Open Sci-
ence Framework (OSF) repository at DOI 10.17605/osf.io/mxwj7. ID labels in tree files were
translated into full species names using the custom Python script rename_leaves_v1.0.py avail-
able on GitHub.
Gene-based phylogeny. Concatenated species trees were estimated in IQ-Tree2 using
UCEs as independent loci (genes). Confidence levels were calculated using 1,000 ultrafast
bootstrap (UFBoot) replicates and topologies tested by the Shimodaira–Hasegawa test (SH-
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Fig 1. Dung beetle tree. (A) Species tree inferred using 1,497 UCEs and the 50% complete dataset; dry-preserved historical museum specimens are
indicated in bold. Collapsed branches are proportional to the number of samples in each lineage. Hollow dots indicate fully supported nodes and
nodes with numbers indicate bootstrap values <100. (Oni) Onitini; (Ont) Onthophagini + Oniticellini; (Onc) Oniticellini; (M2, M1) Madagascan
endemic lineages; (Sc) Scarabaeini; (Ph) Phanaeini; (Aus) Australasian endemic genera. For details, see S1 Table. (B) Dorsal view of the holotype of
Neosisyphus rotundatus before and after DNA extraction using the archival protocol.
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aLRT) [46,47]. The best substitution models were automatically selected using ModelFinder
(-m mfp option) implemented in IQ-Tree2 under the Bayesian Information Criterion [48].
To reduce the risk of overestimating branch support with UFBoot owing to severe model
violations, we used a hill-climbing nearest-neighbor interchange (NNI, -bnni option) [49]
topology search strategy to optimize each bootstrap tree. As phylogenetic models rely on
Fig 2. Morphology of Onychothecus tridentigeris.Dorsal habitus of male (A) and female (B); ventral view of female
(C); right wing in dorsal view, with radial posterior vein 1 (RP1) indicated (D); left elytron in lateral view, indicating
the numbered elytral striae and the lateral carina (E); hind tarsus, with the modified terminal tarsomere concealing the
claws (F); right protibia of male, in dorsal view (G); aedeagus in lateral (H) and dorsal (I) views; endophallites (J)
(abbreviations follow Tarasov & Ge
´nier (2015) [33]).
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various simplifying assumptions to ease computations (e.g., treelikeness, reversibility and
homogeneity of substitution models), estimations of some genomic regions can severely vio-
late model assumptions, causing biases in phylogenetic estimates of tree topologies [50]. To
test these violations on each locus, we also applied the test of symmetry with the option --symt-
est-remove-bad [50]. Partitions (concatenated analyses) and genes (species trees analyses) with
ap-value 0.05 for the test of symmetry were removed from downstream analyses [50].
Partition-based phylogeny. We also recovered concatenated species trees with parti-
tioned genomic regions. The datasets were partitioned with PFinderUCE-Sliding-Window
Site Characteristics (SWSC-EN), an entropy-based method developed specifically for UCE
data [51]. To implement the SWSC-EN method, we used Phyluce to generate a concatenated
Nexus file with the location of each UCE locus as character sets. With the SWSC-EN Python
3.6 script, configuration files were created to be processed by Partitionfinder 2.1.1 [52] and
Python 2.7. As Partitionfinder2 works only with Phylip alignments, we converted the
concatenated Nexus file to Relaxed Phylip format using Geneious 2022.2.1 [53]. The partition-
ing scheme was then generated with Partitionfinder2 with linked branch lengths, a GTR+G
model of evolution, an Akaike information criterion with correction (AICc) [54] for model
selection and a variant of the relaxed hierarchical clustering search algorithm https://osf.io/
3fpg4/ [21,55].
Morphological examination
As a complement to molecular inference of the phylogenetic position of Onychothecus and
related taxa, we studied the morphology of two available specimens of O. tridentigeris (depos-
ited in MNHN) in detail. Morphological terminology and protocols follow Tarasov & Dimi-
trov (2016) [28] and Tarasov & Ge
´nier (2015) [33]. Specimens were examined under a Leica
S9D stereomicroscope (Leica Microsystems GmbH, Germany). Photographs were taken with
a Canon MP-E 65 mm, f/2.8, 1–5×macro lens mounted on a Canon EOS 5D (Canon Inc.,
Japan) camera and then stacked using the StackShot (Cognisys Inc., USA) automated system.
Results
UCE data
We obtained a mean of 1.86×10
7
paired-end reads per sample. Our results revealed that
shorter fragments from museum samples were effectively integrated into DNA libraries, result-
ing in the recovery of a substantial number of UCE loci for phylogenomic analyses (Fig 3A,S1
Table). Specifically, samples for which DNA was extracted using the archival DNA protocol
yielded the highest number of recovered loci (2,264), followed by alcohol-preserved samples
extracted using the commercial kit (1,620 loci) and UCE data retrieved from GenBank
genomes (909 loci; S1 Table and Fig 3B). Interestingly, older specimens, such as O. tridentigeris
and N. rotundatus, yielded a similar number of recovered loci compared to the more recently
collected wet-preserved samples extracted with the commercial kit. (Table 1 and S1 Table).
Prior to data filtering, the full concatenated alignment (96 tips) contained 3,160 UCE loci
and 269,808 parsimony-informative sites distributed across 675 Kbp (S3 Table). The 50p data-
set contained 1,497 UCE loci with a mean of 79.97 parsimony-informative sites per locus (S4
Table) and the 70p dataset contained 289 UCE loci with a mean of 60.54 parsimony-informa-
tive sites per locus (S5 Table). The conspicuous disparity among unfiltered, 50p and 70p data-
sets is due to the large proportion of missing data present in the genomes retrieved from
GenBank, which mostly served as outgroup taxa in our study [29] (S2 Table and S1–S5 Figs).
UCE data from GenBank-represented species was mostly obtained from existing draft
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genomes. These genomes, having been assembled in different sizes and showing discrepant
levels of contiguity, were the primary contributors to the aforementioned missing data.
Phylogenomics
Increasing the completeness threshold during construction of data matrices significantly
decreased the number of UCE loci and overall bootstrap support (Fig 1,S1–S4 Figs and S4 and
S5 Tables). Because phylogenomic studies generally do not benefit from filtering out loci hav-
ing an increased proportion of missing data [45], we focused on the 50p dataset, which resulted
in an optimal trade-off between the highest overall bootstrap support and SH-values (Fig 1
and S1 and S2 Figs) and the number of recovered loci (S1 Table).
Our phylogenetic trees were well-supported, with only a few nodes of moderate depth hav-
ing poor support (see Fig 1 and S1 and S2 Figs). Notably, Scarabaeinae formed a monophyletic
group, with Frankenbergerius Balthasar and Sarophorus Erichson representing a basal lineage
that is sister to the remaining Scarabaeinae. The Afrotropical genus Epirinus Dejean was iden-
tified as the sister to the remaining Sisyphini. The historical specimen of N. rotundatus consis-
tently clustered within the tribe Sisyphini across all analyses, with robust bootstrap support
(BS: 100; Fig 1A and 1B).
In all analyses, the Madagascan Helictopleurus species formed a sister clade to the other
Oniticellini (clade Onc in Fig 1), represented solely by Euoniticellus Janssens in our tree. Slight
variations in the grouping of certain Helictopleurus species resulted from analysis of the 70p
dataset, likely due to limited genomic information and/or short branches in the clade’s back-
bone (Fig 1 and S1–S4 Figs). We recovered the Oniticellini + Helictopleurus clade as nested
within Onthophagini (clade Ont in Fig 1). The enigmatic species O. tridentigeris was consis-
tently inferred in all analyses as the sister taxon to the genera belonging to the tribe Coprini
(BS: 98; Figs 1A and 2).
Other dung beetle tribes were also recovered as monophyletic (Fig 1), namely, Onitini
(clade Oni), Scarabaeini (clade Sc), and Phanaeini (clade Ph). The Madagascan “Canthonini”
were grouped into two lineages: Apotolamprus +Nanos (clade M2) and Arachnodes +Epilissus
(clade M1). The lineage M2 was sister to Scarabaeini + Gyronotus, while M1 was sister to
Fig 3. Summary of UCE data resulting from two DNA extraction methods (archival extraction protocol in red and standard extraction in blue) and
beetle genomes from GenBank (in green, only in B). Violin plots illustrate the kernel density and boxplots display the median and variation. (A)
Distribution of read length generated per sample, demonstrating that the density of shorter reads was generally higher from archival extractions. (B)
Distribution of the number of UCE loci per sample, demonstrating that the greatest density of samples that generated large numbers of captured loci
resulted from archival extractions.
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Coprini. Additionally, the Australasian endemic genera also formed a monophyletic group
(clade Aus in Fig 1).
Discussion
How specimens are captured, killed and preserved prior to being dry-mounted are likely to be
crucial factors contributing to DNA quality and subsequent phylogenomic analyses based on
these data. In this case, no such information is available for the historical dry-preserved speci-
mens that we extracted, yet it is reasonable to assume a variety of preservation treatments- as is
common in any museum collection. For example, the species of Helictopleurus were collected
in the early 2000s by Ilkka Hanski’s team at the Finnish Museum of Natural History, who
probably optimized field sampling protocols to limit DNA damage for anticipated evolution-
ary studies. Indeed, these Helictopleurus specimens resulted in generating a correspondingly
large amount of DNA sequence data—an average of 2,410 UCEs per sample (see Results and
S1 Table). Conversely, it was expected that the older specimens Onychothecus and Nesosisy-
phus, likely collected without molecular-oriented awareness (e.g., by using DNA-damaging
killing compounds such as ethyl acetate, widely used by entomologists), would result in signifi-
cantly diminished DNA data. However, both specimens yielded substantial numbers of UCEs
(1,692 and 1,592, respectively) that were sufficient to phylogenetically place them, with strong
support, within our scarab beetle tree. This therefore highlights the feasibility of applying our
workflow to retrieve and sequence DNA for phylogenomics even from specimens of unknown
preservation history.
Phylogenetic relationships
All resultant topologies were generally consistent with previous morphological [33] and
molecular analyses based on individual genes [28,33,56] and preliminary UCE data [29].
The basal position of the Afrotropical lineage comprising Frankenbergerius +Sarophorus,
as well as the monophyly of Onitini, Scarabaeini, Phanaeini and Onthophagini + Oniticellini,
are consistent with previous findings [28,33,56]. Additionally, the paraphyly of Onthophagini
with Oniticellini nested within it has also been supported by previous analyses [28,57–59].
The Old World tribe Onitini (clade Oni in Fig 1) was found to be sister to the Afrotropical
genus Xinidium Harold, consistent with morphological and mitochondrial data [33,59],
whereas other molecular data tend to place Onitini as sister to Onthophagini + Oniticellini
[28,58] or Sisyphini [56]. Similar discordance between molecular and morphological data was
observed in the relationship between Afrotropical Gyronotus Lansberge and the Old World
tribe Scarabaeini (clade Sc in Fig 1). Molecules suggest a remote relationship [28], while mor-
phology supports close affinities [33]. The splitting of Madagascan “Canthonini” into two line-
ages, Apolamprus +Nanos (clade M2) and Arachnodes +Epilissus (clade M1), has also been
supported by previous studies [28,60]. Additionally, earlier molecular analyses have consis-
tently supported the monophyly of the Australasian endemic genera (clade Aus in Fig 1)
included in this study [28,56]. The relationships of the enigmatic taxa sequenced from histori-
cal specimens are discussed in detail below.
Mauritian Nesosisyphus.This study offers the first insights into relationships of the
endemic Mauritian genus Nesosisyphus, which has not previously undergone phylogenetic
analysis. Our grouping of N. rotundatus (Fig 1A) within the tribe Sisyphini [28,61] was
expected and is also supported by morphological synapomorphies that clearly indicate that
Nesosisyphus is a member of this tribe. Therefore, the phylogenetic result based on UCE data
obtained from the old holotype of N. rotundatus strongly agrees with our initial hypothesis
[32].
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Nesosisyphus rotundatus is a flightless roller dung beetle, uniquely known by the six speci-
mens that make up the type series collected during the early 1940s from the southern slope of
Mount Ory in Mauritius [32]. All subsequent collecting efforts on the island, which have
included sampling at the type locality, have resulted in the discovery of three additional Mauri-
tian endemic species of Nesosisyphus (Losacco et al., in prep.), failed to relocate this species.
Given this and considering the rapid loss of indigenous habitats and biodiversity in Mauritius
in general, due to anthropogenic habitat destruction and the introduction of exotic species [62,
63], we regard this species as potentially extinct (Losacco et al., in prep). One of the achieve-
ments of our study has been to unlock genomic data from this enigmatic species for further
investigation. An additional benefit of having genomic data available for this species is that fur-
ther eDNA studies (e.g., metagenomics) could utilize this data to locate the species and deter-
mine its distribution using indirect DNA sources, such as soil, through an eDNA approach.
Oriental Onychothecus.This extremely rare genus comprises four species distributed in
southeastern Asia (Figs 1and 2): China (Yunnan), Myanmar, Thailand, Laos and Vietnam
[64,65]. It is remarkable for displaying secondary sexual dimorphism that is unusual within
scarab beetles—females bear a cephalic horn and males are hornless (the reverse is overwhelm-
ingly more common in the superfamily Scarabaeoidea)—in addition to having unknown hab-
its, diet and general biology. Onychothecus has not yet been classified (=incertae sedis) into any
of the existing tribes of the subfamily Scarabaeinae [28]. Only a single previous phylogenetic
analysis incorporating this genus exists [66], based upon morphological data that identified it
as sister to the genus Paraphytus, having a disjunct Afrotropical and Oriental distribution.
Paraphytus belongs to the most basal lineage of Scarabainae [28] that also includes the Afrotro-
pical genera Frankenbergerius and Sarophorus, which we have included in the present analyses.
Our resulting phylogeny indicates that Onychothecus does not belong to that basal lineage,
being instead recovered as sister to the clade containing the genera Copris Geoffroy, Water-
house and Microcopris Balthasar, belonging to the tribe Coprini sensu Tarasov & Dimitrov
(2016) [28] (Fig 1A and S1–S4 Figs). Consequently, based on our results, we assign Onychothe-
cus to the tribe Coprini and discuss this assignment in a separate section below. Oriental Ony-
chothecus. This extremely rare genus comprises four species distributed in southeastern Asia
(Figs 1and 2): China (Yunnan), Myanmar, Thailand, Laos and Vietnam [64,65]. It is remark-
able for displaying secondary sexual dimorphism that is unusual within scarab beetles—
females bear a cephalic horn and males are hornless (the reverse is overwhelmingly more com-
mon in the superfamily Scarabaeoidea)—in addition to having unknown habits, diet and gen-
eral biology. Onychothecus has not yet been classified (=incertae sedis) into any of the existing
tribes of the subfamily Scarabaeinae [28]. Only a single previous phylogenetic analysis incor-
porating this genus exists [66], based upon morphological data that identified it as sister to the
genus Paraphytus Harold, having a disjunct Afrotropical and Oriental distribution. Paraphytus
belongs to the most basal lineage of Scarabainae [28] that also includes the Afrotropical genera
Frankenbergerius and Sarophorus, which we have included in the present analyses. Our result-
ing phylogeny indicates that Onychothecus does not belong to that basal lineage, being instead
recovered as sister to the clade containing the genera Copris Geoffroy, Litocopris Waterhouse
and Microcopris Balthasar, belonging to the tribe Coprini sensu Tarasov & Dimitrov (2016)
[28] (Fig 1A and S1–S4 Figs). Consequently, based on our results, we assign Onychothecus to
the tribe Coprini and discuss this assignment in a separate section below.
Madagascan Helictopleurus.The genus Helictopleurus comprises approximately 65 species,
all endemic to Madagascar (Fig 1A) and primarily occurring in forest habitats [60,67]. We
extracted and sequenced DNA from nine dry-preserved and two alcohol-preserved specimens
from museum collections, having been collected between 2003 and 2010. Sequence data for
one additional species was included from a previously published study [68]. Our phylogenetic
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analyses (Fig 1A and S1–S4 Figs) confirm previous results, demonstrating the monophyly of
the genus Helictopleurus, its sister relationship to the genus Euoniticellus and that the Helicto-
pleurus +Euoniticellus clade falls within the clade containing the tribes Onthophagini + Oniti-
cellini [57,58,69]. However, when compared to earlier molecular phylogenies based on
individual genes, our analyses resulted in slight variations in the interspecific relationships
within Helictopleurus [68,69]. Enhancing taxon sampling in future studies and potentially
integrating previously published single-gene data will help achieve more robust results.
The fact that our results, including newly sequenced data from museum specimens of vary-
ing ages (Table 1) and preservation, produced robust results that are consistent with existing
phylogenies [28,29], demonstrates the effectiveness of the proposed archival DNA approach
in combination with UCE sequencing. Such consistency is of particular significance because
concerns about sequence data obtained from historical specimens being contaminated or of
poor quality and, consequently, obfuscating or impeding phylogenetic inference, appear not to
have been borne out in our study.
Tribal transfer of Onychothecus to Coprini
Genus Onychothecus Boucomont, 1912
•Onychothecus Boucomont, 1912: original description; as member of Scatonomini Lacordaire,
1856 synonym of Deltochilini Lacordaire, 1856: sensu Bouchard et al. (2011) [70].
•Onychothecus; Balthasar (1963) [71]: as member of Pinotini Kolbe, 1905 synonym of Ateu-
chini Perty, 1830: sensu Smith (2006) [72].
•Onychothecus; Tarasov & Dimitrov (2016) [28]: as incertae sedis.
•Type species: Onychothecus ateuchoides Boucomont, 1912.
The tribe Coprini is distributed in both the Old and New World and includes five genera:
Copris,Litocopris,Microcopris,Pseudocopris Ferreira and Pseudopedaria Felsche. The tribe
lacks unique apomorphies allowing for unequivocal diagnosis. Instead, it is characterized only
by a combination of six characters [28].
We examined the morphology of two specimens of Onychothecus tridentigeris in detail,
including the first-known male of the species (Fig 2A and 2G–2J). Our phylogenetic analyses
strongly support the position of Onychothecus as a sister taxon to a clade containing the five
genera making up the Coprini. Based upon this evidence, two alternative taxonomic actions
were considered: either creating a new tribe to accommodate Onychothecus or assigning it to
Coprini. We have chosen the latter option and herein treat the genus Onychothecus as a mem-
ber of the tribe Coprini. In our opinion, this action is justified to maintain stability in the clas-
sification of Scarabaeinae.
Although the general habitus of Onychothecus resembles that of many other members of
the tribe Coprini, its morphology stands out within the Scarabaeinae in general. Specifically,
Onychothecus exhibits dorsally excavated protibial apices, terminal tarsomeres that conceal the
tarsal claws and, most strikingly, ‘inverse sexual dimorphism’, wherein the female is the
horned sex. Additionally, it possesses characters that have not previously been adopted to diag-
nose Coprini, including laterally carinate elytra, an absence of the posterior sclerite of the
wing, and an absence of the posterior ridge of the hypomera. These observations therefore
oblige revision and expansion of the morphological diagnosis of the tribe Coprini. We present
the new diagnosis of Coprini in Table 2.
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Conclusion
We successfully obtained genomic data that allowed for the evolutionary positioning of several
dung beetle species represented by unique historical specimens deposited in important natural
history museum collections (Table 1). Our results were consistent with previous phylogenetic
studies [28,29,33,56] and demonstrate that combining a minimally destructive and low-cost
archival DNA extraction, with subsequent target enrichment of DNA libraries for sequencing
a curated set of beetle UCE loci, is an efficient museomics tool for phylogenomics (see S1 and
S2 Tables). The proposed extraction protocol should also combine well with Anchored Hybrid
Enrichment sequencing. By being able to capture small fragments of degraded DNA even
from the limited quantity of source tissue available in old museum specimens (Fig 3), we have
demonstrated a favorable trade-off between preserving specimen morphology and generating
informative genomic-level data. Our customized extraction protocol can be performed using
standard equipment commonly available in molecular laboratories within two days, including
an overnight digestion step. Because the procedure is designed to capture small amounts of
fragmented DNA, prone to cross-contamination, simultaneous handling of a large number of
samples is not recommended and the protocol is therefore optimized for 4–6 samples in each
extraction batch (see protocol) to minimize this risk. We believe that the method’s strength is
that it is particularly applicable when extractions from old specimens deposited in museum
collections are necessary. Because many taxa are rare and known only by unique or very few
valuable specimens held in museums, non-destructive museomics methods, such as the one
we have described, are essential to allow for such (often inordinately interesting) taxa to be
included in phylogenomic studies.
Supporting information
S1 File. Step-by-step protocol, also available on protocols.io. This protocol follows the gua-
nidine treatment protocol by Straube et al. (2021) [22], based on Dabney et al. (2013) [30] and
Rohland et al. (2004) [31].
(PDF)
Table 2. Updated diagnosis of the tribe Coprini. The combination of characters 1–6 constitutes a diagnosis of
Coprini that includes Onychothecus; for details see Tarasov & Dimitrov (2016) [28]. Characters 7–10 (marked with *)
refer to autapomorphies for Onychothecus.
Character Onychothecus other Coprini genera
1. Wing apex, posterior sclerite absent (Fig 2D) present
2. Number of elytral striae 10 (Fig 2E) 10
3. Elytral stria 8 carinate (Fig 2E) not carinate
4. Superior right peripheral (SRP) endophallite not ring-shaped (Fig 2J) not ring-shaped
5. Hypomera, anterior ridge reaches lateral
margin
present present
6. Posterior longitudinal hypomeral ridge absent usually present
7. Last tarsomere concealing tarsal claws (Fig 2F–
2G)
not concealing tarsal claws
8. Protibial apex excavated dorsally (Fig 2G) not excavated dorsally
9. Parameres asymmetric (Fig 2H–2I) usually symmetric
10. Sexual dimorphism only females with cephalic horn often males with cephalic
horn
https://doi.org/10.1371/journal.pone.0309596.t002
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S1 Table. Sample and sequencing summary information. Genus and species information
along with associated taxonomic details, repository, lab codes, and genetic data.
(XLSX)
S2 Table. Consumables and permanent material. Detailed information on plastics, chemicals
and permanent material used in this study to perform the archival DNA extraction protocol.
(XLSX)
S3 Table. Summary statistics of genetic data. Alignment-specific metrics, detailing various
statistical measures for the UCE loci concatenated alignment.
(XLSX)
S4 Table. Summary statistics of genetic data. Alignment-specific metrics, detailing various
statistical measures for UCE loci alignments from 50p dataset.
(XLSX)
S5 Table. Summary statistics of genetic data. Alignment-specific metrics, detailing various
statistical measures for UCE loci alignments from 70p dataset.
(XLSX)
S1 Fig. Gene-based phylogeny. Phylogeny reconstructed with 50p dataset containing all taxa
assessed in this study and bootstrap/SH values.
(TIF)
S2 Fig. Partition-based phylogeny. Phylogeny reconstructed with 50p dataset containing all
taxa assessed in this study and bootstrap/SH values.
(TIF)
S3 Fig. Gene-based phylogeny. Phylogeny reconstructed with 70p dataset containing all taxa
assessed in this study and bootstrap/SH values.
(TIF)
S4 Fig. Partition-based phylogeny. Phylogeny reconstructed with 70p dataset containing all
taxa assessed in this study and bootstrap/SH values.
(TIF)
S5 Fig. Matrix showing the presence of loci (black) for each sample. The absence is shown
in white. The amount of missing data is highlighted with red arrows and rectangles for UCEs
captured from genomes from Genbank.
(TIF)
Acknowledgments
We are grateful to the Coleoptera curators of collaborating natural history museums for the
loan of rare specimens under their care: Max Barclay (The Natural History Museum, London)
and Olivier Montreuil (Muse
´um national d’Histoire Naturelle, Paris). We thank the Malagasy
Institut pour la Conservation des Ecosystèmes Tropicaux (MICET) for their help in acquiring
research permits and logistical support during fieldwork in Madagascar. We also acknowledge
the generous technical support given to us by Louise Lindblom, head of the DNA lab, Univer-
sity Museum of Bergen, regarding the archival DNA extraction protocol. Finally, from the
Finnish Museum of Natural History, Helsinki, we thank all members of the Tarasov lab and
the Coleoptera team for their constructive suggestions and discussions, in addition to the staff
of the DNA lab, especially the head of the DNA Lab, Gunilla Ståhls, for their support.
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Author Contributions
Conceptualization: Fernando Lopes, Sergei Tarasov.
Data curation: Fernando Lopes, Nicole Gunter, Conrad P. D. T. Gillett, Giulio Montanaro,
Michele Rossini, Federica Losacco, Gimo M. Daniel, Sergei Tarasov.
Formal analysis: Fernando Lopes, Giulio Montanaro.
Funding acquisition: Nicole Gunter, Sergei Tarasov.
Investigation: Fernando Lopes, Nicole Gunter, Conrad P. D. T. Gillett, Giulio Montanaro,
Michele Rossini, Federica Losacco, Gimo M. Daniel, Sergei Tarasov.
Methodology: Fernando Lopes, Nicolas Straube, Sergei Tarasov.
Project administration: Fernando Lopes, Sergei Tarasov.
Resources: Conrad P. D. T. Gillett, Sergei Tarasov.
Supervision: Sergei Tarasov.
Validation: Fernando Lopes, Giulio Montanaro, Sergei Tarasov.
Visualization: Fernando Lopes, Giulio Montanaro, Federica Losacco, Sergei Tarasov.
Writing – original draft: Fernando Lopes, Nicole Gunter, Conrad P. D. T. Gillett, Giulio
Montanaro, Federica Losacco, Gimo M. Daniel, Nicolas Straube, Sergei Tarasov.
Writing – review & editing: Fernando Lopes, Nicole Gunter, Conrad P. D. T. Gillett, Giulio
Montanaro, Sergei Tarasov.
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