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From museum drawer to tree: historical DNA phylogenomics clarifies the systematics of rare dung beetles (Coleoptera: Scarabaeinae) from museum collections

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Abstract

Although several methods exist for extracting and sequencing historical DNA originating from drypreserved 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 internationally important natural history museums in London, Paris and Helsinki. The focal specimens comprise enigmatic dung beetle genera ( Nesosisyphus, Onychotechus and Helictopleurus ) that 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 suggested the reclassification of Onychotechus (previously incertae sedis ) within the tribe Coprini. Our approach demonstrates the feasibility 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/v1 Highlights We combined custom low-cost archival DNA extractions and Ultraconserved Element phylogenomics DNA from rare museum specimens of enigmatic dung beetles revealed their phylogenetic connections Genomic data was obtained from the holotype of a potentially extinct monoinsular endemic species Genomic data allowed a rare and enigmatic species of previously unknown affinity to be classified The morphology of museum specimens remained intact following non-destructive DNA extraction Abstract Figure
Graphical Abstract
From museum drawer to tree: historical DNA phylogenomics clarifies the systematics of rare dung
beetles (Coleoptera: Scarabaeinae) from museum collections
Fernando Lopes,Nicole Gunter,Conrad P.D.T. Gillett,Giulio Montanaro,Michele Rossini,Federica Losacco,Gimo M. Daniel,Nicolas
Straube,Sergei Tarasov
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
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Highlights
From museum drawer to tree: historical DNA phylogenomics clarifies the systematics of rare dung
beetles (Coleoptera: Scarabaeinae) from museum collections
Fernando Lopes,Nicole Gunter,Conrad P.D.T. Gillett,Giulio Montanaro,Michele Rossini,Federica Losacco,Gimo M. Daniel,Nicolas
Straube,Sergei Tarasov
We combined custom low-cost archival DNA extractions and Ultraconserved Element phylogenomics
DNA from rare museum specimens of enigmatic dung beetles revealed their phylogenetic connections
Genomic data was obtained from the holotype of a potentially extinct monoinsular endemic species
Genomic data allowed a rare and enigmatic species of previously unknown affinity to be classified
The morphology of museum specimens remained intact following non-destructive DNA extraction
.CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a
preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted November 1, 2023. ; https://doi.org/10.1101/2023.10.27.564347doi: bioRxiv preprint
From museum drawer to tree: historical DNA phylogenomics clarifies
the systematics of rare dung beetles (Coleoptera: Scarabaeinae) from
museum collections
Fernando Lopesa,,Nicole Gunterb,Conrad P.D.T. Gilletta,Giulio Montanaroa,Michele Rossinia,c,
Federica Losaccoa,Gimo M. Danield,Nicolas Straubeeand Sergei Tarasova
aFinnish Museum of Natural History - University of Helsinki, Pohjoinen Rautatiekatu 13, Helsinki, 00100, Uusima, Finland
bThe Cleveland Museum of Natural History, 1 Wade Oval Drive, Cleveland, 44106, Ohaio, USA
cUniversity of Padova, Via dell’Università 16, Padua, 35020, Padua, Italy
dNational Museum, Bloemfontein, 36 Aliwal Street, Bloemfontein, 9301, Free State, South Africa
eUniversity Museum Bergen, Allégaten 41, Bergen, 5020, Vestland, Norway
A R T I C L E I N F O
Keywords:
Museomics
Archival DNA
UCE
Mauritius
Madagascar
Scarabaeidae
A B S T R A C T
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 internationally important natural history museums in London, Paris and
Helsinki. The focal specimens comprise enigmatic dung beetle genera (Nesosisyphus,Onychotechus
and Helictopleurus) that 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
suggested the reclassification of Onychotechus (previously incertae sedis) within the tribe Coprini.
Our approach demonstrates the feasibility 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/v1
1. Introduction
Museomics, a term encompassing procedures allowing
access to and analysis of the historical genomic data pre-
served in biological specimens deposited in natural his-
tory museums, is providing unprecedented opportunities
to investigate evolutionary histories (Miller et al.,2009;
Raxworthy and Smith,2021). Together with concomitant
advances in high-throughput sequencing technologies and
bioinformatics, museomics has paved the way for the ex-
ploitation of an ever-broader diversity of taxonomic and
temporal sampling (Orlando et al.,2015;Burrell et al.,
2015). Importantly, by enabling access to genomes already
preserved in existing museum specimens, museomics can
circumvent the need for costly, laborious, and unpredictable
bespoke fieldwork, in order to achieve taxon sampling ob-
jectives (Raxworthy and Smith,2021;Orlando et al.,2015).
Museomics is also compatible with physically preserving the
morphological integrity of specimens, when non-destructive
DNA extraction methods are employed. This is of paramount
Corresponding authors
fernando.vieiralopes@helsinki.fi (F. Lopes)
ORCID(s): 0000-0002-1246-2777 (F. Lopes); 0000-0002-8355-0862 (N.
Gunter); 0000-0002-1487-4003 (C.P.D.T. Gillett); 0000-0003-0836-1364 (G.
Montanaro); 0000-0002-1938-6105 (M. Rossini); 0000-0002-3754-7179 (F.
Losacco); 0000-0003-3602-5034 (G.M. Daniel); 0000-0001-7047-1084 (N.
Straube); 0000-0001-5237-2330 (S. Tarasov)
importance to natural history museums and the scientific
community because it ensures that intact voucher specimens
will remain available for study by future generations (Fong
et al.,2023;Weirauch et al.,2020). Indeed, the importance
of museomics can only heighten as the necessity for inclu-
sion of recently extinct species within phylogenies becomes
increasingly inevitable (Toussaint et al.,2021). Progress
in insect museomics has already greatly contributed to the
study of insects - Earth’s most diverse organisms (Eggleton,
2020;Fong et al.,2023). However, despite notable achieve-
ments, challenges still remain (Raxworthy and Smith,2021).
Specifically, DNA in many dry-preserved museum spec-
imens is fragmented and prone to contamination, whilst
the comparatively small amount of tissue present in small
insects can further limit the success of DNA extractions
(Patzold et al.,2020).
In recent years, a variety of molecular methods have been
developed to obtain historical DNA data at a genome-wide
scale (Orlando et al.,2015;Burrell et al.,2015), includ-
ing approaches exploiting both whole-genome (shotgun)
and reduced-genome sequencing (Jin et al.,2020;Toussaint
et al.,2021;Twort et al.,2021). Many widely used meth-
ods rely on standard DNA extractions using commercial
DNA kits, followed by the construction of DNA libraries
based on hybridization approaches that combine restriction
enzyme fragmentation and RNA probe capture. For instance,
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Historical DNA and Dung Beetles
HyRAD uses a double enzymatic restriction of DNA extracts
from fresh samples (containing well-preserved DNA) to
produce RNA probes that serve as baits for capturing ho-
mologous fragments from historical (more degraded) DNA
libraries (Suchan et al.,2022;Gauthier et al.,2020). How-
ever, standard DNA extraction, typically undertaken with
commercially available kits, is optimized for high molec-
ular weight DNA, only ineffectively capturing low-weight
short fragments, which are precisely those expected from
degraded historical samples. Furthermore, reduced genome
approaches exploiting restriction enzymes require a compar-
atively large initial amount of source DNA, not easily ob-
tained from small insect specimens (Sproul and Maddison,
2017). Moreover, those methods tend to be costly when ex-
tensively 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
methods 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 distantly related or due to DNA fragmentation at
restriction sites (especially in poorly preserved samples),
both possibilities that can lead to missing data (Cerca et al.,
2021).
In phylogenetic studies with historical DNA, cost-effective
methods such as Ultra Conserved Elements (UCEs) and
Anchored Hybrid Enrichment (AHE) are rising in popularity
due to their ability to target specific informative loci within
a focal group (Faircloth,2017;Call et al.,2021;Faircloth
et al.,2012;Mayer et al.,2021). While standard extraction
from dry-preserved specimens may yield adequate DNA
for UCE and AHE sequencing (Gustafson et al.,2020;
Mayer et al.,2021), its success varies based on preservation.
Therefore, exploring more sensitive extraction methods is
essential, especially since their application to entomological
collections remains poorly investigated (Call et al.,2021).
In this study, we aim to bridge this gap by assess-
ing a cost-effective (10 per sample) archival DNA ex-
traction protocol (Straube et al.,2021) tailored for his-
torical insect specimens, specifically for UCE-sequencing.
We applied this protocol to explore the phylogenetic rela-
tionships of eleven species and subspecies of dung beetles
(Coleoptera: Scarabaeinae) represented by historical speci-
mens from three museums: The Natural History Museum,
London (NHMUK); the Muséum National d’Histoire Na-
turelle, Paris (MNHN); and the Finnish Museum of Natural
History, Helsinki (MZHF).
The specimens are of diverse ages and represent enig-
matic species of questionable phylogenetic assignment (Ta-
ble 1). The oldest specimen, the holotype from NHMUK of
Nesosisyphus rotundatus collected in 1944, is a potentially
extinct species from Mauritius, not previously included in
molecular phylogenies. The extremely rare Oriental genus
Onychothecus, with uncertain taxonomic affinity (Tarasov
and Dimitrov,2016) and previously lacking DNA data,
was represented by a specimen from MNHN, collected in
1985. Finally, nine poorly-known taxa belonging to the en-
demic Madagascan genus Helictopleurus were represented
by specimens from MZHF collected between 2003–2010.
Our archival DNA extraction protocol yielded high quality
DNA for successful UCE-sequencing using the recently
designed probe set for scarab beetles (Gustafson et al.,
2023). To elucidate the phylogenetic position of the selected
enigmatic species, we expanded our taxon sampling by
sequencing additional alcohol-preserved dung beetles using
a standard DNA extraction protocol with a commercial kit.
In the following sections, we discuss the phylogenetic place-
ment of the focal species based on our results and provide
necessary taxonomic changes. We also explore the broader
application of the proposed extraction protocol to a wide
range of historical specimens of insects and other taxa.
2. Material and methods
2.1. Taxon sampling
We compiled a dataset of UCE sequences from 96 bee-
tles (Table S1), 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 and
available on GenBank (Gustafson et al.,2023). 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 Sil-
phidae). 59 of the newly 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,Onychotechus and
Helictopleurus) were selected from the dry collections of
three museums, and formed the focal taxa in our study (Table
1, Fig. 1A).
2.2. DNA extraction: archival DNA protocol
Our optimized protocol is available at Protocols.io.
Briefly, the protocol is a customization of the archival DNA
extraction protocol and Guanidine treatment described by
Straube et al. (2021) which, in turn, was influenced by
the studies of Dabney et al. (2013) and Rohland et al.
(2004). The new approach was first proposed for wet-
preserved 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 (Table S2
and at Protocols.io). The method uses an extension reservoir
attached to a commercial silica spin column, 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
(Straube et al.,2021). We further customized this protocol
as a non-destructive extraction protocol using dry-preserved
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Historical DNA and Dung Beetles
Table 1
Dry-preserved scarab dung beetle specimens from natural his-
tory museum collections, used in historical DNA extractions.
Natural History Museum, London (NHMUK); Muséum National
d’Histoire Naturelle, Paris (MNHN); and Finnish Museum of Nat-
ural History (MZHF).
Species Sampling Year Museum Origin
Nesosisyphus rotundatus Vinson, 1946 [holotype] 1944 NHMUK Mauritius
Onychothecus tridentigeris Zelenka, 1992 1985 MNHN Thailand
Helictopleurus fasciolatus fasciolatus (Fairmaire, 1898) 2003 MZHF Madagascar
Helictopleurus sinuatocornis (Fairmaire, 1898) 2003 MZHF Madagascar
Helictopleurus perrieri (Fairmaire, 1898) 2006 MZHF Madagascar
Helictopleurus undatus (Olivier, 1789) 2008 MZHF Madagascar
Helictopleurus nicollei Lebis, 1960 2008 MZHF Madagascar
Helictopleurus near furcicornis Lebis, 1960 2008 MZHF Madagascar
Helictopleurus neuter (Fairmaire, 1898) 2009 MZHF Madagascar
Helictopleurus fasciolatus obscurus Lebis, 1960 2009 MZHF Madagascar
Helictopleurus fasciolatus pseudofasciolatus Montreuil, 2007 2010 MZHF Madagascar
beetle specimens from several museum entomological col-
lections, as specified in step 4 of the protocol. In short, we
optimized the way we used the samples for the lysis step
by not destroying body parts. DNA extractions from dry-
preserved museum specimens (Table S1) were undertaken
in a dedicated ’clean room’ for historical samples at MZHF.
Nesosysiphus rotundatus, a monoinsular endemic species
from Mauritius that is known only from two specimens, was
represented by its holotype, deposited in NMHUK (Fig. 1C).
The tiny specimen, one of the smallest scarab dung beetles
in the world (4 mm), was collected by J. Vinson in 1944
(Vinson,1946). This specimen was carefully relaxed and
disarticulated; only the prothorax (with exposed internal
tissues) with attached forelegs (but not the head) was used as
a source of DNA during the digestion step of the extraction
(Fig. 1C), 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,
without visible external deterioration, and the specimen
was successfully reassembled (Fig. 1C). Onychothecus tri-
dentigeris 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). From this specimen,
we destructively used an entire leg during the extraction (Fig.
2B,C), which resulted in 17.40 ng of DNA that generated
1,692 UCE loci.
2.3. DNA extraction: alcohol-preserved specimens
DNA was extracted from wet-preserved museum spec-
imens using the QIAamp DNA Micro Kit (QIAGEN) fol-
lowing the manufacturer’s protocol. 2 𝜇l of the resulting
purified DNA from both dry-preserved (historical) and wet-
preserved specimens was quantified on a Qubit fluorom-
eter 4.0 (Thermo Fisher Scientific) using high-sensitivity
reagents.
2.4. Library preparation and sequencing
We used the tailored UCE Scarabaeinae probe-set Scarab
3Kv1 (Gustafson et al.,2023), a genome reduction ap-
proach for beetle genomes combining the Coleoptera 1.1Kv1
probeset (Faircloth,2017) and Scarabaeinae-specific tar-
geted loci (Gustafson et al.,2023), to generate sequence
data for reconstruction of phylogenies (Fig. 1and Figs.
S1-S4). This probe set contains 25,786 probes targeting
3,174 loci (Gustafson et al.,2023). Library preparation
and enrichment were performed at RAPiD Genomics LLC
(Gainesville, FL, U.S.A.) for Illumina sequencing, applying
their high-throughput workflow with proprietary chemistry.
DNA was sheared to a mean fragment length of 500 bp
and A-tailed, followed by the incorporation of unique dual-
indexed Illumina adaptors and PCR enrichment. Libraries
were sequenced on an Illumina HiSeq 2500 (2 ×150 pair-
end).
2.5. Data Processing
Demultiplexing and trimming were performed by RAPiD
Genomics LLC using Illumina bcl2fastq2 2.20 (Illumina,
2017). Our UCE datasets were assembled using the package
Phyluce 1.7.3 (Faircloth,2016) following our workflow
available on GitHub. Raw demultiplexed reads were first
cleaned using Illumiprocessor 2.0 (Faircloth,2013) to re-
move residual adapter contamination. Cleaned reads were
inspected for quality using FastQC (Andrews et al.,2010)
and assembled into contigs in Spades 3.15.4 (Bankevich
et al.,2012). The Scarab 3kv1 UCE probes were matched to
the assembled contigs in Phyluce, with a minimum identity
of 80% and coverage of 80×to avoid off-target contaminat-
ing sequences (Gustafson et al.,2023;Bossert and Danforth,
2018). UCE loci were then extracted from the sequenced
data. We harvested UCE loci from the available whole beetle
genomes on GenBank (Gustafson et al.,2023) (Table S1)
using Phyluce and combined them with our newly sequenced
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Historical DNA and Dung Beetles
data. The UCE loci were aligned in MAFFT (Katoh and
Standley,2013), 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 (Faircloth,2016). The resulting alignments were
parsed to a parallel wrapper around Gblocks to eliminate
poorly aligned positions and divergent regions using the
settings: b1=0.5, b2=0.85, b3=8, b4=10 (Castresana,2000;
Talavera and Castresana,2007). Summary statistics for the
generated datasets were computed using the program AMAS
(Borowiec,2016).
2.6. Phylogenomics
For phylogenomic analyses, we constructed data ma-
trices for species trees and Maximum-Likelihood (ML)
concatenated-based inferences with 50% and 70% complete
data, allowing up to 50% and 30% missing taxa for each
locus, respectively (Molloy and Warnow,2018;Gustafson
et al.,2020). Hereafter, the 50% and 70% complete datasets
will be called 50p and 70p datasets. Full and partitioned
UCE alignments are available on the Open Science Frame-
work (OSF) repository at DOI 10.17605/osf.io/mxwj7. ID
labels in tree files were translated into full species names us-
ing the custom Python script rename_leaves_v1.0.py avail-
able on GitHub.
2.6.1. Species tree analysis
We recovered phylogenies accommodating for gene tree
heterogeneity due to Incomplete Lineage Sorting (Mad-
dison,1997;Edwards,2009) considering each UCE loci
as an independent gene. Species trees were estimated in
IQ-Tree 2.0.7 (Minh et al.,2020), with confidence levels
calculated using 1000 ultrafast bootstrap (UFBoot) repli-
cates, and topologies tested by the Shimodaira–Hasegawa
test (SH-aLRT). The best substitution models were automat-
ically selected using ModelFinder (-m mfp option) imple-
mented in IQ-Tree under the Bayesian Information Criterion
(Kalyaanamoorthy et al.,2017).
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) topology search strategy to optimize each bootstrap
tree. As phylogenetic models rely on various simplifying
assumptions to ease computations (e.g., treelikeness, re-
versibility, and homogeneity of substitution models), esti-
mations of some genomic regions can severely violate model
assumptions, causing biases in phylogenetic estimates oftree
topologies (Naser-Khdour et al.,2019). To test these viola-
tions on each locus, we also applied the test of symmetry
with the option --symtest-remove-bad. Partitions (concate-
nated analyses) and genes (species trees analyses) with p-
value 0.05 for the test of symmetry were removed from
downstream analyses (Naser-Khdour et al.,2019).
2.6.2. Concatenated-based analysis
We also constructed ML phylogenies from the concate-
nated 50p and 70p datasets using IQ-Tree, using the same
parameters mentioned above for species trees.
The datasets were partitioned with Sliding-Window Site
Characteristics (SWSC-EN), an entropy-based method de-
veloped specifically for UCE data (Tagliacollo and Lanfear,
2018). To implement the SWSC-EN method, we used Phy-
luce 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
used with Partitionfinder 2.1.1 and Python 2.7 (Lanfear
et al.,2017). As Partitionfinder2 works only with Phylip
alignments, we converted the concatenated Nexus file to
Relaxed Phylip format using Geneious 2022.2.1 (Kearse
et al.,2012). The partitioning scheme was then generated
with Partitionfinder2 with linked branch lengths, a GTR+G
model of evolution, an Akaike information criterion with
correction (AICc) for model selection, and a variant of
the relaxed hierarchical clustering search algorithm (supple-
mentary data) (Lanfear et al.,2014;Gustafson et al.,2020).
2.7. Morphological Examination
As a complement to molecular inference of the phyloge-
netic position of Onychothecus and related taxa, we studied
the morphology of two available specimens of Onychothecus
tridentigeris (deposited in MNHN) in detail. Morphological
terminology and protocols follow Tarasov and Dimitrov
(2016) and Tarasov and Génier (2015). Specimens were
examined under a Leica S9D stereomicroscope. Photographs
were taken with a Canon MP-E 65 mm, f/2.8, 1–5×macro
lens mounted on a Canon EOS 5D camera, and then stacked
using the Stackshot (Cognisys Inc.) automated system.
3. Results
3.1. UCE data
We obtained a mean of 1.86×107paired-end reads per
sample. Our results revealed that shorter fragments from
museum samples were effectively integrated into DNA li-
braries, resulting in the recovery of a substantial number
of UCE loci for downstream phylogenomic analyses (Fig.
3, Table S1). Specifically, samples for which DNA was ex-
tracted using the historical DNA protocol yielded the high-
est 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; Table S1 and Fig. 3B). Interestingly, older samples,
such as O. tridentigeris and N. rotundatus, exhibited a simi-
lar number of recovered loci compared to the more recently
collected wet-preserved samples extracted with the commer-
cial kit. (Table 1and Table S1).
Before data filtering, the full concatenated alignment
(96 tips) contained 3,160 UCE loci and 269,808 parsimony-
informative sites distributed across 675,2 Kbp (Table S3).
The 50p dataset contained 1,497 UCE loci with a mean
of 79.97 parsimony-informative sites per locus (Table S4),
and the 70p dataset contained 289 UCE loci with a mean
of 60.54 parsimony-informative sites per locus (Table S5).
The conspicuous disparity among unfiltered, 50p and 70p
datasets is due to large proportion of missing data present in
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Historical DNA and Dung Beetles
Figure 1: (A) Species tree inferred using 1,497 UCEs and the 50% complete dataset; dry-preserved historical museum specimens are in
bold. (B) The part of the tree showing the relationships within Helictopleurus. (C) The holotype of Neosisyphus rotundatus before and
after DNA extraction using the proposed archival protocol. Node numbers in (A) and (B) indicate bootstrap values < 100, while all other
nodes have full bootstrap support.
the genomes retrieved from GenBank, which mostly served
as outgroup taxa in our study (Gustafson et al. (2023) - Table
S2 and Figs. S1-S5).
3.2. Phylogenomics
Increasing the completeness threshold during the con-
struction of data matrices significantly decreased the number
of UCE loci and overall bootstrap support (Fig. 1, Figs.
S1-S4 and Table S4S5). Because phylogenomic studies
generally do not benefit from filtering out loci with an
increased proportion of missing data (Molloy and Warnow,
2018), we focused on the 50p dataset, which resulted in
an optimal trade-off between the highest overall bootstrap
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Historical DNA and Dung Beetles
Figure 2: Morphology of Onychothecus tridentigeris Zelenka, 1992. 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, with elytral striae numbered and
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 and Génier (2015)).
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Historical DNA and Dung Beetles
Figure 3: Summary of UCE data resulting from two DNA extrac-
tion methods (archival extraction protocol in red and standard ex-
traction 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.
support and SH-values (Fig. 1and Figs. S1-S2), and the
number of recovered loci (Table S1).
Our phylogenetic trees based on the concatenated align-
ment (ML tree) were generally well-supported, with only a
few nodes of moderate depth having poor support (see Fig.
1and Figs. S1-S2). Notably, Scarabaeinae formed a mono-
phyletic group, with Frankenbergerius and Sarophorus rep-
resenting a basal lineages which is sister to the remaining
Scarabaeinae. The Afrotropical Epirinus was identified as
the sister taxon to the remaining Sisyphini. Additionally, the
Australasian genera appeared to form a monophyletic group,
as too did the clade consisting of the tribes Onthophagini and
Oniticellini.
The oldest historical specimen of N. rotundatus from
Mauritius consistently clustered within the tribe Sisyphini
across all analyses with strong bootstrap support (BS: 100;
Fig. 1A and C). The enigmatic species, O. tridentigeris from
the Oriental Region, consistently emerged as the sister taxon
to the genera of the tribe Coprini in all trees (BS: 98;
Fig. 1A and Fig. 2). All Madagascan Helictopleurus species
formed a sister clade to Oniticellini in all analyses as well
(Figs. 1A and B). Some slight variations in the grouping
of certain Helictopleurus species were observed in the 70p
datasets, likely due to reduced genomic information and/or
short branches in the backbone of the clade (Fig. 1and S1-
S4).
4. Discussion
4.1. Phylogenetic relationships
All resultant topologies were generally consistent with
previous morphological (Tarasov and Génier,2015) and
molecular analyses based on individual genes (Tarasov and
Génier,2015;Tarasov and Dimitrov,2016;Gunter et al.,
2016) and UCE data (Gustafson et al. (2023), but containing
only limited taxon sampling).
The present phylogeny, whilst encompassing a relatively
taxonomically diverse set of dung beetles, is not compre-
hensive enough to justify discussion of general phyloge-
netic relationships and their taxonomic consequences within
Scarabaeinae. Therefore, we leave that broader discussion
to forthcoming phylogenomic studies that will incorporate
substantially larger taxon sampling. Instead, we focus on
assessing the relationships among the enigmatic taxa herein
sequenced from historical specimens.
Mauritian Nesosisyphus.This study offers the first in-
sights into the relationships of the endemic Mauritian genus
Nesosisyphus that has not previously undergone phyloge-
netic analysis. Our placement of N. rotundatus (Fig. 1A,
C) within the tribe Sisyphini (Daniel et al.,2020;Tarasov
and Dimitrov,2016) was expected and is also supported
by morphological synapomorphies that clearly indicate that
Nesosisyphus is a member of this tribe. Therefore, the phy-
logenetic result based on UCE data obtained from the old
holotype of N. rotundatus strongly agrees with our initial
hypothesis (Vinson,1946).
Nesosisyphus rotundatus is a flightless roller dung bee-
tle, uniquely known by the two specimens that make up
the type series collected during the early 1940s from the
southern slope of Mount Ory in Mauritius (Vinson,1946).
All subsequent collecting efforts on the island, which have
included sampling at the type locality, and have resulted in
the discovery of three additional Mauritian 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 introduc-
tion of exotic species (Safford,1997;Monty et al.,2013),
we regard this species as potentially extinct (Losacco et al.,
in prep). One of the achievements of our study has been to
unlock genomic data from this enigmatic species for further
investigation.
Oriental Onychothecus.This extremely rare genus
comprises four species distributed in southeastern Asia
(Figs. 1and 2): China (Yunnan), Myanmar, Thailand, Laos,
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Historical DNA and Dung Beetles
Table 2
Updated diagnosis of the tribe Coprini. The combination of charac-
ters 1–6 constitutes a diagnosis of Coprini that includes Onychothe-
cus; for details see Tarasov and Dimitrov (2016). Characters 7–10
(marked with ) refer to autapomorphies of 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-G) not concealing tarsal claws
8. Protibial apex excavated dorsally (Fig. 2G) not excavated dorsally
9. Parameres asymmetric (Fig. 2H-I) usually symmetric
10. Sexual dimorphism only females with cephalic horn often males with cephalic horn
and Vietnam (Ochi and Kon,1998;Schoolmeesters,2023).
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 overwhelmingly
more common in the superfamily Scarabaeoidea), in addi-
tion to having an unknown diet, habits, and general biology.
Onychothecus has not yet been classified (=incertae sedis)
into any of the existing tribes in the subfamily Scarabaeinae
(Tarasov and Dimitrov,2016). Only a single previous phy-
logenetic treatment of the genus exists (Montreuil,1998),
based upon morphological data, which identified it as a sister
to the genus Paraphytus that has a disjunct Afrotropical
and Oriental distribution. Paraphytus belongs to the most
basal lineage of Scarabainae (Tarasov and Dimitrov,2016)
that also includes the Afrotropical genera Frankenbergerius
and Sarophorus, which we 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,Litocopris,
Microcopris, belonging to the tribe Coprini sensu Tarasov
and Dimitrov (2016). Consequently, based on our results,
we assign Onychothecus to the tribe Coprini and discuss this
placement in a separate section below.
Madagascan Helictopleurus.The genus Helictopleurus
comprises approximately 65 species, all endemic to Mada-
gascar (Figs. 1A-B) and primarily occurring in forest habi-
tats (Wirta et al.,2010;Miraldo et al.,2011). We extracted
and sequenced DNA from nine dry-preserved specimens
from museum collections, having been collected between
2003 and 2010, in addition to two alcohol-preserved speci-
mens. Sequence data for one additional species was included
from a previously published study (Rossini et al.,2021). Our
phylogenetic analyses (Figs. 1A-B, S1-S4) confirm previous
results, demonstrating the monophyly of the genus Helicto-
pleurus, its sister relationship to the genus Oniticellus, and
that the Helictopleurus +Oniticellus clade falls within the
clade containing Onthophagini + Oniticellini (Breeschoten
et al.,2016;Tarasov and Solodovnikov,2011;Wirta et al.,
2008). However, when compared to earlier molecular phy-
logenies based on individual genes, our analyses resulted
in slight variations in the interspecific relationships within
Helictopleurus (Rossini et al.,2021;Wirta et al.,2008).
Enhancing taxon sampling in future studies and potentially
integrating it with prior single-gene data will help achieve
more robust results.
The fact that our results, including newly sequenced
data from museum specimens of varying ages (Table 1)
and preservation, produced robust results that are consistent
with existing phylogenies (Gustafson et al.,2023;Tarasov
and Dimitrov,2016), demonstrates the effectiveness of the
proposed historical DNA approach in combination with
UCE sequencing. Such consistency is of particular signifi-
cance because concerns about sequence data obtained from
historical specimens being contaminated or of poor quality,
and consequently obfuscating or impeding phylogenetic in-
ference, appear not to have been borne out in our study.
4.2. Tribal transfer of Onychothecus to Coprini
Genus Onychothecus Boucomont, 1912
Onychothecus Boucomont, 1912: original description; as member
of Scatonomini Lacordaire, 1856 synonym of Deltochilini Lacor-
daire, 1856: sensu Bouchard et al. (2011).
Onychothecus;Balthasar (1963): as member of Pinotini Kolbe,
1905 synonym of Ateuchini Perty, 1830: sensu Smith (2006).
Onychothecus;Tarasov and Dimitrov (2016): as incerta 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, and Pseudopedaria. The tribe
lacks unique apomorphies allowing for unequivocal diagno-
sis. Instead it is characterized only by a combination of six
characters (Tarasov and Dimitrov,2016).
We examined the morphology of two specimens of Ony-
chothecus tridentigeris in detail, including the first-known
male of the species (Figs. 2A and 2G-J). 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
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Historical DNA and Dung Beetles
taxonomic actions were be 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 member of the tribe Coprini.
In our opinion, this action is justified in order to maintain
stability in the classification of Scarabaeinae.
Although the general habitus of Onychothecus resem-
bles that of many other members of the tribe Coprini, its
morphology stands out within the Scarabaeinae in gen-
eral. 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 pos-
sesses characters that have not previously been adopted
to diagnose 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 morpholog-
ical diagnosis of the tribe Coprini. We present the new
diagnosis of Coprini in Table 2.
4.3. Conclusion
We successfully obtained genomic data that allowed for
the phylogenetic placement of several species represented
by unique historical specimens deposited in important nat-
ural history museum collections (Table 1). Our results
demonstrate that combining a minimally destructive and
low-cost archival DNA extraction (10 per sample),
with subsequent target enrichment of DNA libraries for
sequencing a curated set of beetle UCE loci, is an efficient
museomics tool (see Tables S1-S2). The proposed extraction
protocol should also integrate well with AHE sequencing.
Even from the limited quantity of source tissue available
in old museum specimens, by being able to capture small
fragments of degraded DNA (Fig. 2), we demonstrated a
remarkably 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 labo-
ratories within two days, including an overnight digestion
step. The protocol is optimized for 4–6 samples in each
extraction batch (see protocol). Because the procedure is
designed to capture small amounts of fragmented DNA,
prone to cross-contamination, simultaneous handling of a
larger number of samples is not recommended, in order 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 in order to allow for such (often
inordinately interesting) taxa to be included in phylogenomic
studies.
Funding
This research received funding from the Research Council
of Finland (grant 331631) and a 3-year grant from the
University of Helsinki to ST. Networking was supported by a
Finnish Museum of Natural History Pentti Tuomikoski Fund
award to CG. NG was supported by the National Science
Foundation (USA, grant DEB-1942193).
Acknowledgements
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 Mu-
seum, London) and Olivier Montreuil (Muséum 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 for their
logistical support during fieldwork in Madagascar. We also
acknowledge the generous technical support given to us by
Louise Lindblom, head of the DNA lab, University Museum
of Bergen, in relation to the historical 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.
Authorship contribution statement
Fernando Lopes: Conceptualization of this study, data
curation, methodology, investigation, analyses, visualiza-
tion, writing - original draft preparation. Nicole Gunter:
Funding acquisition, data curation. Conrad P.D.T. Gillett:
Resources, data curation, writing - review & editing. Giulio
Montanaro: Data curation, investigation, writing - original
draft preparation. Michele Rossini: Data curation. Federica
Losacco: Data curation, visualization. Gimo M. Daniel:
Data curation. Nicolas Straube: Data curation, writing -
review & editing. Sergei Tarasov: Funding acquisition,
resources, conceptualization of this study, data curation,
methodology, investigation, visualization, writing - original
draft preparation.
Data Availability
The data presented in this study can be accessed at Open Sci-
ence Framework and Protocols.io via the two following hy-
perlinks: DOI 10.17605/osf.io/mxwj7 and DOI 10.17504/pro-
tocols.io.81wgbybqyvpk/v1. Raw sequencing data can be
found at NCBI BioProject PRJNA1031114.
Declaration of Competing Interest
The authors declare that they have no known competing
financial interests or personal relationships that influenced
the work reported in this article.
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... It would be helpful if future phylogenetic studies including Phanaeus species obtain sequences de novo, because in several cases is not possible to determine which of the currently recognized species pertain to the previously published sequences (i.e., Price 2009;Gillett & Toussaint 2020). It would be advisable to implement phylogenomic tools, but particularly Ultraconserved Elements, which even allow the study of rare and old museum specimens (Daniel & Davis 2023;Lopes et al. 2023a). ...
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