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Draft genome from facultatively parthenogenetic
Opiliones indicates frequent mitonuclear sequence
transfer and novel full-length insertions
Sarah Stellwagen
University of North Carolina at Charlotte
Mercedes Burns ( burnsm@umbc.edu )
University of Maryland, Baltimore County
Research Article
Keywords: genome assembly, Opiliones, polyploidy, long-read sequencing, facultative parthenogenesis
Posted Date: January 11th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-3846124/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Additional Declarations: No competing interests reported.
Draft genome from facultatively parthenogenetic Opiliones indicates
frequent mitonuclear sequence transfer and novel full-length insertions
Sarah Stellwagen1,2,*and Mercedes Burns2,†
1Department of Biological Sciences, University of North Carolina at Charlotte and 2Department of Biological
Sciences, University of Maryland, Baltimore County
January 9, 2024
Abstract
Background: Facultative parthenogenesis and intra-population mixed ploidy are rare in animals. These unique
characteristics allow opportunities to investigate the relationship between sexual modality and ploidy. We have
completed a draft genome of the Japanese harvester ("daddy-longlegs") Leiobunum manubriatum, a species which
reproduces sexually and asexually, and with mixed diploid and tetraploid populations in some areas. Results: We
combined Oxford Nanopore’s MinION long-read sequencing platform with Dovetail Hi-C scaffolding to assemble the
haploid genome for the diploid race, which is approximately 336 MBp after collapsing heterozygous sequence. The
assembly’s completeness was measured using BUSCOs from Eukaryota (complete: 92.6%), Arthropoda (complete:
96.9%), and Arachnida (complete: 95.3%). We searched raw sequence reads and the draft genome for nuclear
mitochondrial DNA (numt) sequences. While only one complete mitochondrial genomic transfer was found in the draft
genome, there are at least 12 complete numts across 9 reads within the raw sequencing data that were lost during the
assembly process. Conclusions: The genome of the L. manubriatum diploid race is an invaluable resource not only for
opilionid research, but also for facilitating studies investigating the evolution of their unique reproductive mode and
mixed ploidy. To our knowledge, this is the first published genome of a wild-derived facultative parthenogen. Future
work will leverage this resource in comparative genomics and transcriptomics of L. manubriatum to understand the
connection between ploidy and sexual strategy.
Key words: genome assembly; Opiliones; polyploidy; long-read sequencing; facultative parthenogenesis
Introduction
Sexual reproduction is the dominant mode of reproduction, however many animals and plants exhibit parthenogenesis, or the
production of viable offspring without fertilization of an egg. Particularly in plants and arthropods, approximately one out of
every 1000 multicellular eukaryotic taxa exhibit parthenogenesis [1]. Polyploidy, the condition where an organism has more than
two sets of chromosomes, is often associated with asexuality [2, 3]. The Daphnia pulex complex of water fleas has both obligate
asexual and cyclically sexual/asexual populations, in addition to polyploidization of some lineages [4]. Populations of New Zealand
freshwater snail Potamopyrgus antipodarum, which include sexual and asexual lineages, have variations in ploidy level and genome
size [5]. The spiny leaf insect Extatosoma tiaratum exhibits facultative parthenogenesis, where females can produce offspring
asexually, or, if mated, sexually [6].
Two species of Japanese opilionids (also known as "harvesters" or "daddy-longlegs") belonging to the Leiobunum curvipalpe
group exhibit facultative parthenogenesis: L. manubriatum (Figure 1) and L. globosum. These species are endemic to northern Japan,
with L. manubriatum’s range extending through the Japanese Alps and overlapping on the range of L. globosum in Aomori, Akita, and
Hokkaido Prefectures. While L. globosum maintains tetraploidy in all individuals [7], those of L. manubriatum have intra-population
diploidy and tetraploidy, or a single cytotype in some populations [8]. Both ploidies can reproduce sexually or asexually, but it is
not currently known whether mated females can produce unfertilized eggs even after mating. The number of chromosomes for the
mixed-sex diploid race of L. manubriatum is reported to be 2n=24, with the all-female tetraploid race varying between 2n=4x=ca.
48 (46-49) [7].
Nuclear mitochondrial sequence (numt) refers to areas of the nuclear genome that contains sequence which originated in
the mitochondrial genome [9]. Numts tend to be relatively fragmented, and to our knowledge, there is only one reported full-
length mitogenome insertion to the nuclear genome, discovered in tarsiers [10]. This insertion covered the complete 17,004 bp
Compiled on: January 9, 2024.
Draft manuscript prepared by the author.
1
2|Journal of XYZ, 2020, Vol. 00, No. 0
Figure 1. Adult Leiobunum manubriatum male (left) and female from Shomyo Falls, Japan, copulating. Photo credit: Sarah Stellwagen
mitogenome with an additional 862 bp overlap of the D-loop region and partial 16S rRNA [11, 10]. The largest human numt covers
approximately 90% of the mitochondrial genome [12]. In arthropods, honeybees have the highest percentage of reported numts,
though their numts are relatively short, the longest being 3,335 bp and spread across a 25 kb area of the nuclear genome [13].
Mitochondrial genomes average around 17 kb [14], and duplications can confuse assembly efforts when aligning sequences ob-
tained through short read technologies [15, 16]. Furthermore, nuclear insertions of mitochondrial sequence can lead to problems
with phylogenetic reconstruction if numts are inadvertently amplified [17] or present in only a subset of taxa. Long-read sequenc-
ing, a necessary tool for overcoming the challenges of repetitive genomics, has exploded on the scene, however there are only a few
studies that have used long-read sequencing to study mitochondrial biology. These studies have used this new technology to gain
insight into heteroplasmy and mitogenome rearrangement [18], mitochondrial DNA variant analysis [19], and new computational
pipelines to assemble mitochondrial genomes [20].
Here, we describe the de novo sequencing and assembly of the genome of the diploid race of harvester species L. manubriatum
using the long-read sequencing platform from Oxford Nanopore Technologies combined with Dovetail scaffolding. This draft
genome is the first of a facultatively parthenogenetic harvester species, and the second of Opiliones following the genome of the
widely distributed, human-associated species Phalangium opilio, [21]. The L. manubriatum nuclear genome has unusually large
numt insertions and documents the first incidence of multiple full-length transfers in animals. Facultative parthenogenesis is
rare in organisms, but provides an interesting case study on the benefits and consequences for the evolution of sex. The genome
assembled is moreover singular in that it is, to our knowledge, the first published genome of a wild-derived facultative parthenogen
(but see [22] and [23] for captive-bred examples). L. manubriatum presents a unique combination of polyploidy and facultative
asexuality, and understanding the complexity of their genomic make-up will allow a deeper insight into the maintenance of sex.
Methods
Sample Collection and Extraction
Adult female L. manubriatum specimens were collected from forest around Hirayu Campground (Figure 2) on July 11, 2014 and
August 3, 2019 and Sh¯
omyo Falls (Figure 2) visiting area on July 11, 2014 and August 3-4, 2019. Specimens collected in 2014 were
stored in 100% ethanol. Specimens collected in 2019 were immediately transported live to Tottori University, Tottori, Japan for
DNA extraction. In order to reduce the amount of contaminating DNA from gut flora and parasites (e.g. gregarines), the gut of each
specimen was dissected and removed. High molecular weight DNA was then extracted from the remaining tissue of each specimen
using the MasterPure Complete DNA and RNA Purification Kit following the DNA Purification section protocol (cat.no. MC89010).
DNA samples were then transported to the University of Maryland, Baltimore County, Maryland, USA for further processing and
sequencing.
Stellwagen and Burns |3
Figure 2. Map of Japan showing L. manubriatum distribution (tan shading) and collecting sites (Sh¯
omyo Falls and Hirayu Campground).
Table 1. Leiobunum manubriatum nuclear genome assembly statistics. BUSCO scores are complete (single plus duplicated).
Genome Assembly Value
Nanopore Sequencing Statistics
Number of Reads (Q10) 7,958,356
Number of Bases (bp) 46,760,569,792
Assembly Statistics
Assembly Size (bp) 336,872,803
%CG 37.48
Number of Contigs 3,399
Longest Contig (bp) 71,836,609
N50(bp) 27,489,741
L50(bp) 4
Protein-coding genes 24,032
BUSCO Scores
Assembly, Arthropoda 96.9%
Annotation, Arthropoda, transcripts 93.6%
Annotation, Arthropoda, proteins 93.6%
Nanopore Sequencing
The DNA from 29 specimens was used for sequencing. Extracted DNA was combined to reach 10 ug pooled samples and loaded onto
a Sage Science BluePippin cassette (cat.no. BLF7150 or BPLUS10) and run with a 10, 15, or 20 kbp high pass threshold overnight, or
prepped without size selection. The resultant samples were then cleaned using Agencourt AMPure XP beads (cat.no. A63881) and
eluted overnight to several days in water. Clean DNA was then used in Oxford Nanopore’s 1D Genomic DNA by Ligation protocol
(SQK-LSK109). A total of 11 runs were completed using SpotON Flow Cells (R9.4; cat.no. FLO-MIN106) and the resultant fast5
files were basecalled using Oxford Nanopore’s program Guppy 3.4.4+a296acb, and filtered to include only those with a Q-score of
10 or higher. Adapter sequences were then trimmed using Porechop v0.2.4 (Porechop, RRID:SCR_016967).
4|Journal of XYZ, 2020, Vol. 00, No. 0
De novo Nuclear Genome Assembly
Trimmed reads were assembled using Canu v1.9 (Canu, RRID:SCR_015880) [24] with default parameters. The raw draft assembly
was then further scaffolded by Dovetail HiRise. The resulting draft assembly was then polished using Nanopore’s Medaka v1.0.1
program. Purge Haplotigs v1.1.1 [25] was then used on the polished assembly to remove heterozygous haplotype contigs that were
assembled separately with a=50.
The final assembly is 336 Mbp from 3,399 contigs, with an N50 of 27,489,741 bp (Table 1; NCBI Project: PRJNA814647). Half
of the genome is represented by 4 contigs (L50). The genome recovered 96.9% of the 1,066 BUSCO [26] arthropod genes (Table 1;
Single: 94.7.8%, Duplicated: 2.2%, Fragmented: 0.8%, Missing: 2.3%). These BUSCO scores are excellent compared to recent spi-
der assemblies, for example the chromosome-scale Argiope bruennichi genome, which used Illumina, PacBio, and Hi-C sequencing,
recovered 91.1% complete arthropod BUSCOs [27]. The Dysdera silvatica genome, which used Illumina paired end and mate pair
sequencing in addition to both PacBio and Oxford Nanopore sequencing, recovered 69.1% complete BUSCOs [28]. While chromo-
some scale genome organization is not currently feasible with Oxford Nanopore alone, this sequencing strategy can outperform
completeness estimates compared to mixing various technologies that achieve chromosome scale resolution.
The polishing program Medaka (Oxford Nanopore) combined with Purge Haplotigs [25] to reduce heterozygous haplotype
contigs greatly improved BUSCO completeness metrics, while reducing duplications (Figure 1). The first round of Medaka polishing
increased complete BUSCOs by nearly 10%, however duplications also increased by 4%. Fragmented and missing BUSCOs were
also greatly reduced, and only slightly increased (<1%) after purging haplotigs. Purge Haplotigs greatly duplicated BUSCOs by over
10% without severely affecting the number of complete BUSCOs (<1% reduction). A final round of Medaka polishing improved all
metrics (<1%).
De novo Mitochondrial Genome Assembly and Numt Analysis
We extracted reads containing mitogenomic sequence from the raw Nanopore data using published CO1 sequence. As there was an
abundance of both small and extremely large reads containing mitonuclear sequence, we used reads that were between 16-18kb to
assemble the mitochondrial genome. We assumed the mitogenome was within this range, as this range had the largest number of
sequences and is the typical size for metazoan mitogenomes. Similar to that of the nuclear genome, we used Canu [24] to assemble
the mitochondrial genome, followed by polishing with Medaka. The final mitogenome is 16,999 bp and contains common genes
found within the mitogenomes of eukaryotes (Figure 3).
To isolate nuclear contigs that contain mitogenomic sequence, we used Geneious’s annotation feature to search the final draft
assembly’s 3,399 contigs for mitogenomic sequence with a 25% similarity or greater with the 13 coding genes or 2 rRNAs. We
found 1009 numts (989 coding sequences and 20 rRNAs) within 222 contigs, totalling 293,992 bp. One contig (Contig 171) contains
a full-length mitogenomic insertion, however while the contig is verifiable using raw long reads, it is clear additional sequence
was inserted during assembly that cannot be found in the long read data. Assembly data from before purging shows an additional
full numt insertion that could be fully confirmed using raw long reads (Figure 3(B). These examples demonstrate the difficulty in
balancing the removal of extraneous sequence while retaining important information.
We also searched the raw Nanopore data for reads >50 kbp that contained a 50 bp match to any portion of the mitogenome.
We found 118 long reads containing mitogenomic sequence, some of extreme length and 9 with complete mitogenomes (Figure 4).
However, these complete numt reads are not incorporated into the final assembly.
Interestingly, the similarity of the numt coding genes and rRNAs from raw reads that map to contigs is lower that that of the
contig itself. Raw reads that are ostensibly actual mitogenomic reads have typically 95% similarity or higher (less than 100% due
to sequencing error) when compared to the polished mitogenome, while raw numt reads have typically 80-90% similarity. It is
likely that genomic contigs are being corrected with fragmented mitochondrial reads, a potential problem for accurate genome
assembly.
Annotation
Several datasets were used to guide annotation of the L. manubriatum draft genome. First, Genemark-ES (GeneMark, RRID:SCR_011930)
and SNAP [29] were trained to identify protein coding genes. Second, as a transcriptome for L. manubriatum has not yet been gener-
ated, publicly available transcriptome RNA-seq reads from the related species Leiobunum verrucosum (accession num: SRR1145701)
were downloaded and assembled using Trinity v2.10.0 (Trinity, RRID:SCR_013048) [30, 31]. Third, protein databases from several
arthropods were downloaded from NCBI and used as references for homology prediction (SupTableX). After two rounds of training
using Genemark and SNAP, we used the L. verrucosum transcriptome assembly and custom protein database, to guide annotation of
the L. manubriatum genome using Maker v3.01.03 (MAKER, RRID:SCR_005309)[32, 33, 34]. The BUSCO scores for the final anno-
tation using the arthropod gene group against predicted transcripts was 92.4% and predicted proteins was 92.3%. Furthermore,
the mean AED score from Maker was 0.32, which suggests a well annotated genome.
Discussion
Genome Size
We verified the size of the L. manubriatum genome using Illumina HiSeq short-read sequencing data in GenomeScope [35]. Our
genome size estimate is somewhat smaller than the only other publicly available nuclear genome resource for Opiliones [21], which
estimates a haploid count of ∼500 Mbp. Spider genomes average ∼2.5 Gbp, but have a broad range from 0.74 - 5.7 Gbp [36]. Garb
et al. (2018) [37] have noted a need for the resolution of additional arachnid genomes in order to answer evolutionary questions
about gene duplication and its role in arachnid functional diversity. Indeed, the assembly of P. opilio[21] lacks whole genome
Stellwagen and Burns |5
CO1
ATP8
CO2
ATP6
ND3
CO3
ND2
ND5
ND4
ND4L
ND6
CYTB
ND1
16s
12S
Ala
Arg
Asn
Ser
Glu
Phe
Gly
Lys
Asp
Tyr
Cys
Trp
Met
Val
Gln
Ile
Leu
Ser
Pro
Thr
His
Leiobunum manubriatum
16,999 bp
10 20 30 40 50 60 71.9
kbp
19.4 kbp
at least 70% similarity
31.1 kbp 21.4 kbp
5 10 15 20 25 30 34.8
kbp
86/92
54-85/92
83-92/96
85-90/94
89/94
90/96
90/95
87/93
89/95
91/95
89/94
90/93
90/96
89/95
83-92/93
83/93
83/87
83/96
88/93
58-91/95
87/92
55-88/62
A
B
C
supporting reads
%
Figure 3. Mitchondrial genome sequence of Leiobunum manubriatum (A) The mitogenome consists of 13 genes, 2 rRNAs, and 20 tRNAs. (B) An example of a nuclear
contig that contains a complete mitochondrial genome transfer with mitogenome alignment chart above. Green indicates good alignment, yellow indicates poor
alignment, and red indicates alignment gaps between the numt and mitogenome. Dotted lines represent genomic sequence that is not mitochondrial in origin.
Percentages indicate similarity of mitogenome sequences to read average/contig. Arrowed lines represent supporting raw sequencing reads that align with the
nuclear contig. Arrows indicate that the read extends beyond the contig ends. (C) An example of an extreme-length read of continuous mitochondrial sequence.
Arrowed lines indicate the direction of the mitochondrial sequence.
duplications found in other arachnid lineages. More importantly, ongoing genome evolution research in arachnids will benefit
from improved assemblies that incorporate long reads [37] such as in this work.
6|Journal of XYZ, 2020, Vol. 00, No. 0
Figure 4. Raw Nanopore reads >50kb that contain a full-length mitogenome. Grey boxes indicate continuous mitogenomic sequence that includes, in order, all
major genes and both rRNAs.
Nuclearized Mitochondrial Genes
We found evidence of numerous transfers of mitochondrial DNA into the nuclear genome of L. manubriatum. This finding is not
uncommon for multi-cellular eukaryotes, which vary in numt abundance based on transfer frequency and the efficiency of nuclear
gene purge [38]. However, our finding of complete mitochondrial genomes with limited interspersion of nuclear sequence appears
to be entirely undocumented for any arthropods (but see [10] for a mammalian example). These large blocks invite ongoing
research as to the mechanism of mitochondrial gene transfer, as well as the potential and implications for functionality of these
genes, which we discuss here.
Numt creation: more common in parthenogens?
While numts are common in the eukaryotic genome, little research has focused on the mechanisms responsible for their initial
transfer, nor on the frequency of these transfers. Notably, chloroplastic DNA is rarely found in the nuclear genomes of plants,
suggesting that organelle type may be potentially significant in the evolution of genome nuclearization. The typically small
reported size of numts further suggests nuclearization is a rare event followed by generations of recombination that serve to further
fragment mitochondrial genes transferred to the nuclear genome [38]. However, we posit asexually-reproducing organisms, like
L. manubriatum, are potentially more likely to have genomes with many large numts. This is because facultative parthenogenesis,
as hypothesized to occur in L. manubriatum, relies on meiotic errors such as nondisjunction to develop. These errors may create
the germ line instability necessary to disrupt cytoplasmic separation and pull mitochondrial genes into the reforming nuclear
envelope. Alternatively, organelles may incorrectly segregate to polar bodies formed during oogenesis, and later be reintroduced
to the oocyte in asexual syngamy. Parthenogens are known to have larger genomes than closely related sexual species, but this
is due to multiple reasons. With fewer opportunities to clear so-called "junk" DNA through outcrossing, parthenogenetic taxa
accumulate transposable elements and extreme nonsynonymous mutations at higher rates than sexual species [39, 40]. Following
enablement of parthenogenetic reproduction, the genomes of parthenogens frequently double due to the same meiotic errors
enabling the reproductive mode itself. Thus, mitochondrial nuclearization is probably an additional contributor to the larger size
of parthenogenetic genomes.
Numt maintenance: could numts be beneficial to fitness?
The large numts that we identified in the L. manubriatum nuclear genome were in some cases indistinguishable from the actual
mitochondrial genome. This could be due to the recentness of the genomic transfers, with insufficient time in the lineage we
sampled to break down the sequence of the numts via mutations and recombinatory events. However, the potential that these
numts have been selectively maintained in the L. manubriatum genome, and even potentially transcription-active, opens a score of
possibilities for novel genomic evolution. What would be the evolutionary benefit of numts within the nuclear genome? Answers to
this question are dependent upon the direction and content of the transfer. Recent studies on human mitochondrial haplogroups
have identified large numts whose presence resembles biparental transmission of mitochondrial DNA [41]. This is significant be-
cause, aside from a few rare cases [42, 43], mitochondria are nearly entirely maternally transmitted in animals. With an occasional
influx of mitochondrial DNA entering the nucleus due to meiotic instability, the potential for the creation of a rescue reservoir of
functional mitochondrial genes is formed. Such a reservoir would be extremely beneficial for obligate or facultatively partheno-
genetic organisms, which are more likely to accumulate standing deleterious mutations than sexual species. This mechanism
could additionally enable paternal transmission of mitochondrial genes. If these genes are functional, paternally-derived numts
could furthermore provide genetic rescue specifically in facultative parthenogenetic species like L. manubriatum, which experience
at least infrequent sexual reproduction.
Practical Concerns for Genome Scaffolding
Mitochondrial sequence is commonly found in the nuclear genome [44], and we have shown that in some cases these sequences
may be indistinguishable from the mitochondrial source. This impacts the function of programs such as GenomeScope [35], which
excludes high copy number genes from genome size estimates via kmer coverage limits. However, genome scientists may rarely
examine numts, and their presence tends to be treated more as a nuisance than as a source of evolutionary information [45].
In the age of long-read sequencing, we propose that some review of the raw reads from mitochondrial sequences is justified,
particularly as the abundance of mitochondria ensures that reads from numts with internal nuclear sequence, or many mutations,
will be comparatively few and therefore possible to isolate and review by hand, as we have done here (Figure 3). The analysis of
Stellwagen and Burns |7
fully scaffolded long-read sequences must include identification of the numts incorporated within them and separation of true
mitochondrial sequence in order to identify reproductive mode or meiotic instability. The numts recovered may differ in the
recentness of their transfer, their size, and their maintenance of expression. This last factor can impacted by the location of a
numt within the nuclear genome; therefore, we primarily discuss concerns with numt detection here.
Numts that have been recently formed are more likely to be complete copies of the mitochondrion, as they have not yet been
impacted by recombination or mutation. This sequence may be more likely to be expressed, as well. This means that the numt will
share a high percentage of sequence similarity with the mitochondrial genome. Similarly, numts that are large and/or complete
may also be improperly corrected by the mitochondrial genome during scaffolding influence because of their similarities to the
source. Reducing genome size to match that of external predictions may also lead to the removal of true numt sequence, as they
are often tagged as repetitive or collapsed, as demonstrated here.
Nuclear assembly with large or very complete numts should first filter by percent identity of sequenced reads to the mitochon-
drial genome. If the assembly goals do not include analysis of numts, a cutoff value can be employed to remove all high copy reads
from mitochondrial assemblies to ensure that the mitochondrial genome does not influence the nuclear consensus sequence by
erroneously correcting any numts. If, however, there is interest in studying the numts, filtration to remove mitochondrial reads
with a length equal to or shorter than the mitochondrial genome should be performed to ensure that numts are not corrected.
Reads containing internal sequence that does not map to the mitochondrion could later be isolated and returned to the pool of
fragments for assembly. This procedure would therefore preserve numts for downstream study.
Data Availability
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/NCBI GenBank under the project number PRJNA814647.
Declarations
List of abbreviations
If abbreviations are used in the text they should be defined in the text at first use, and a list of abbreviations should be provided
in alphabetical order.
BUSCO = Benchmark Universal Single Copy Ortholog
Competing Interests
The author(s) declare that they have no competing interests.
Ethics Approval and Consent to Participate
Not applicable.
Consent to Publication
Not applicable.
Funding
This research was supported in part by a UMBC START grant to M.B. and in part by a National Science Foundation IOS grant to
both authors (M.B.: 2113665; S.S.: 2113666). The funding bodies had no role in the design of the study and collection, analysis,
and interpretation of data, nor in the decision to publish.
Author’s Contributions
M.B conceived of and the planned the experiments; M.B and S.S. collected specimens and extracted DNA; S.S. conducted sequencing
runs and assembled the genome; M.B. and S.S co-wrote the manuscript.
Acknowledgements
We thank Dr. Nobuo Tsurusaki for supporting our collection efforts in Japan, including making housing arrangements, driving
to the collecting sites, and allowing generous use of his laboratory. Preliminary analyses were conducted with the UMBC High
Performance Computing Facility.
8|Journal of XYZ, 2020, Vol. 00, No. 0
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