ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 11, pp. 1723-1740 © Pleiades Publishing, Ltd., 2025.
Russian Text © The Author(s), 2025, published in Biokhimiya, 2025, Vol. 90, No. 11, pp. 1843-1861.
1723
Gene Order in Mitochondrial DNA Affects Abundance
of their Transcripts (A Case of Marine Nematodes)
Olga V. Nikolaeva
1
, Anna S. Ovcharenko
1,2
, Polina V. Khorkhordina
1,2
,
Tatyana S. Miroliubova
1,3
, Nataliya S. Sadovskaya
1
, Victoria A. Scobeyeva
1,2
,
Nadya P. Sanamyan
4
, Elena G. Panina
5
, Kirill V. Mikhailov
1,6
, Leonid Yu. Rusin
6
,
Alexei V. Tchesunov
1,2
, and Vladimir V. Aleoshin
1,2,6,a
*
1
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,
119234 Moscow, Russia
2
Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia
3
Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, 119071 Moscow, Russia
4
Kamchatka Branch of the Pacific Institute of Geography,
Far Eastern Branch of the Russian Academy of Sciences, 683000 Petropavlovsk-Kamchatsky, Russia
5
Zoological Institute, Russian Academy of Sciences, 199034 Saint Petersburg, Russia
6
Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences,
127051 Moscow, Russia
a
e-mail: Aleshin@genebee.msu.su
Received July 10, 2025
Revised November 13, 2025
Accepted November 14, 2025
Abstract— Mitochondrial genomes of most animals contain the same set of genes, with all or many protein-cod-
ing genes (PCGs) arranged in the same order, forming conserved blocks termed syntenies. Some syntenies
have been preserved for hundreds of millions of years and are found in both vertebrates and invertebrates.
This evolutionary conservation indicates functional role for PCG arrangement; however, biochemical and/or
physiological mechanisms by which the gene order in mtDNA affects viability are unknown. Among animals,
there are taxa that have completely lost conserved syntenies in mtDNA. Canonical animal syntenies in mtDNA
have not been reported in nematodes, until some were recently discovered in the previously unstudied nema-
tode taxa, including the marine family Thoracostomopsidae (Nematoda, Enoplida). We sequenced the complete
mitochondrial genomes of three thoracostomopsid species, determined gene order, and their expression levels
from the RNA-seq data for all available family representatives. We found that six species of the Thoracosto-
mopsidae there are three distinct patterns of PCG arrangement, and the relative mRNA levels correlate with
the gene order rather than species phylogeny. We hypothesize that the influence of PCG translocations on
their expression levels underlies the long-term preservation of mitochondrial syntenies among animals.
DOI: 10.1134/S0006297925602114
Keywords: mtDNA, mitogenome, transcriptome, genome sequencing, RNA-seq, molecular evolution, phylogeny,
nematodes, Enoplia, Thoracostomopsidae, Enoplolaimus, Marimermis, Thoracostomopsis, Trileptium
* To whom correspondence should be addressed.
INTRODUCTION
Mitochondrial genome (mitogenome) of most
multicellular animals is a single circular DNA mole-
cule of about 15kbp [1], which contains 37 genes en-
coding seven subunits of respiratory chain complexI,
one subunit of complex III, three subunits of com-
plex IV, two subunits of ATP synthase (13 mitochon-
drial protein-coding genes, mtPCGs), two ribosomal
RNAs (rRNAs), and 22 transfer RNAs (tRNAs). Typical-
ly, there are almost no gaps between the genes, except
for one extended (1-1.5 kbp) non-coding region.
NIKOLAEVA et al.1724
BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025
Both strands of animal mitochondrial DNA (mtD-
NA) can encode genes, but the strands often differ in
the gene number and purine/pyrimidine ratio. Based
on nucleotide composition, the strands are designated
as heavy (H) and light (L). Regulatory elements con-
trolling mtDNA replication and transcription are usu-
ally located in the non-coding region. In humans and
other mammals, this region contains three strand-spe-
cific transcription promoters: two for the L-strand
and one for the H-strand [2-4]. In other vertebrates,
such as Xenopus laevis and chickens, the non-cod-
ing region contains only one bidirectional promoter
[5]. In lancelets, the extended non-coding region is
absent, and regulatory elements are apparently scat-
tered throughout the mtDNA [6]. In invertebrates, oth-
er transcription promoter organization patterns are
observed: the nematode Caenorhabditis elegans has
one transcription initiation site on the H-strand and
none on the L-strand [7], since all genes are locat-
ed on the H-strand [8]; in Drosophila melanogaster,
more than five transcription initiation sites are found
throughout the mtDNA [5]. In any case, there are few-
er promoters in mtDNA than genes, and transcripts
are polycistronic.
During maturation of polycistronic transcripts,
each tRNA is released due to hydrolysis at the 5′-
and 3′-ends by RNase P and RNase Z, respectively
[9], which also leads to the release of monocistronic
mRNAs and rRNAs [10, 11]. The origination of ma-
ture mRNAs from a few or even a single polycistronic
transcript would seem to imply an equimolar ratio of
all mRNAs. However, in reality, the mRNA pool for
complex I proteins is usually lower than for complex
IV, and mRNA levels for the proteins that form com-
plexes in an equimolar ratio may differ substantial-
ly [12]. It can be hypothesized that deviation from
the expected equimolar ratio is explained by random
factors, for example, due to different mRNA stability.
However, randomness cannot explain the similar im-
balance in different species. A similar ratio of mRNAs
between the same mitochondrial genes is character-
istic of both vertebrates and invertebrates [13, 14],
although small variations are observed even within
one species depending on sex, age, and physiological
state [12, 15, 16]. In some species, the ratio of tran-
scripts changes dramatically compared to the typical
animal pattern; these species also show more or less
significant differences in the order of genes in mtDNA
[15, 17].
Considering that processing of a long polycis-
tronic precursor into a monocistronic mRNAs occurs
before translation, the order of genes initially seems
insignificant. However, contrary to expectations, the
order of gene arrangement in the mitogenomes of
many animal taxa, such as vertebrates, is highly con-
served [18]. In other taxa, it is more variable, as in
mollusks [19], bryozoans [20], mites [21], tunicates
[22], thrips [17, 23], and others. However, both stable
and variable mitogenomes usually contain conserved
gene blocks (syntenies). Four conserved gene blocks
were identified in the mtDNAs of Bilateria, within
which composition and arrangement of genes remain
constant [24]. The oldest syntenies [24] are preserved
in vertebrates and invertebrates, i.e., since the Cam-
brian period (over 550 million years). Such conserva-
tion indicates functional importance of the gene or-
der. It could be assumed that a certain order of genes
is coordinated with regulation of their transcription
and, ultimately, with the ratio of mitochondrial pro-
teins. However, for example, seven mtDNA-encoded
subunits of complex I that are incorporated in the
complex in an equimolar ratio, are not grouped into
a single conserved block but are split among four
different blocks [24]. Three cox1–cox3 genes encod-
ing complex IV proteins, cox1–cox3, are also located
in two different blocks. Conservation of the order of
mtPCGs in mtDNA appears to be a genuine paradox
considering variability of the tRNA gene positions.
Transfer RNA genes are located between the mtPCGs
and rRNA genes, where they are necessary as mark-
ers for processing [10], but most of them are not as-
signed to conserved blocks and change their location
in the mitochondrial genome much more frequently
than the mtPCGs and rRNA genes [25]. Thus, selection
exerts significantly greater pressure on the location
of mtPCGs than on the location of tRNA genes.
The mystery of preservation of the gene order
in mtDNA is compounded by the existence of species
that have completely lost conserved gene blocks, ap-
parently without visible consequences – functional
constraints that have operated for hundreds of mil-
lions of years seem to have been relaxed in these spe-
cies. Paradoxically, individual taxa that “abandoned”
the Cambrian legacy in the form of conserved blocks,
have acquired specific gene orders that in turn remain
preserved fixed it over a long evolutionary times [26-
28]. The only known factor to date associated with the
changes in the genetic map of animal mtDNA is an
increased rate of molecular evolution [1, 24]. Until re-
cently, roundworms (nematodes) could be considered
as one of such taxa. More than 200 complete mitog-
enomes of soil and parasitic nematodes are known;
almost all of them are similar to each other in the
gene order, but without a single synteny in common
with the other animals [8, 29]. Almost all of them be-
long to the giant, largest in the phylum Nematoda, but
isolated terrestrial evolutionary branch – Rhabditia.
Taxonomic representation of the nematode
mtDNA has begun to change in recent years due to de-
velopments in the genomic sequencing of microscopic
organisms and publication of the first mitogenomes
of the uncultured free-living marine nematodes [30].
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BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025
In these mitogenomes, some of the traditional bilat-
erian gene blocks are found, and for two species of
the family Thoracostomopsidae, similar but slightly
different genetic maps are shown [30], making these
species a promising model for searching for biochem-
ical and physiological consequences of the changes in
gene order in mtDNA. The free-living Thoracostomop-
sidae inhabit silty and sandy bottom sediments in the
intertidal, sublittoral, and bathyal zones throughout
the World Ocean [31, 32]. Currently, the family in-
cludes 3 subfamilies (Thoracostomopsinae, Trilepti-
inae, and Enoplolaiminae), 16 genera, and 193 spe-
cies [33]. Based on the structure of the buccal cavity
and gut content, the thoracostomopsids are classified
as predators: they feed on other nematodes, cyano-
bacteria, diatoms, rotifers, oligochaetes, turbellarians,
and copepods [31]. According to the recent molecular
data, parasites of marine invertebrates of the genus
Marimermis are included in the family Thoracosto-
mopsidae [32, 34, 35].
MATERIALS AND METHODS
Materials. The study included a sample of six
nematode species from all three subfamilies of the
Thoracostomopsidae family. Specimens of Enoplolai-
mus vulgaris de Man, 1893 (~100 individuals) and
Thoracostomopsis (Th.) barbata Ditlevsen, 1918 (~10
individuals) were collected from the littoral and sub-
littoral zones of the Kandalaksha Bay of the White Sea
in August 2023. A specimen of Marimermis maritima
Rubtzov & Platonova, 1974 was extracted from the
coelomic cavity of the sea urchin Strongylocentrotus
polyacanthus caught near the Matua Island (Kuril
Islands, Pacific Ocean) in August 2016. Before the
DNA and RNA extraction, the M. maritima specimen
was stored in 95% ethanol. Data on Enoplolaimus
lenunculus Wieser, 1959 (SRR24201686), Trileptium
(T.) ribeirensis Vilas-Boas, Silva, Alves, Castro & Pin-
heiro-Junior, 2016 (SRR24201700) [30], and an uniden-
tified Thoracostomopsidae Gen. sp. KK-2019 isolate
(SRR8943408) [36] were obtained from the NCBI SRA
database.
Sequencing, assembly, and annotation of mi-
tochondrial genomes. Genomic DNA libraries were
prepared using an Accel-NGS WGA kit (Swift Biosci-
ences, Inc., USA). RNA extraction from E. vulgaris was
performed using a RNAqueous-Micro Total RNA Isola-
tion Kit (Thermo Fisher Scientific, USA), from Th. bar-
bata using an Arcturus PicoPure Isolation Kit (Thermo
Fisher Scientific), and from M. maritima using an in-
nuPREP DNA/RNA Mini Kit (Analytik Jena, Germany).
Complementary DNA (cDNA) library preparation was
performed using an oligo-dT primer and the SMART-
Seq v4 Ultra Low Input RNA Kit (Takara Bio, Japan)
with 12 (E. vulgaris and M. maritima) or 15 (Th. bar-
bata) PCR cycles. Sequencing of the three thoracosto-
mopsid species was performed using an Illumina
NovaSeq 6000 (E. vulgaris, M. maritima) or Illumina
NextSeq (Th. barbata) platforms. For E. vulgaris, 47.3
million (DNA) and 42.2 million (cDNA) paired-end
reads were obtained; for Th. barbata we obtained
34.4 million (DNA) and 25.4 million (cDNA) paired-
end reads; for M. maritima we obtained 29.6 million
(DNA) and 36.3 million (cDNA) paired-end reads. Qual-
ity of the obtained reads, as well as of the reads from
NCBI SRA, was assessed using FastQC [37]. Adapters
and incorrect terminal nucleotides were removed us-
ing Trimmomatic [38]. Assembly was performed using
SPAdes [39] with k-mer lengths of 55, 77, 99, and 123.
Target contig search was performed locally using the
BLAST package [40]. Mitochondrial genomes were as-
sembled using NOVOPlasty [41]. Mitogenome annota-
tion was performed using MITOS [42]; proteins were
predicted according to the invertebrate mitochondri-
al genetic code. Read mapping to mitogenomes was
performed using Bowtie2 [43]. Visualization of map-
ping and search for post-transcriptional modifications
were performed using Tablet [44].
Comparative analysis of mitochondrial genome
expression. Read coverage of mitogenomes was as-
sessed using BEDtools [45] based on alignments pro-
duced with Bowtie2. After obtaining coverage data for
each nucleotide, coverage plots were constructed in
MS Excel. To study differential expression, we used
total coverage of each gene by reads normalized by
length (TPM; transcripts per million) calculated in
RSEM [46] for trimmed reads. The atp8 and rrnL
genes were not used due to the very low and very
high coverage, respectively. Based on the TPM data,
principal component analysis was performed using
STATISTICA10 (TIBCO Software Inc., USA); Spearman
and Kendall rank correlation analysis was performed
in the R environment.
Phylogenetic analysis. To determine direction of
evolution of the gene order, phylogenetic relationships
of the six studied species were examined. For this pur-
pose, nuclear 18S and 28S rRNA genes were obtained
from the genomic assemblies, including E. lenunculus,
T. ribeirensis, and the KK-2019 isolate, since only for
these markers there is a representative sample of
Thoracostomopsidae in the NCBI GenBank database.
Alignments were performed using MAFFT [47] with
manual correction in BioEdit [48] and subsequent
concatenation. For the reconstruction with mitochon-
drial proteins, we concatenated the alignments of 12
proteins featuring the six Thoracostomopsidae repre-
sentatives and Enoplus communis, which was used
as an outgroup. Phylogenetic trees were construct-
ed using IQ-Tree 3.0.1 [49] and MrBayes 3.2.6 [50].
Nucleotide evolution model was selected according
NIKOLAEVA et al.1726
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Table 1. Nucleotide composition of thoracostomopsid mitochondrial DNA
Species GC, % A, % T, % G, % C, % AT-skew GC-skew
Enoplolaimus vulgaris 28 30 42 14 14 −0.17 0
Thoracostomopsis barbata 17 35 48 9 8 −0.15 0.06
Trileptium ribeirensis 20 34 46 10 10 −0.15 0
Enoplolaimus lenunculus 21 33 46 10 11 −0.16 −0.05
Isolate KK-2019* 20 38 42 11 9 −0.05 0.1
Marimermis maritima 35 30 35 17 18 −0.08 −0.03
Note. * Incomplete genome.
to the Bayesian information criterion (BIC) [51] in
IQ-Tree: for 18S rRNA, the GTR + F + R3 model; for
28S rRNA, the TVMe + I + G4 model; for mitochondrial
proteins, Mtrev + G4. For the Bayesian inference with
MrBayes, the following parameters were used: nst = 6,
ngammacat = 6, ngen = 3,000,000, burnin = 50% (for
nucleotide sequences) and aamodelpr = fixed(mtrev),
ngammacat = 4, ngen = 200,000, burnin = 50% (for
amino acid sequences). Trees were visualized using
MEGA 7 [52].
RESULTS
Structure of thoracostomopsid mitochondrial
genomes. The mitochondrial genome of E. vulgaris
is organized into a single circular DNA molecule of
13,843 bp, containing 37 genes (13 mtPCGs, 2 rRNA
genes, and 22 tRNA genes). All genes are located on
one strand. Four types of start codons are predict-
ed for mtPCGs: ATA (10 genes), ATG (1 gene), ATC
(1 gene), and TTA (1 gene). Eleven mtPCGs terminate
with the stop codon TAA, and 2 with TAG.
The mitochondrial genome of Th. barbata is or-
ganized into a single circular DNA molecule of 14,399
bp, containing the same set of 37 genes commonly
present in animals. Thirty-five genes are located on
one strand, while trnY and trnD are located on the
other strand. Based on the alignment of translated
mtPCGs, four types of start codons are predicted:
ATA (6 genes), ATG (3 genes), ATT (3 genes), and GTT
(1 gene). Twelve mtPCGs terminate with the stop co-
don TAA, and 1 gene with TAG.
The mitochondrial genome of M. maritima is
organized into a single circular DNA molecule of
14,284 bp, containing 37 genes. All genes are located
on one strand. Three types of start codons are pre-
dicted for mtPCGs: ATA (6 genes), ATG (4 genes), and
ATT (3 genes). Eight mtPCGs terminate with the stop
codon TAA, and 5 genes with TAG.
The mitochondrial genome of the KK-2019 isolate
was assembled into an unclosed contig of 15,589 bp,
containing 29 genes, of which 13 are mtPCGs. Eight
tRNA genes were not found. All identified genes are
located on one strand. Five types of start codons are
predicted for mtPCGs: ATA (8 genes), GTA (1 gene),
TTA (1 gene), ATT (1 gene), and TGG (1 gene). Start of
the nad5 gene was not found. Twelve mtPCGs termi-
nate with the stop codon TAA, and 1 with TAG.
Nucleotide composition (GC) and AT- and GC-skew
in the mtDNA of the six thoracostomopsid species are
presented in Table 1.
Nucleotide composition of the Th. barbata mtDNA,
with GC content of 17%, approaches the lowest value
for animals. The GC content of M. maritima is 35%.
Atwofold difference in the GC content among the spe-
cies of the same family is quite unusual. In all spe-
cies, thymidine predominates over adenosine in the
coding strand of mtDNA, while cytosine and guanine
are present in almost equal amounts.
Order of mitochondrial genes. Among the six
thoracostomopsid species, three variants of mtPCG
and rRNA gene order are observed (Fig. 1). The dif-
ferences between E. vulgaris, Th. barbata, and T. ri-
beirensis affect only tRNA genes. The same applies to
E. lenunculatus and the KK-2019 isolate, representing
another variant of mtPCG arrangement. The gene or-
der in M. maritima is unique.
In the mitochondrial genomes of thoracostomop-
sids, conserved gene blocks typical for Bilateria [24]
are partially preserved, but they are unusual for
nematodes. In four species, the block 2 (nad4l–nad4–
nad5) is completely preserved, and in E. lenuncula-
tus and the KK-2019 isolate, two of the three genes
from this block are adjacent. Although the block 1 is
fragmented, all six species retain the canonical pair
atp6–cox3 from this block. The block 3 is fragment-
ed in all species, but adjacency of two of its three
genes is preserved (rrnL–nad1 in E. lenunculatus
and in the KK-2019 isolate, nd1–rrnS in the others).
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Fig. 1. Evolution of the order of mitochondrial genes in nematodes of the family Thoracostomopsidae. Genes are marked
according to the conserved blocks typical for Bilateria [24]. tRNA genes that maintain a neighboring position relative to at
least one of the mtPCGs in different species are shown by a single-letter code.
The block 4 (nad6–cytb) is preserved in five species;
in M. maritima, the gene order is changed to cytb–
nad6.
In all six species, two tRNA genes, trnW and
trnG, are located between nad3 and cox2; another
four tRNA genes retain a neighbor at the 5′- or 3′-end
(Fig.1). Position of the remaining tRNA genes is more
variable and is specific for each species.
In all six species, the non-coding region is locat-
ed near the beginning of the nad1 gene, although its
position relative to the neighboring tRNA genes and
the atp8 gene differs. In E. lenunculus and the KK-
2019 isolate, the non-coding region, together with the
nad1 gene, has moved to a completely different place
on the genetic map.
Polarization of gene order evolution. To un-
derstand how changes in the order of mtDNA genes
affect their expression, it is desirable to know direc-
tion of these changes, i.e., which of the three variants
observed in modern Thoracostomopsidae is ancestral.
It would be imprudent to a priori consider the most
common variant as ancestral, since it could have aris-
en in one of the evolutionary lines better represented
in the database.
We reconstructed phylogeny of the Thoracosto-
mopsidae family based on the nuclear 18S and 28S
rRNA genes, for which the largest amount of repre-
sentatives are available. Two reconstruction methods,
Bayesian and maximum likelihood, led to the similar
topologies with the same arrangement of the six spe-
cies of interest (Fig. 2).
Species with the same genetic map (E. vulgaris,
Th. barbata, and T. ribeirensis) do not form a mono-
phyletic group relative to the species with a differ-
ent map (Fig. 2). One of them, T. ribeirensis, together
with Enoploides species, represents an early branch
of Thoracostomopsidae, which is consistent with the
results of previous studies on phylogeny of this family
[32, 35, 53, 54]. The simplest scenario of evolution
(minimum number of gene permutations) is obtained
if we assume that the gene order of T. ribeirensis,
E. vulgaris, and Th. barbata was inherited from the
common ancestor of the family. Let us call this order
plesiomorphic. The order of mtPCGs of M. maritima
and E. lenunculus differs from the plesiomorphic one
and was acquired by them independently (Fig.1). Let
us call these maps apomorphic. The order of mtPCGs
in the KK-2019 isolate and E. lenunculus coincide,
which is consistent with the relatedness of these spe-
cies (Fig. 2). Thus, the arrows in Fig.1 show not only
the method of formal transformation of the map, but
actual direction of evolution of the gene order.
Mapping of DNA and RNA reads to mitochon-
drial genomes. Coverage of the mitochondrial ge-
nomes by DNA sequencing (DNA-seq) reads fluctuates
within a certain range without clear attachment to
the gene boundaries (Fig. 3). However, in Th. barbata
and M. maritima, the decrease followed by a sharp
NIKOLAEVA et al.1728
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Fig. 2. Phylogenetic tree of the Thoracostomopsidae family based on concatenated 18S and 28S rRNA genes (a) and mi-
tochondrial proteins (b). Numbers at the nodes correspond to Bayesian posterior probability values (first number) and
bootstrap support (parametric bootstrap) for the maximum likelihood tree (second number). Values of 1/100 are replaced
by
●
. Supports below 0.50 or 50 are indicated by a dash; values −/− are omitted. Species for which the mitogenome was
sequenced are highlighted in bold;
★
species with different order of mtPCGs; gray background highlights representatives
of the subfamilies Thoracostomopsinae and Trileptiinae.
increase in the coverage is observed in the non-coding
region. For the non-coding region of Th. barbata, the
increase in coverage could be associated with short
tandem repeats in this area. In other species, decrease
in the coverage is observed in the non-coding region.
Proportion of the RNA sequencing (RNA-seq)
reads mapping to the mitochondrial genome is 0.13%
for Th. barbata, 22.9% for M. maritima, and 32% for
E. vulgaris. Coverage of the mitochondrial genomes
by the RNA-seq reads, unlike DNA-seq, is highly het-
erogeneous both within one gene and at the level of
different genes (Fig. 4). The rrnL gene has the high-
est coverage in all species, while coverage of the rrnS
gene is significantly lower and comparable with the
coverage of mtPCGs. It is expected that rRNA reads
should primarily originate from molecules making up
mature ribosomes, and, therefore, should be present
in the cell in an equimolar ratio. However, “deficien-
cy” of the 12S rRNA has been previously detected in
different species, including when cDNA sequencing
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Fig. 3. Coverage of Thoracostomopsidae mitochondrial genomes by the DNA-seq reads. tRNA genes are shown in single-
letter code.
NIKOLAEVA et al.1730
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Fig. 4. Coverage of mitochondrial genomes of Thoracostomopsidae by RNA-seq reads. Dashed line indicates a break point
in the KK-2019 isolate assembly.
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Table 2. Proportions of polyadenylated mitochondrial transcripts (as % of coverage at the modified nucleotide)
in the six species of Thoracostomopsidae
Gene E. vulgaris Th. barbata T. ribeirensis E. lenunculus KK-2019 M. maritima
atp6 15.8 5.9 1.7 2.7 – –
cox1 26.9 33.0 0.5 7.4 – 0.2
cox2 2.7 45.8 – – 1.8 1.3
cox3 10.7 – 33.5 44.7 – 0.2
cytb – – 39.0 19.5 – 0.2
nd1 – – 49.2 6.7 – 0.1
nd2 – – 40.1 – – 1.4
nd3, site 1 – – – 4.0 – –
nd3, site 2 – – 19.4 4.0 – –
nd4 28.2 – 23.9 2.4 – 1.2
nd4l – – 1.6 0.1 – 0.2
nd5 3.0 – 41.7 6.4 6.8 1.7
nd6 1.2 – 5.4 – 0.5 –
rrnS ––––4.8–
rrnL – 27.3 ––––
trnD –––5.7––
trnL2 –––––0.3
trnR ––––2.5–
trnS1 ––1.0–––
trnV –––––1.1
trnY ––0.3–––
was performed without enrichment of poly-A tran-
scripts with long-fragment sequencing and using ran-
dom primers [55, 56]; so, it is unlikely that in the case
of thoracostomopsids it is explained by the library
preparation artifacts. The lowest coverage is observed
in the regions with tRNA genes, which is consistent
with the tRNA punctuation model [10]. Among the
mtPCGs, the highest level of coverage is observed for
cox1, cox2, and cox3, and the lowest for atp8. An ex-
ception is the KK-2019 isolate with a relatively low
level of cox1 mRNA and high level of nad4. Coverage
noticeably decreases towards the beginning of each
gene. This is explained by more active degradation
of mRNAs from the 5′-end, as well as enrichment of
libraries with 3′-ends of sequences due to the cDNA
preparation method that employs an oligo-dT primer.
The RNA of Th. barbata and M. maritima, extract-
ed from the specimens stored in ethanol for several
years, were more degraded.
Mapping of cDNA reads revealed from 4 (Th. bar-
bata) to 13 (T. ribeirensis) polyadenylation sites, to-
taling 51 sites (Table 2). mRNA of every protein is
polyadenylated, except for atp8 (modifications of
which may not have been detected due to low cover-
age) in at least two thoracostomopsid species. Thus,
the dicistronic mature mRNAs, characteristic of an-
imal mitochondrial transcriptomes, are apparently
not present in thoracostomopsids. In E. lenuncu-
lus, in addition to polyadenylation of the 3′-ends of
mRNAs, an additional polyadenylation site was found
NIKOLAEVA et al.1732
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Table 3. TPM values for mtPCGs of six Thoracostomopsidae species (rounded to whole numbers)
Gene Th. barbata E. lenunculus T. ribeirensis E. vulgaris KK-2019 M. maritima
cox1 370,422 209,913 120,679 181,041 52,565 68,263
cox2 120,095 50,387 281,626 153,501 48,014 111,296
cox3 251,191 14,364 233,143 178,235 78,702 72,122
cytb 41,575 39,715 41,248 44,589 75,505 52,037
atp6 20,383 132,559 113,650 165,097 186,906 40,601
nad1 111,753 20,458 30,490 12,505 105,815 35,397
nad2 106 2,478 35,902 6,270 28,354 70,957
nad3 3,147 48,833 17,181 100,595 81,445 306,290
nad4 51,011 82,396 43,730 66,985 104,962 53,063
nad4l 4,120 113,462 16,825 15,171 47,107 128,139
nad5 24,456 35,682 20,528 13,527 68,424 14,362
nad6 1,741 249,753 44,998 62,484 122,201 47,473
Sum 1,000,000 1,000,000 1,000,000 1,000,000 1,000,000 1,000,000
in thecoding frame of the nad3 gene. In the individual
libraries, polyadenylated rRNAs and tRNAs are pres-
ent. A total of six polyadenylated tRNAs were found:
trnD, trnL2, trnR, trnS1, trnV, and trnY. In T. ribei-
rensis, one modified dicistronic transcript rrnL–trnS1
was found: some cDNA reads capture poly-A at the
3′-end of trnS1, and reverse reads map to the 3′-end
of the neighboring rrnL gene. The reads produced by
the Illumina platform are not long enough to properly
detect unprocessed polycistronic precursors of mature
RNAs (pre-mRNAs) [55], nevertheless, in another spe-
cies, the horsehair worm Parachordodes pustulosus,
similar methods revealed three dicistronic products in
the presence of 23 polyadenylation sites [57]. Appar-
ently, maturation of pre-mRNAs differs among distant
species.
Polyadenylation sites in the species with the same
order of mtPCGs do not coincide. This difference is
unlikely to stem from the lack of coverage: 11 polya-
denylation sites in M. maritima are confirmed by a
total of 8 million reads mapped to the mtDNA, while
13 sites in T. ribeirensis were found using ~1 million
mapped reads. For Th. barbata, however, underesti-
mation of polyadenylation sites is likely due to the
low read depth of the cDNA library (0.03 million
reads mapped to mtDNA).
Proportion of the modified site to the total cov-
erage at of the corresponding nucleotide in the cDNA
library is low for genes in M. maritima. In other
species, this proportion varies from less than 1%
to more than 40% within a single library (Table 2).
There is no direct correlation between the number
of transcripts of a particular gene and the degree
of polyadenylation of their ends. We consider these
two circumstances as important indirect evidence of
suitability of the obtained libraries for transcriptome
comparison. In the cDNA libraries constructed using
oligo-dT primers, enrichment of polyadenylated tran-
scripts can be expected. In the obtained libraries, the
ratio of different cDNAs does not correlate with the
degree of their polyadenylation, therefore, this ratio
reflects contribution of factors related to the construc-
tion methodology and the actual ratio of transcripts
in vivo.
No other modifications, such as non-templated
addition of CCA to the acceptor stem of tRNA, char-
acteristic of animal mitochondria, were detected in
thoracostomopsids.
Differential expression of mitochondrial
genes. An accepted measure of differential expres-
sion assessment is the TPM value. We obtained TPM
values for mtPCGs (Table 3) and performed principal
component analysis (Fig. 5).
Species with plesiomorphic genetic map – E. vul-
garis, Th. barbata, and T. ribeirensis – contribute the
most to the first principal component and form a dis-
tinct cluster in the projection onto the plane of the
first two principal components. The first principal
GENE ORDER IN MITOCHONDRIAL DNA 1733
BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025
Fig. 5. Principal component analysis of normalized cDNA
quantity (TPM) of 12 mtPCGs of six nematode species of
Thoracostomopsidae (three variants of the genetic map).
Abbreviations: E.v., Enoplolaimus vulgaris; T.r., Trileptium
ribeirensis; T.b., Thoracostomopsis barbata; E.l., Enoplolai-
mus lenunculus; KK-2019, Thoracostomopsidae gen. sp., iso-
late KK-2019; M.m., Marimermis maritima.
component explains 39% of total variability in the
expression of mitochondrial genes for the species in-
cluded in the analysis, i.e., we can speak of a simi-
lar pattern of mtPCG mRNAs in the species with the
plesiomorphic genetic map. Importantly, the species
with the plesiomorphic genetic map are not the clos-
est relatives (Fig.2). The species E. lenunculus and the
KK-2019 isolate, which have another variant of the
genetic map, also form their own cluster on the plane
of the first two principal components. The species
M. maritima, with a unique gene order, has a pattern
of expression that differs from all others, including
its closest relative Th. barbata. Thus, we observe sim-
ilarity in the patterns of expression of mitochondrial
genes in the species with similar genetic maps that
transcends their phylogenetic relationships.
As an alternative method of transcriptome com-
parison, we applied rank correlation analysis, where
we ranked mtPCGs according to TPM in each of the
transcriptomes, and calculated pairwise (by species)
Spearman (r
s
) and Kendall (τ) rank correlation coef-
ficients (Table 4).
In both types of analyses, Spearman and Kendall,
correlations in the ranks are statistically significant
at p < 0.05 for one pair of species out of three with
the plesiomorphic order of mtPCGs. When calculating
significance, we considered it unnecessary to apply
a correction for multiple comparisons (Bonferroni or
similar) for 15 pairwise comparisons. With a sample
size that small, a significant proportion of the results
could be false negatives, so we focused not on sig-
nificance of the correlation, but on the value of its
coefficient, expressing the strength of the relationship
between the quantities. Between any species with the
plesiomorphic gene order, the correlation is higher
or at least not lower than in the species with the
apomorphic gene order. Given reproducibility of the
results obtained by different methods, we cautiously
propose the existence of correlation in the expression
of mtPCGs in the species with the same gene order,
regardless of their degree of relatedness.
DISCUSSION
Distortions in the natural ratio of various RNAs in
a generated cDNA library can be attributed to several
factors – primarily, selective RNA degradation, selec-
tive cDNA synthesis, and context-dependent sequenc-
ing effects. The underlying causes are predominantly
associated with the inherent molecular properties
of RNAs, including secondary structure features, the
presence of specific nuclease target motifs or motifs
that are difficult for reverse transcriptase or sequenc-
ing polymerase to pass through, and the presence and
length of the polyadenylated region. The findings
Table 4. Spearman rank correlation coefficients (r
s
) and Kendall rank correlation coefficients (τ) for mtPCGs
ranked by their TPM values
Species E. vulgaris Th. barbata T. ribeirensis E. lenunculus KK-2019 M. maritima
E. vulgaris 1 0.545 0.706* 0.462 0.224 0.294
Th. barbata 0.424 1 0.573 −0.028 −0.021 −0.042
T. ribeirensis 0.485* 0.394 1 0.217 0.133 −0.007
E. lenunculus 0.394 −0.061 0.121 1 0.301 −0.014
KK-2019 0.152 0 0.121 0.212 1 −0.497
M. maritima 0.182 −0.03 0.091 0 −0.364 1
Note. Above the diagonal with bold values are r
s
values, below the diagonal are τ values. Gray cells refer to the species with
plesiomorphic arrangement of mtPCGs. * Statistically significant correlation at p < 0.05.
NIKOLAEVA et al.1734
BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025
offactor and rank analyses, as presented in the paper,
lend support to the hypothesis that the relative level
of cDNA reads is contingent on the relative position
of genes in mtDNA. It is challenging to conceive of a
scenario in which selective RNA degradation, selective
cDNA synthesis, or context-dependent sequencing ef-
fects could result in the observed dependence. This
evidence was obtained in a system of fairly closely
related species, which renders this and similar sys-
tems promising for detailed study of the mitogenome
variability and its manifestation through differential
expression of RNAs in the mitochondrion. A key is-
sue of fundamental and applied significance is to
elucidate the mechanisms of selective regulation of
genes that are transcribed in a single round towards
a common polycistronic pre-mRNA. The correlation
between gene order (i.e., configuration of genes on a
genetic map) and their expression levels suggests an
indirect relationship, whereby the relocation of one
gene affects expression of another, indicating a mul-
tifaceted regulatory mechanism, potentially operating
at the level of transcription or post-transcriptional
modifications (i.e. maturation, or processing) of the
polycistron.
Transcriptional regulation. The lifecycle of mi-
tochondrial RNAs unfolds within the giant, microscop-
ically discernible complexes, known as mitochondri-
al RNA granules [4, 58, 59], where the coordinated
processes of transcription, RNA maturation, and mi-
tochondrial ribosome assembly are orchestrated. Ac-
cording to some estimates, up to 300 nucleus-encoded
mitochondrial proteins are involved in ensuring the
cycle [2], and transcription of the mitogenome is reg-
ulated in a complex manner by several transcription
factors with chromatin-modulating activity, which
have multiple binding sites along the length of mtDNA
and change binding activity during ontogeny [5, 60-
62]. The binding sites of certain factors and helicases
are associated with G-quadruplexes, non-canonical 3D
structures that primarily form on the heavy strand of
mtDNA and exhibit a non-random, conservative distri-
bution throughout the genome [63]. The involvement
of these structures in the regulation of transcription
and replication of mtDNA, chromatin remodeling, for-
mation of mutation and deletion breakpoints [64-66]
has been demonstrated, indicating their role in gene
regulation at a higher-order level of mtDNA organi-
zation [67]. The activity of mitochondrial RNA poly-
merase is modulated by components of this system,
impacting the start, speed, and termination of tran-
scription. It stands to reason that alterations in the
order of genes bearing conserved regulatory regions
could modify the regulatory context of the mitochon-
drial chromosome, potentially leading to impaired
or even arrested transcription of a part of the poly-
cistron, followed by downregulation of downstream
genes. Meanwhile, elevated expression levels are an-
ticipated at start of the polycistron, with its first gene
expected to exhibit reduced processing dependency.
Post-transcriptional regulation. The maturation
of polycistronic pre-mRNA relies on the specific endo-
nuclease-mediated excision of tRNA genes to generate
processed fragments with free 3′-ends, which normally
undergo polyadenylation by the mitochondrial poly(A)
polymerase MTPAP [68]. This highlights the role of
polyadenylation and tRNA positioning on the genetic
map as related and potentially important factors in
the regulation of mitochondrial genes. Polyadenylation
is known to contribute to transcript stabilization at
several levels: directly by protecting the coding part
of the 3′-end from degradation by exonucleases [68]
and indirectly via involvement of poly(A)-tails in com-
plex processes of translation regulation (breakdown
of aberrant mRNA–mitoribosome complexes [69];
mRNA handoff to the ribosome for translation ini-
tiation [70]), routing of processed mRNAs [71], and
overall folding and stabilization of the mitochondri-
al transcriptome by RNA-binding chaperone proteins
[72]. A variation in polyadenylation sites during
gene rearrangement might exert pleiotropic effects,
including gene degradation at 3′-end of the common
transcript in a fraction of molecules. Notably, the
function of poly(A)-tails could be species-, tissue-,
and transcript-specific, depending on their presence
or length in different biological contexts, suggesting
their role in various, primarily undefined, regulatory
mechanisms. For instance, the knockdown of MTPAP
in human cell cultures compromises the stability of
mRNAs in the cox1, cox2, cox3, and atp6 genes, while
it does not affect nad3 and even increases the level
of nad1 [73, 74]. Polyadenylation occurs in normal
mitochondria for some tRNAs and rRNAs [73]. Infruit
flies, the 12S rRNA carries a short poly(A)-tail [68],
suggesting its involvement in the regulation of this
transcript, although its primary function remains
poorly understood and may differ from that in pro-
tein-coding genes.
It is important to acknowledge that the expres-
sion estimates, as presented in this study, primarily
reflect the processed genes with functional polyade-
nylation sites due to poly(A) enrichment-based library
construction. Upstream genes (i.e., those without free
3′-ends) may appear underrepresented, and the more
so the greater is the gene’s distance from the poly-
cistronic 3′-end, whereas the content of such genes
depends on their order along the polycistron and its
length.
The orientation of the punctuation tRNA genes
plays its own pivotal role in the maturation of the
common transcript and, consequently, in achieving
functional polyadenylation: the transcription strand
must be sense for both tRNA and its flanking genes.
GENE ORDER IN MITOCHONDRIAL DNA 1735
BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025
Should the genetic map rearrangement cause altered
tRNAs orientation, their primary structures will not
fold into hairpins required for recognition and exci-
sion by endonucleases [73, 74], the upstream genes
will not form free 3′-ends and will not become polyad-
enylated, which will ultimately disrupt their regulation
(as well as affect representation in a poly(A)-library).
It is also of note that the composition of tran-
scripts (revealed in RNA-seq data) does not accurate-
ly reflect the composition of their encoded proteins,
since translation efficiency also depends on the gene
order. A free and polyadenylated 3′-end may be re-
quired to complete a functional stop codon from the
incomplete T or TA codons in the mitogenome [74]
(all stop codons in thoracostomopsids are complete);
the ribosome binding efficiency at 5′-untranslated re-
gions of neighboring genes in the polycistronic tran-
script may require proper sequence overlap with an
upstream gene [75, 76], which can change upon gene
relocation.
Given the intricacy of the mechanisms that link
the mitochondrial gene order and their expression,
we believe that chromosomal rearrangements are
associated with high lethality risks and require si-
multaneous compensatory changes in the molecular
systems involved. This renders them rare events in
which discrete changes are fixed in mitochondrial
evolution for over millions of years, i.e., at the level
of higher taxa [26-28]. Early mitogenome rearrange-
ments are of particular interest for research into the
factors of stability of the mitochondrial system. The
first evidence in animal species at a relatively small
evolutionary distance is presented in this work.
Taxonomic and morphological conclusions.
The obtained cladogram (Fig. 2) allows us to draw
some conclusions about the morphological evolution
and taxonomy of Thoracostomopsidae.
In the cladogram, two species assigned to the
same genus Enoplolaimus, E. vulgaris and E. lenun-
culus, turned out to be on different branches of the
thoracostomopsid tree. The type species of the genus
E. vulgaris groups with its closest relative Enoplolai-
mus attenuatus, several unidentified Enoplolaimus
species, and Mesacanthion, as well as with Trileptium
sp. 3 (the latter appears to be a misidentification).
E. lenunculus (isolates from two studies [30, 35] and
unpublished MG599045) and the unidentified tho-
racostomopsid KK-2019 [36] are combined into a large
group with large number of Epacanthion species; this
group also includes several species of Mesacanthion,
Mesacanthoides, and Enoploides. There may be two
possible explanations for the grouping of species of
different genera together: (1) morphological features
of the genus Enoplolaimus are not apomorphies, and
the genus Enoplolaimus itself is not monophyletic;
(2)taxonomic identifications of thoracostomopsid spe-
cies in the GenBank database are partially erroneous.
Let us consider both possible options.
(1) According to the latest review of the Tho-
racostomopsidae family [33], Enoplolaimus is charac-
terized by the position of the anterior ten setae at
the level of the posterior edge of the head capsule.
In Mesacanthion and in Epacanthion, the anterior
setae are located at the level of the anterior edge or
the middle of the head capsule. Enoploides differs
from Enoplolaimus and Mesacanthion in the shape of
mandibles. In Epacanthion, the mandibles are of in-
termediate structure. Relationship between the slight
displacement of sensilla or changes in the shape of
mandibles, although clear from the point of view of
morphological taxonomy, has not been tested for the
ability to reflect phylogeny.
(2) The probability of erroneous taxonomic iden-
tification always exists, and it could increase signifi-
cantly if the species is identified from the specimens
from an area remote from the type locality. In our
case, this is Enoplolaimus lenunculus, described from
Puget Sound (USA) and then collected for molecular
genetic research in China, on the opposite shore of
the Pacific Ocean. For the specimens not identified
to the species level, and there are many such tho-
racostomopsids in the GenBank database, accuracy
of the genus determination could also be questioned.
Based on the published illustrations (Figs. 6-8
in the article by Meng et al. [35]), arrangement of
setae in the Chinese isolate of E. lenunculus corre-
sponds to the diagnosis of the genus Enoplolaimus.
Aconflict arises with its distance on the phylogenetic
tree from the type species of the genus, E. vulgaris.
Either E. lenunculus should be transferred from the
genus Enoplolaimus to preserve monophyly of the
latter, but then morphological diagnosis of the ge-
nus would have to be revised, or the scope of the
genus Enoplolaimus would have to be expanded to
almost all thoracostomopsids, but then a revision of
the significance of morphological features would also
be required. Regardless of the problems of taxonomy,
E. lenunculus and the KK-2019 isolate have synapo-
morphies in the arrangement of genes in mtDNA and
belong to one clade (Fig.2), which did not prevent the
KK-2019 isolate from acquiring unique differences in
the pattern of mtPCG expression (low level of cox1
and high level of nad4). In the absence of molecular
data on the type species of the genus Trileptium, we
are inclined to consider the genus Neotrileptium [32]
as a synonym of Trileptium. According to the latest
review [33], the genus Metenoploides is recognized
as invalid, but this opinion probably needs to be re-
vised, since the AS1357 isolate, assigned to the genus
Metenoploides [53], is located on the phylogenetic tree
separately from most species of the genus Enoploides
(Fig. 2a).
NIKOLAEVA et al.1736
BIOCHEMISTRY (Moscow) Vol. 90 No. 11 2025
According to the phylogenetic reconstruction, di-
vision of Thoracostomopsidae into three subfamilies
has not been observed. Two of them, Thoracostomop-
sinae and Trileptiinae, turn out to be branches within
Enoplolaiminae. The first two subfamilies are char-
acterized by clear morphological features (fusion of
mandibles into a spear and sharp reduction of the
stoma, respectively), which could well have arisen
as an evolutionary specialization in two phylogenetic
branches of Enoplolaiminae. No apomorphies at the
morphological level have been proposed for Enoplo-
laiminae, which is consistent with their paraphyly
revealed at the genetic level.
The phylogenetic reconstruction based on 12 mi-
tochondrial proteins (Fig. 2b), as well as on the rRNA
genes [32, 34, 35], confirms belonging of the species
M. maritima to the family Thoracostomopsidae, previ-
ously referred to the order Marimermithida.
CONCLUSION
Nematodes of the family Thoracostomopsidae
preserve some of the syntenies in the mitochondrial
genetic maps commonly seen in other animal phyla,
in contrast to the nematodes of the subclass Rhabdi-
tia, which have lost these syntenies entirely. Three
variants of the genetic map, which involve rearrange-
ments of protein-coding and rRNA genes, were discov-
ered in six species of Thoracostomopsidae. One of the
variants is recognized as plesiomorphic, as the spe-
cies possessing the variant are paraphyletic relative to
the species with the alternative gene orders. The two
alternative variants arose in evolution independent-
ly of each other. Factor analysis and rank analysis
based on the normalized gene coverage by cDNA
reads (TPM) revealed positive correlations between
the expression levels of mitochondrial protein-coding
genes in the species with the same gene arrangement,
regardless of their relatedness. This indicates the ex-
istence of a yet unknown mechanism by which the
gene order in mtDNA influences the transcription or
mRNA maturation, and ultimately the Darwinian fit-
ness. Phylogenetic analysis confirms the inclusion of
marimermithid M. maritima in Thoracostomopsidae
but refutes the traditional delineation of subfamilies
within Thoracostomopsidae.
Abbreviations
mtDNA mitochondrial DNA
mtPCG mitochondrial protein-coding genes
pre-mRNA polycistronic precursor of mature RNAs
TPM transcripts per million
rRNA ribosomal RNA
tRNA transfer RNA
RNA-seq RNA sequencing
DNA-seq DNA sequencing
cDNA complementary DNA
Acknowledgments
The authors are grateful to Yi-Chien Lee for providing
mtDNA assemblies of E. lenunculus and T. ribeirensis;
to V. Yu. Shtratnikova, M. A. Kulbachnaya, D. A. Knore,
and an anonymous reviewer for constructive criticism
of the manuscript; to S. N. Lysenkov for assistance in
statistical analysis. Computer calculations were per-
formed using equipment purchased with the funds
provided by the state assignment of Lomonosov Mos-
cow State University; animal collection was carried
out under state assignment no.122031100275-4.
Contributions
O. V. Nikolaeva, A. S. Ovcharenko, N. S. Sadovskaya,
K. V. Mikhailov, and V. V. Aleoshin – conducted com-
putational experiments; T. S. Miroliubova – conduct-
ed laboratory work; N. S. Sadovskaya, V. A. Skobeeva,
and A. S. Ovcharenko – conducted statistical analy-
sis; N. P. Sanamyan, E. G. Panina, L. Yu. Rusin, and
A. V. Tchesunov – conducted field work; P. V. Khork-
hordina, L. Yu. Rusin, A. V. Tchesunov, and V. V. Aleo-
shin – conducted phylogenetic analysis; all authors–
prepared the text and illustrations.
Funding
Field work and material collection was carried out
with support of the Zoological Institute of Russian
Academy of Sciences (“Taxonomy, biodiversity and
ecology of invertebrates of the Russian and adja-
cent waters of the World Ocean, continental ponds
and wetlands”, no. 122031100275-4). Analysis of the
structure of mitochondrial genomes and transcrip-
tomes was carried out with financial support of the
Russian Science Foundation (grant no. 19-74-20147).
Phylogenetic analysis was carried out with financial
support of the Russian Science Foundation (grant
no.25-74-20009).
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects that are regulated by ethics
committee.
Conflict of interest
The authors of this work declare that they have
noconflicts of interest.
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