The mitochondrial genome of
Calypogeia is 159,061–163,057 bp in length (
Table 1,
Figure 1) and is slightly shorter than the closest related species with a known mitogenome structure:
P. purpurea (168,526 bp) [
36]. The length of the mitochondrial genome of
Calypogeia is most similar to the mitogenome of
A. pinguis (164,989 bp) [
37]. The two other known mitochondrial genomes of the liverwort species,
T. lacunosa [
12] and
M. polymorpha [
38], differ more and are composed of 151,983 and 186,609 bp, respectively. The GC content in the studied genome (45%) is similar to other liverworts (42–45%) [
12,
36].
Seventy genes have been identified in the
Calypogeia mitogenome: 42 protein-coding genes 25 tRNAs and three rRNAs (
Table 2). Chondriomes of liverworts, similar to moss and hornwort mitogenomes, are reported to be rather static in gene content and order [
12,
13,
50] and even with respect to pseudogene contents and retroposed pseudogene pieces [
12]. The gene order in
Calypogeia mitogenome is identical to the four aforementioned liverwort mitogenomes, although the gene content is slightly different between them. Mitogenomes of
M. polymorpha,
P. purpurea,
A. pinguis and
Calypogeia are very similar in gene composition. The differences occur mainly in the content of transfer RNA genes.
M. polymorpha has only one copy of the
trnRucu, but contains two more tRNA genes:
trnRucg and
trnTggu. One copy of the
trnRucu has probably given rise to
trnRucg [
12]. The
trnT gene is a part of the
trnA-
trnT-
nad7 gene cluster, whose different forms were identified by Wahrmund et al. [
51] in liverwort mitogenome evolution. In leafy (jungermanniid) liverworts and in simple thalloid (metzgeriid),
trnT is lacking between
trnA and
nad7, whereas in
Blasia pusilla, representing a sister lineage to all other complex thalloid (marchantiid) liverworts, this gene occurs in conserved
Chara-like version. The
trnT gene is also present in
M. polymorpha and other complex thalloid (marchantiid) liverworts, but its sequence is inverted compared to
Blasia. Furthermore, in the
A. pinguis mitochondrial genome, there is only one copy of the
trnYgua in contrast to other liverwort mitogenomes [
36]. Another difference concerns the
rtl gene. In all aforementioned mitogenomes, this gene is functional with nucleotide sequence similarity > 84%, whereas in
P. purpurea, it may be a pseudogene because of the high level of sequence divergence and several indels in the open reading frame [
36]. However, a big part of the reading frame in
rtl is intact, so this gene in
P. purpurea may be still functional. The other dissimilarities occur between mitogenomes of the above four species and
T. lacunosa. In the mitochondrial genome of the latter, either some genes of the cytochrome c biogenesis (
ccmC,
ccmFN) are missing or some of them are pseudogenized (
ccmB,
ccmFC). Another conspicuous dissimilarity concerns the
nad7 gene, which is only functional in
T. lacunosa [
12]. In most hornworts and liverworts, this gene is missing or occurs as a pseudogene with a degenerated structure [
52,
53,
54], which is reflected in
Calypogeia mitogenome and in the other sequenced mitochondrial genomes of liverworts. The only liverwort species with functional
nad7 are
Treubia and
Haplomitrium [
52] belonging to Haplomitriopsida, a sister clade to the rest of the liverworts: Marchantiopsida and Jungermanniopsida [
55] with the inactive
nad7 gene.
Ninety-four spacers and 24 introns (
Table 3) have been found across the entire mitochondrial genome. The different length (1000–1300 bp) of the
nad5-
nad4 spacer, containing the inverted sequence of the second
cob intron in
M. polymorpha, was recognized in different liverwort groups. Almost the entire
cob intron sequence is inserted in the
nad5-
nad4 spacer in marchantiid, while an internal region of this intron sequence copy is deleted among metzgeriid and jungermanniid taxa [
56]. The above findings are also supported by the current study, because the
nad5-
nad4 spacer in
Calypogeia has a structure typical of jungermanniid liverworts.
In the
Calypogeia mitogenome, 22 introns (
Table 3) are located in protein-coding genes, one in the
rrn26 gene and one in the
trnS gene. Seven genes (
nad2,
nad3,
nad4,
nad5,
rpl2,
rps14 and
atp9) contain one intron. The genes
cox2,
cox3 and
nad4L have two introns, whereas the coding sequence of the
cob gene is divided into three introns. The largest number of introns is localized in the
cox1 gene. However, surprisingly, only six introns occur in
Calypogeia, whereas nine introns exist in the
cox1 gene of thalloid liverworts sequenced to date [
12,
35,
36,
37]. The
cox1 gene of
Calypogeia lacks the cox1i395g1, cox1i624g1 and cox1i729g1 introns. The
atp1 gene has also lost two introns (atp1i989g2 and atp1i1050g2) and become in
Calypogeia intronless (
Figure 2). The CDS (protein-coding sequence) structure of both genes has not been affected. The intron set among species within each of the three major lineages of bryophytes is reported to vary slightly [
12]. The intron number in the previously sequenced liverwort mitogenomes ranges from 28 in
T. lacunosa to 30 in
M. polymorpha, not including introns in pseudogenes. In liverworts, three cases of changes in the intron number have been detected to date (marked with numbers (1)–(3)). The rrn18i1065gII intron present in
M. polymorpha is lacking in
P. purpurea [
35,
36],
A. pinguis [
37] and in the examined genus
Calypogeia (1). The intron set of
T. lacunosa differs most compared to the other liverworts. Apart from the absence of the mentioned rrn18i1065gII intron (1), it lacks one intron in the
nad4L gene (2) and, as in
Calypogeia, two introns of the
atp1 gene (3) [
12].