1. Introduction
Otters belong to the subfamily Lutrinae within the family Mustelidae and have recently radiated from terrestrial weasel-like ancestors and successfully thrive in semi-aquatic habitats.
P. brasiliensis and
L. canadensis are two otter species. The giant otter (
P. brasiliensis), which belongs to the genus
Pteronura, is the largest and unique species of otter living in freshwater habitats [
1]. The North American river otter (
L. canadensis), belonging to the genus
Lontra, is mainly distributed in the North American watersheds [
2] and can occupy different habitats from the sea to freshwater habitats, mountain streams, and desert canyons [
2]. These two species were often identified by their morphological characteristics [
2,
3,
4]. Their phylogenetic status was also determined using their morphological characteristics or partial fragments of the mitochondrial genome [
4,
5,
6], as their mitochondrial genomes are still unknown up to now. Therefore, we assembled the mitochondrial genomes of these two species for the first time to provide important genetic resources for future study, and to determine their phylogenetic status. Additionally, these mitochondrial genomes may be important genetic resources for protecting these two species in the future.
The mitochondrion is the center of energy metabolism [
7], and its genome is independent of the nuclear genome. Mammalian mitochondrial genomes are double-stranded circular molecules approximately 16 kB nucleotide in length [
8]. One strand of the mitochondrial genome is the heavy (H) strand, which is rich in guanine. The other strand, called the light (L) strand, is cytosine-rich [
8,
9]. The mammalian mitochondrial genome usually contains 22 transfer RNA (tRNA) genes, 13 protein-coding genes (PCGs), 2 ribosomal RNA (rRNA) genes, and a major non-coding control region (D-loop) [
8,
10].
The mitochondrial genome is maternally inherited; its molecular size is small, the sequencing procedures are simple, and the recombination rate is low. Therefore, they are often used to analyze phylogenetic relationships, genetic diversity, and evolutionary adaptations [
11,
12]. Mitochondrial genome evolution has been previously shown to be related to niche adaptation in animals. The Mustelidae mitochondrial genome had undergone divergent evolution among animals adapted to different niches [
13]. The Cetartiodactyla mitochondrial genome also displayed divergent evolutionary patterns during the process of niche adaptation [
14]. Positive selection signals in the mitochondrial genomes of Vesicomyidae species had revealed evidence for their adaptive to deep-sea environments [
15]. Studies on the mitochondrial genome of domesticated animals, such as dogs, cattle, and yaks had shown relaxed selection patterns when compared with their wild relatives under the adaptation to domesticated environments [
16,
17,
18]. Additionally, the mitochondrial genome of Tibetan loaches displayed more non-synonymous mutations than that of non-Tibetan loaches in order to adapt to the environment of the Tibetan plateau [
19]. Based on this previous research, this study hypothesized that the mitochondrial genome of otters might have divergent evolutionary patterns in order to adapt to semi-aquatic environments when compared to their terrestrial Mustelidae close relatives.
In this study, we assembled and annotated the complete mitochondrial genome of P. brasiliensis and L. canadensis based on high-quality raw genome sequencing data for the first time to explore their structural characteristics and provide genetic resources for protecting these two important species. The mitochondrial genomes of the two genera of otters have not been previously studied. Contrastingly, in order to investigate how the otters adapt to the semi-aquatic habitats, nine otters representing all the seven genera of Lutrinae and ten Mustela who were the closest relatives of otters in Mustelidae were selected to explore the evolutionary characteristics of otters’ mitochondrial genome. Therefore, this study aimed to reveal the adaptive evolution of otters to semi-aquatic niches from the mitogenomics perspective and provide genetic resources for the protection of these two otter species.
4. Discussion
Mitochondria are the key cellular organs that provide energy for the life activities of animals through OXPHOS [
33,
34,
35]. The mammalian mitochondrial genome is independent of the nuclear genome and is a double-stranded circular molecule, approximately 16 kb in length [
8]. One strand of the guanine-rich mitochondrial genome is called the heavy (H) strand, and the other cytosine-rich strand is called the light (L) strand [
8,
9]. The mitochondrial genome is maternally inherited and often used to analyze phylogenetic relationships and evolutionary adaptations [
11,
12]. Additionally, it is extremely important in conservation genetics research. Therefore, we assembled two mitochondrial genomes for the two otter species that needed to be protected and studied.
Characteristics of the mitochondrial genome may differ among different animal groups. Herein, the mitochondrial genomes of
P. brasiliensis and
L. canadensis were 16,395 and 16,500 bp, respectively, in length and contained 37 genes (13 PCGs, 22 tRNA genes, and 2 rRNA genes) and a control region (D-loop). Among these, 28 genes were in the heavy strand and 9 other genes were in the light strand. The initiation codon for most PCGs was ATG, and the common termination codon was TAA. These characteristics were consistent with those of the mitochondrial genome’s characteristics of other otters [
36,
37,
38,
39,
40]. The overall AT content of the heavy strands of these two mitochondrial genomes was higher than the GC content, indicating AT-rich characteristics. The base compositions were skewed similarly to those of other vertebrate mitochondrial genome sequences [
41,
42,
43]. The AT content was higher than the GC content in most of the mitochondrial genomes of otters (
Table 4). In bacteria, the GC skew represents the footprint of genome evolution driven by DNA replication [
44]. Whether there was a relationship between the GC skew of the mitochondrial genomes and the otter species evolution remains unknown.
Among the 22 tRNAs, the
tRNASER2(GCT) lacked the dihydrouridine hairpin structure, and all the remaining 21 tRNAs had a canonical cloverleaf structure. The
tRNASER was found to lack a canonical cloverleaf structure in several animals [
37,
40,
42,
45]. Several studies have demonstrated that the lack of a dihydrouridine arm or thymidine-pseudouridine-cytidine (TψC) loop in
tRNASER might not affect its normal function [
46,
47]. This suggested that
tRNASER2(GCT) was able to perform normal functions in these two otter species.
The results of the phylogenetic analysis showed that all otters were clustered into one clade, whereas the Mustela species were clustered in another clade.
P. brasiliensis was the earliest otter species to diverge from the Mustela species, which formed the genus
Pteronura. Subsequently,
L. canadensis branched out from
P. brasiliensis.
E. lutris and
H. maculicollis individually formed one single clade each, followed by
L. canadensis, which formed the genera
Enhydra and
Hydrictis.
L. lutra and
L. sumatrana clustered into one clade, belonging to the genus
Lutra. These evolutionary relationships were consistent with the results of previous traditional studies on otter classification [
48].
L. perspicillata,
A. cinerea, and
A. capensis were clustered into one clade. Several previous studies had demonstrated that
L. perspicillata and
A. cinerea clustered into one clade [
39,
49,
50,
51,
52,
53], which was inconsistent with the traditional classification [
48]. We inferred that this might be the result of hybridization between
L. perspicillata and
A. cinerea [
49].
Otters are semi-aquatic mammals, with important characteristics that distinguish them from other terrestrial Mustelidae species. Habitat and locomotive styles have been shown to exert a certain influence on the evolution of animal mitochondrial genomes. For example, ecological specialization exerted selective constraints on the mitochondrial genomes of Mustelidae [
13]. The mitochondrial genome of some domesticated lineages, such as dogs, cattle, yaks, pigs, and silkworms, and weakly locomotive mollusks, birds, and mammalian lineages showed a relaxed evolutionary selection pattern [
16,
17,
18,
54,
55,
56,
57]. Different habitats and lifestyles affect the evolutionary style of fish mitochondrial genomes [
43,
58]. Additionally, the evolution of the Cetartiodactyla mitochondrial genome displayed divergent patterns during the process of niche adaptation [
14]. We analyzed the evolutionary patterns of the otters’ mitochondrial genomes through the method of comparative mitogenomics to clarify the influence of semi-aquatic habitats. The result of root-to-tip ω values demonstrated that the mitochondrial genome PCGs of the nine otters were mainly under purifying selection. This was consistent with the results of previous studies in other animals [
59]. Furthermore, the ω values of
ND1,
ND4, and
ND4L were higher in otters than in terrestrial Mustelidae, whereas
ND2,
ND6, and
COX1 had lower ω values in otters. Additionally, the studies on Tibetan loaches found some differences in mitochondrial genome PCGs’ ω values when compared with the plain species [
19]. Our results also showed that
ATP8 had the highest ω values in all of the 13 PCGs in these 21 species.
ATP8 is suggested to have a high evolutionary rate in several animals [
13,
14,
56].
ATP8 plays an important role in metabolism, respiratory electron transport, and heat production [
60]. The high evolutionary rate of
ATP8 in animals may allow for several further beneficial substitutions, which may be advantageous for animals to adapt to different ecological niches [
61,
62]. Contrastingly, six (
ND1,
ND4,
ND4L,
ND5,
COX3, and
CYTB) of the thirteen PCGs of the mitochondrial genome were rapidly evolving in the otter branch. This suggested that the otter group accumulated more nonsynonymous mutations than other terrestrial Mustelidae animals in these six genes. The high number of nonsynonymous mutations might result in a few beneficial amino acid changes, which may help otters adapt to semi-aquatic habitats [
19,
61,
62,
63]. Studies on galliform birds and loaches found that high-altitude species had large dN/dS for the 13 concatenated mitochondrial PCGs [
19,
64] because of their high energy demands. Combined with the root-to-tip ω values, we predict that the three genes (
ND1,
ND4, and
ND4L) likely evolved more quickly in otters than in terrestrial Mustelidae species. We inferred that these rapidly evolving genes are related to otters adapting to semi-aquatic habitats.
We conducted a PIC analysis to eliminate the impact of evolutionary relationships on the mitochondrial genome evolution. A significant correlation between evolutionary rates and habitats for ND2, ND4, and ND4L was observed. This suggested that habitat type markedly influenced the evolutionary rate of these three genes in otters and terrestrial Mustelidae species, and mitochondrial gene evolution in otters might be correlated with their adaptation to semi-aquatic habitats.