The taxonomy of two uncultivated fungal mammalian pathogens is revealed through phylogeny and population genetic analyses

Ever since the uncultivated South American fungal pathogen Lacazia loboi was first described 90 years ago, its etiology and evolutionary traits have been at the center of endless controversies. This pathogen infects the skin of humans and as long believed, dolphin skin. However, recent DNA analyses of infected dolphins placed its DNA sequences within Paracoccidioides species. This came as a surprise and suggested the human and dolphin pathogens may be different species. In this study, population genetic analyses of DNA from four infected dolphins grouped this pathogen in a monophyletic cluster sister to P. americana and to the other Paracoccidioides species. Based on the results we have emended the taxonomy of the dolphin pathogen as Paracoccidioides cetii and P. loboi the one infecting human. Our data warn that phylogenetic analysis of available taxa without the inclusion of unusual members may provide incomplete information for the accurate classification of anomalous species.

www.nature.com/scientificreports/ These studies placed the DNA sequences from infected dolphins within Paracoccidioides species and away from that of L. loboi in humans. Based on these reports, it was predicted that the etiologic agent of the uncultivated skin pathogen of dolphins was probably a new Paracoccidioides species [20][21][22][23] . Moreover, because the uncultivated human pathogen shared common phenotypic features with that of dolphins, it was suggested the human pathogen possibly a Paracoccidioides species as well 12,23 . Therefore, Vilela and Mendoza 12 introduced P. brasiliensis var. ceti as a new variety in the genus, to differentiate the dolphin pathogen from the one causing skin disease in humans.
To investigate the patterns of genetic diversity between the uncultivated skin pathogen of dolphin, Paracoccidioides species and L. loboi, we conducted phenotypic, phylogenetic and population genetic analyses using rDNA (ITS) and partial DNA coding sequences extracted from four dolphins swimming USA coastal areas with the disease and homologous DNA sequences in the data base. In these analyses the pathogen of dolphin grouped in a monophyletic cluster sister to P. americana and in turn, both species were sister to P. brasiliensis, P. restrepiensis and P. venezuelensis; whereas, the human pathogen formed a monophyletic cluster sister to P. lutzii. Based on this finding we have emended the taxonomy of both pathogens, now known as P. cetii and P. loboi.

Results
Phenotypic traits. The phenotypic traits of Paracoccidioides species in this study grouped these pathogens into two clusters (Table 1). Except for a common Gp43 antigen, recognized by the anti-Gp43-IgG antibodies (brown cell) present in infected hosts species (Table 1), the remaining 19 phenotypic traits showed P. americana, P. brasiliensis, P. lutzii, P. restrepiensis and P. venezuelensis sharing identical phenotypic traits (blue cells). Paracoccidioides cetii and P. loboi displayed contrasting phenotypic features with the above species (green and yellow cells), but similar traits with some differences between them (yellow cells). For instance, P. cetii is found causing disease in dolphins in many oceans (yellow cell) and, although P. loboi shares some phenotypes with Paracoccidioides species (blue cells), it displays numerous features in common with P. cetii (green cells). Figure 1 was assembled utilizing clinical and laboratorial data collected in our facilities. The phenotypic traits included their cultivated or uncultivated nature, morphological features in the infected tissues, and the capacity of causing systemic or subcutaneous infections. These phenotypic traits grouped the seven species in this study into two clusters (Fig. 1a,b). One cluster contains all known Paracoccidioides species causing systemic paracoccidioidomycosis in humans (Fig. 1a), whereas the other cluster comprised P. cetii and P. loboi causing subcutaneous infection in dolphins and humans (Fig. 1b). Figure 1c shows that loss of the ability to grow in culture is a paraphyletic trait in phylogenetic analysis among Paracoccidioides species.
Principal component analysis (PCA). The Gp43 partial DNA sequences (P. americana, n = 9; P. brasiliensis, n = 15; P. cetii n = 5; P. loboi n = 16; P. lutzii, n = 10; P. restrepiensis n = 10; P. venezuelensis, n = 5) (Fig. 2) (Table S1) using three components (PC1, PC2 and PC3) grouped Paracoccidioides species into five populations corresponding to some of the above species (Fig. 2). The three principal components accounted for 71.0%, 12.6%, and 6.7%, respectively, with cumulative variation explaining 90.3% of the interrogated DNA sequences, indicating the relationship of individuals present in each cluster is reliable (Fig. 2). Paracoccidioides loboi (pink) and P. lutzii (yellow) were discriminated by PCA, placing them in independent clusters on the lower right section of Fig. 2. Paracoccidioides americana (green) and P. cetii (blue) DNA sequences were also discriminated into two clusters on the upper left section of the graphic (Fig. 2). The remaining species including P. brasiliensis, P. restrepiensis and P. venezuelensis (red) formed a single cluster located at the lower left section of the graphic.  In culture: hyphae, conidia  Yes  y es  yes  y es  yes  ?  ?  Variable sizes mulƟple budding yeast  yes  y es  yes  y es  yes  n o  n o  Uniforme size yeast usually in long chains  no  no  no  no  no  yes  y es  InfecƟon airborne conidia  y es  yes  y es  yes  y es  ?  ?  Target organ lungs  Y es  yes  y es  yes  y es  no  no  Target organ skin  no  no  no  no  no  yes  y  www.nature.com/scientificreports/ Haplotype analysis. Neighbor-joining haplotype network using 77 Gp43 partial coding DNA sequence (Table S1) (including seven DNA sequences from dolphins in Japan = 3 and USA = 4 [accession numbers in Table S1]), showed P. americana (brown), P. cetii (beige), P. loboi (yellow) and P. lutzii (green) and the remaining Paracoccidioides species in separated clusters (Fig. 3). Haplotype analysis showed a relationship between P. cetii (beige) and P. americana (brown). Thirty-four mutations separated P. cetii and P. americana (Fig. 3). Two DNA sequences recovered from Japanese dolphins (Pcet3 and Pcet2-beige LC537903 and LC057206, only 110 bp of the 266 bp available at NCBI was used) displayed numerous mutations between them and the four USA dolphin DNA sequences. The third DNA sequence recovered from another dolphin in the coastal areas of Japan (Pcet1) was placed linked to the four USA dolphins (Pcew4-7), with 3 missing or extinct haplotypes and eight mutations. In this analysis, P. loboi (yellow) was linked to P. lutzii (green), with numerous mutations separating these two haplotypes (n = 54) and several missing or extinct haplotypes (red empty circles) (Fig. 3). Fifty-two mutations separated P. americana from P. lutzii and only 9 mutations were found between them and the remaining Paracoccidioides species (P. brasiliensis [blue], P. restrepiensis [pink] and P. venezuelensis [green]) (Fig. 3).
Population STRU CTU RE analysis. STRU CTU RE software was used to determine the population structure of the Paracoccidioides species using Gp43 partial coding DNA sequences (see above) (Table S1). In this analysis, the LnP(D) as well as Evanno's ΔK showed 5 as the best K value and then, K = 5 used to build the data (Fig. 4a). The Fst average value was 0.8879 indicating high population structure. Bayesian clustering implemented in STRU CTU RE using the Gp43 (Fig. 4c) (Fig. 4b). The remaining Paracoccidioides species (1Sa, www.nature.com/scientificreports/ 1Sb, PS2, PS3, and PS4 in red) grouped sister to the above clusters (Fig. 4b). The Gp43 partial DNA sequences of one of the three dolphins from Japan (AB811031) clustered together with the four DNA sequences from USA dolphins. However, two other dolphin DNA sequences also from Japan (LC537903 and LC057206) were placed with the Gp43 DNA sequences of P. brasiliensis 1Sa (Fig. 4b, red rectangle). Comparative analysis of the phylogenetic results and that obtained in STRU CTU RE, showed a support for the presence of five populations   www.nature.com/scientificreports/  (Table S1). Phylogenetic analysis using the above partial coding and ITS homologous DNA sequences from dimorphic Onygenales as outgroup showed comparable threes (Fig. 5). ADP ribosylation factor and CHS4 partial DNA sequences showed similar clusters to that reported with the Gp43 DNA sequences (Figs. 4,5). In these trees, P. americana, P. cetii, P. loboi and P. lutzii were placed in monophyletic clusters (Fig. 5). Except for P. loboi and P. lutzii forming two monophyletic clusters, KEX DNA sequences could not discriminate P. cetii from the other Paracoccidioides species (Fig. 5, red rectangle). The evolutionary history of the ITS DNA sequences in this study placed P. cetii (including dolphins in Brazil, Cuba, and the USA), P. loboi, and P. lutzii in three monophyletic clusters, but could not discriminate other Paracoccidioides species (Fig. 5). A poorly supported monophyletic cluster closely related to P. cetii was tentatively labeled as P. americana (Fig. 5, ITS).
Geographic distribution, haplotype network, principal component analysis, and population structure of concatenate Gp43 and ADP-rf DNA sequences. Concatenate data of the Gp43 plus ADP-rf partial coding DNA sequences (59 DNA sequences) (Table IS) using different statistical programs, consistently grouped Paracoccidioides species in this study into five clusters (Fig. 6). Based on recognized areas were cases of human and dolphin disease occur, the geographical distribution of these species is shown in Fig. 6a. The haplotype of P. cetii from dolphins is found around the coastal areas of the Americas (blue + green haplotype) (Japanese cases are not shown), whereas the haplotypes of the other Paracoccidioides species (P. americana = yellow + pink; P. brasiliensis = red + green + yellow; P. lutzii = yellow + pink) are distributed in several South America countries (Fig. 6a). Paracoccidioides loboi (pink + yellow) haplotype located in the Amazon basin (Fig. 6a) and other Latin American countries (not shown). The haplotype-concatenated data showed five haplotype groups comparable to the results using Gp43 DNA sequences. (Figs. 3, 6b). Correspondingly, PCA (Fig. 6c) and STRU CTU RE (Fig. 6d-f) analyses consistently grouped Paracoccidioides species into five populations. The LnP(D) as well as Evanno's ΔK of the Gp43 plus ADP-rf concatenate partial coding DNA sequences was K = 5 used to build the data (Fig. 6d). The triangle plot in Fig. 6f is an analogous result obtained from STRU CTU RE software outputs (Q4 = P. lutzii, Q5 = P. loboi other species, top of the tringle). Similarly, phylogenetic analysis of the concatenate data, inferred by using Maximum Likelihood method and Kimura 2-parameters model, showed analogous results to the analysis using Gp43 DNA sequences (Figs. 4b, 1c). Japanese dolphins lack ADP-rf DNA sequences at the NCBI thus, they were not included in this analysis. Phylogenetic analysis of the concatenated data supports the placement P. americana, P. cetii, P. loboi and P. lutzii in four monophyletic clusters (Fig. 1c).

Figure 5. Evolutionary analyses inferred by Maximum
Likelihood of the ADP-rf, CHS4, KEX, and ITS, DNA sequences respectively, using homologous DNA sequences from well-known dimorphic Onygenales as outgroup (Table S1). In these trees, P. lutzii (yellow bars) consistently grouped as monophyletic clusters sister to P. loboi (pink bars). Except for Kex DNA sequences, grouping P. cetii (red rectangle) with P. brasiliensis (sensu lato) (bluish bar), the other DNA sequences in these analyses (ADP-rf, CHS4, and ITS) grouped P. cetii (blue bars) in monophyletic clusters. Paracoccidioides americana (green bars) clustered as a monophyletic group using ADP-rf and CHS4 DNA sequences and the other Paracoccidioides species (red bars) grouped in a single cluster. Using ITS DNA sequences, a poorly supported cluster was tentatively labelled as P. americana (green bar).   Disease nomenclature. Names such as Jorge Lôbo disease, lobomycosis, and others are retained. The term lacaziosis is no longer appropriate. In addition, since the term paracoccidioidomycosis has been traditionally used to describe systemic infections caused by Paracoccidioides species other than P. cetii and P. loboi, the term "paracoccidioidomycosis loboi" is proposed to emphasize this species is restricted to the subcutaneous tissues.

Discussion
After 90 years of taxonomic uncertainties, using phenotypic, phylogenetic, and population genetics analyses, the two uncultivated fungi causing skin disease in humans and dolphins, long known as Lacazia loboi 8 , are now placed as separate species within the genus Paracoccidioides. Early studies using phenotypic or phylogenetic data alone erroneously placed these two fungal pathogens in different genera and species [3][4][5][6][7][8]12,13,[15][16][17]24,25 . This trend persisted for years 2,13,16,17,25 . For instance, recent studies using several partial DNA sequences recovered from Brazilian humans with skin disease in phylogenetic analyses concluded that the genus Lacazia, the accepted name at that time, was an independent taxon from Paracoccidioides species 16,24,25 . Their phylogenetic data was correct, but their analyses missed the inclusion of DNA from the uncultivated pathogen causing skin disease in dolphins. This was an understandable mistake, since the collection and processing specimens from infected dolphins is highly regulated and the fact that the etiology of dolphins' disease was long believed to be the same as that in humans, as shown in Fig. 1 and Table 1. Although P. cetii has numerous phenotypic differences with Paracoccidioides species (Table 1, Fig. 1), in the pass used to group them in separated clusters 2,3,7,8 , our data showed they share several phylogenetic features in common (Figs. 4, 5 and 6). With the addition of P. cetii DNA sequences, the phylogenetic support of closely related Paracoccidioides species dramatically changed. For example, P. loboi clustered in a monophyletic group sister to P. lutzii, even with the inclusion of homologous dimorphic Onygenales DNA sequences as outgroup (Figs. 4b, 5), whereas the support of monophyletic species within the genus weakened (Figs. 4, 5 and 6). More dolphin DNA sequences from different geographical locations must be sequenced to understand P. cetii´s true evolutionary traits. Several studies reported geographical cryptic speciation among Paracoccidioides species 14,24,[26][27][28] . In those analyses the presence of at least five species within the genus, including P. lutzii, was found 14,15,24,27,29,30 . Recent genome sequencing in phylogenetic analysis tend to validate these findings 26,28,29 . Although the DNA sequences of P. loboi were used in some of the analyses, the human skin pathogen was always placed as an independent genus from that in Paracoccidioides species 16,24,25 . The placement of P. cetii sister to P. americana DNA sequences in this study, indicates the use of phenotypic or phylogenetic characteristics without the inclusion of anomalous species, can lead to inaccuracies in the taxonomic and phylogenetic classification of these type of microbes. For instance, our data, using several statistical tools, consistently showed the presence of different clusters within Paracoccidioides species. In our analyses, P. americana, P. cetii, P. lutzii, and P. loboi were placed in monophyletic groups sister to the remaining Paracoccidioides species (Figs. 2, 3, 4, 5 and 6). Therefore, the addition of P. cetii to the genus Paracoccidioides not only confirmed that the genus has indeed a high level of speciation but, indicates that the concept of species delimitation in this genus must be revisited 12,31 .
Recently, Vilela et al. 16 , using phylogenetic analysis of five different genes, showed P. loboi shared the same ancestor with Paracoccidioides species. The results in our study support their proposal. The main obstacle of this hypothesis at that time was the phenotypic features of P. loboi (Fig. 1). However, if P. loboi and P. cetii (both uncultivated and subcutaneous pathogens) share the same ancestor with other Paracoccidioides species (cultivated and causing systemic infections), the likelihood that the ancestor of Paracoccidioides species could growth in culture, as previously suggested, is a strong possibility 16 . If this concept is correct, when in the evolutionary history of P. cetii and P. loboi they lost the capacity to grow in culture? What evolutionary pressure triggered such a change? Sadly, as is common in neglected pathogens such as P. cetii and P. loboi key questions such as these, remain without an answer. Interestingly, the uncultivated feature found in these two neglected fungi was also reported in a strain of Histoplasma capsulatum infecting monkeys, suggesting that an uncultivated ancestral trait in the Onygenales dimorphic fungi may be at work 32 . However, the evolutionary pressures that triggered such ancestral feature remains an enigma.
The report of new human cases of paracoccidioidomycosis loboi acquired by traveling to endemic areas 2-5,33-36 , suggests P. loboi may has a similar phenotype (hyphae with conidia) to the one displayed by Paracoccidioides species in nature and in culture. Thus, it may be present in specific ecological niches in the endemic areas (around the Amazon basin and other Latin American big rivers) 2,14,15,25  org/ fungal-genome-initi ative/ lacaz ia-loboi-seque ncing), only fragmented genomic information is available for P. loboi, and the genome of P. cetii is yet to be sequence. We hypothesize that the genomes of both uncultivated pathogens may hide important genomic clues that could answer this and other evolutionary questions. Several P. cetii DNA sequences recovered from dolphins captured in Brazil, Cuba, Japan, and the USA are currently available in the database (Table S1) [19][20][21][22][23] . The complete ITS DNA sequences from Brazilian and Cuban dolphins with paracoccidioidomycosis ceti, showed high percentage of identify with the DNA sequences in this study (ITS = 100%) whereas the partial Gp43 DNA sequences from a Japanese dolphin (471 bp) had 98.62% identity with P. cetii DNA sequences from dolphins captured in the Americas. During Gp43 DNA alignment of Japanese and USA dolphins, a five nucleotides gap was consistently present in the DNA sequences of USA dolphins. Moreover, two additional 266 bp GP43 DNA sequences extracted from a Japanese dolphin (Lagenorhynhus obliquidens) with paracoccidioidomycosis ceti showing, 99.62% identity with P. brasiliensis (sensu lato). In our analyses, these two sequences (only 110 bp could be used) clustered also with P. brasiliensis (Fig. 4, red  rectangle). However, the same DNA sequences clustered close to P. cetii in haplotype analysis indicating a fragile relationship (Fig. 3). If P. cetii DNA sequences from Japanese dolphins are accurate, the differences in the genetic makeup of these two populations of uncultivated pathogens is intriguing and deserve further analysis. Our data suggest P. cetii strains causing paracoccidioidomycosis ceti in Japanese and USA dolphins, likely are evolving into two different populations.
According to Teixeira et al. 24 , the estimated time for genetic divergence in Paracoccidioides species was calculated around 33 million years. Although, others have questioned this result 31 , Carruthers et al. 43 , cautioned that the use of linage-specific data usually demonstrate approximate divergence time regardless of the number of loci interrogated. Nonetheless, according to these reports, Paracoccidioides species probably diverged from their ancestor from a fraction of a million of years (P. restrepiensis and P. venezuelensis) to 10-30 million of years (P. lutzii and P. brasiliensis, sensu lato) 24,31 . Conversely, dolphins evolved into aquatic mammals ~ 50 to 30 million years ago, around late Paleocene period (Eocene, Oligocene epochs) 44 . According to fossil records, South America at this time had a large body of water crossing from the north Atlantic Ocean to what is today Bolivia, Brazil, Ecuador, Colombia, Peru and Venezuela 45 , all endemic areas of these species [3][4][5]24,26,29 , that lasted for millions of years. A similar situation occurred in what is today the estuary of the Amazon River. The current location of Paracoccidioides species (including P. loboi), coincide with the locations of such geological periods, and then it is quite possible that during the time following these geological events, an ancestor of P. cetii first encountered dolphins entering these areas. Since humans came to South Americas only ~ 15,000-year ago 46 , likely the ancestor of Paracoccidioides species infected dolphin first and later humans. Whether this event had a role on the pathogenic capabilities of the genus to infect mammals is difficult to determine, nonetheless it is an intriguing possibility.
Working with uncultivated pathogens infecting the skin of mammals is challenging. Not only because collecting specimens from these species (dolphins are protected species and human cases are located in poor remote rural areas) is extremely difficult, but because open lesions usually harbor numerous environmental contaminants, which in the past had led to erroneous conclusions on the classifications of these two anomalous pathogens 2,8,15,16,25,47 . Furthermore, these unusual fungi are not in the list of neglected pathogens, thus discouraging investigators to submit proposals to funding organizations. Previous studies using P. loboi in phenotypic or phylogenetic analyses placed this anomalous pathogen away from the genus Paracoccidioides 2,4,15,16,25 . This study found that the use of phenotypic or phylogenetic approaches without the inclusion of DNA from infected dolphins, likely led previous studies to flawed data 15,16,25 . Thus, the failure of including organisms sharing a common ancestor, based in phenotypic or phylogenetic traits alone, could result in incomplete or incorrect assessment of the investigated populations. This study showed that the interpretation of taxonomic and/or phylogenetic data could be affected by missing neighboring anomalous taxa. Phenotypic traits. Key phenotypic traits of Paracoccidioides species (epidemiology, distribution, etiology, laboratory finding such as histopathology, culture, experimental infections and others), were collected from data available on paracoccidioidomycosis published literature 49,50 . Likewise, the above phenotypic traits of the pathogen of dolphins were also collected from publications on the subject for the past 50 years 2,6,7,12,[18][19][20][21][22][23]35,39 . The data was then used to build Table 1 displaying the most relevant phenotypic features of Paracoccidioides species in this study. Clinical and laboratorial data available in our facilities were also used to visualize the phenotypic traits displayed on Fig. 1 www.nature.com/scientificreports/ DNA isolation, sequencing, and genotyping. The biopsied tissues were originally sent frozen to our laboratory and manipulated according to approved guidelines 48 . Sections of the biopsied tissue containing numerous yeast-like cells by wet mount and/or histopathology were selected for DNA extraction. The dolphin skin fragments were cut into small 2 to 4 mm in diameter cubes and then ground under liquid nitrogen. The DNA from the grounded samples was treated with sodium dodecyl sulfate and digested with RNase A and protein K (Quiagen, Germantown, MD, USA) at 60 °C for 1 h. The DNA from the resulting mix was extracted with phenol/chloroform, dissolved in sterile distilled water and storage at − 80 °C, Double-stranded copies were amplified with AmpliTaq-Gold polymerase (Applied Biosystems, Branchburg, New Jersey, USA) in 25 μl volume reactions. The primers used were as per Vilela et al. 16,17,23  Haplotype and principal component analyses. Based (Table S1). In addition, diversity of haplotypes within the genus Paracoccidioides was also investigated using Gp43 DNA sequences and the concatenate Gp43 plus ADP-rf partial DNA sequences. The data was estimated using the software DnaSP, v7 56  Population STRU CTU RE analysis. Spatial genetic structure was further analyzed using STRU CTU RE 5.2.1 56 which uses Bayesian algorithm to estimate probability of membership 57 . The population structure of each of the Paracoccidioides species in this study, based on single nucleotide polymorphism (SNPs) loci, was investigated using Gp43 partial DNA sequences from 77 individuals and the concatenate data from Gp43 plus ADP-ribosylation factor partial DNA sequences from 59 individuals (Table S1). To identify the number of populations (K) that comprises the structure of the data, the burn-in phase was set at 10,000 with the Markov Chain Monte Carlo iterations and the run duration at 50,000 using the admixture model correlating allele frequencies independently for each run. Ten runs were carried out for each value of K, with ranges from 1 to 10. For each K value, the statistical value delta K was calculated using Evanno et al. 58 computations. The optimal K of the analysis was collected using the STRU CTU RE Harvester (http:// taylo r0. biolo gy. ucla. edu/ struc tureH arves ter/). Based on the LnP(D) and Evanno's ΔK identified 5 as the best K value on both the Gp43 partial DNA sequences and concatenate data from Gp43 DNA plus ADP-ribosylation factor partial DNA sequences. Each Paracoccidioides species genotype was assigned to a cluster (Q) determined by the probability of the software that a particular genotype is belong to the cluster. The cut-off probability for cluster assignment was 0.5 for more than two clusters. According to the optimum K a bar plot (sort by Q) was obtained to display the population structure among the Paracoccidioides spp.

Methods
Phylogenetic analysis. Genetic diversity was also investigated, and phylogenetic trees constructed using dolphin amplified DNA sequences in this study and that available in the database including those from Japanese dolphins and from dimorphic pathogenic Onygenales, used as outgroups (Table S1). The following partial DNA sequences, ADP-rf, CSH4, Gp43, kex, and ITS, were amplified from four USA dolphins and then aligned, using MUSCLE software in MEGA X 53 , with homologous DNA sequences available at the National Center for Biotechnology Information (NCBI) ( Table S1). Phylogenetic trees were constructed using MEGA X 53 . Evolutionary analyses were inferred by Maximum Likelihood and Kimura 2-parameter model 59 . The topologies generated for MP analysis were fully compatible and branches were considered supported when bootstrap values exceeded 70%. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. Codon positions included were 1st + 2nd + 3rd + Noncoding. The Gp43 partial DNA sequence was recently described as a good marker to separate Paracoccidioides species 14,15,30 . Thus, we used 77 Gp43 DNA sequences (including three dolphins. Only 110 bp was used from LC537903 and LC057206 dolphins) and 59 (ADP-rf + Gp43 concatenated data) DNA sequences (dolphin ADP-rf DNA sequences from Japan were not available, thus they were not included in concatenated analysis) to construct the phylogenetic trees. The ADP-rf, CSH4, Kex, and ITS amplified dolphin www.nature.com/scientificreports/ DNA sequences in this study (Table S1), plus available homologous DNA sequences at the NCBI were also used to construct phylogenetic evolutionary trees using the above parameters.

Data availability
Morphological and molecular data analyzed, other than P. cetii in this study, had been previously published 8,9,[12][13][14][15][16][17]23,24,52 . The DNA sequences and final assembly data in the manuscript have been deposited in the NCBI BioProject database under accession code PRJNA714057. Original data in this manuscript is also available by the authors on request.