Phylogenetic relationships of silver crucian carp in Carassius auratus complex based on mtDNA analysis

The phylogenetic relationships of Carassius genus subspecies were investigated based on the data of the variability of nucleotide sequences of the mtDNA cytochrome b (cyt b) and control region (CR). Dendrograms constructed based on the BA, ML, NJ, and MP methods revealed five clusters of the congruent topologies that substantially corresponded to geographical localities and taxonomic conception of the C. auratus complex. An analysis of two mtDNA fragment topologies demonstrated that the island forms of Japanese crucian carps C. cuvieri and C. auratus langsdorfii diverged later compared to the divergence of continental C. auratus forms (4.0?C4.5 mln years ago, by molecular calibration). Among the continental silver crucian carps, C. a. gibelio forms two clusters corresponding to two phylogroups with a mean uncorrected genetic distance p = 0.044. The genealogical combination of haplotypes with the first C. a. gibelio phylogroup was observed in C. auratus clade. According to the data of mtDNA analysis, these subspecies represent sister lineages with a level of intergroup divergence of p = 0.022?C0.036. No genetic differences were observed between diploid (except for the two C. a. gibelio phylogroups) and polyploid C a. auratus, as well as monophyly in polyploid forms. New approaches based on a comparative study of the nuclear markers might help to unravel the origin of gynogenetic forms and phylogenetic relationships within the C. auratus complex.     

Genetic identification of larvae and juveniles reveals the difference in the spawning site among Cyprininae fish species/subspecies in Lake Biwa

To understand the differences in the spawning sites among Cyprininae fishes in Lake Biwa, we conducted periodic sampling of larvae and juveniles at three sites (irrigation ditch, St. 1; river, St. 2; and satellite lake, St. 3). On the basis of species/subspecies identification by using RAPD analysis, we examined the species composition of the larvae and juveniles at these three sampling sites. The number of specimens was 616, 68, and 117 at St. 1, St. 2, and St. 3, respectively. Based on morphological and genetic identification, the specimens were found to include nine fish species/subspecies, namely, Carassius auratus grandoculis, Carassius cuvieri, Carassius auratus langsdorfii, Cyprinus carpio, Sarcocheilichthys sp., Silurus asotus, Oryzias latipes, Odontobutis obscura obscura, and Rhinogobius sp. The species composition at the three sites also differed. Among the Cyprininae fishes, C. auratus grandoculis, C. auratus langsdorfii, and Cyprinus carpio were found in abundance at St. 1; C. cuvieri was not collected from St. 1 but was found at the other two sites, particularly St. 3. Among the other fishes, Rhinogobius sp. was collected at St. 1 and St. 3, whereas the other four occurred only at St. 1. These results suggest that the selection of spawning sites by C. cuvieri differs to a certain extent from that of the other Cyprininae fishes, and the irrigation ditch in the lake is an important habitat for the larvae and juveniles of native fish species.     

Male mate choice in the gynogenetic–sexual complex of crucian carp,<Emphasis Type="Italic"> Carassius auratus</Emphasis>

The asexual all-female Japanese crucian carp, Carassius auratus langsdorfii (Teleostei: Cypriniformes), reproduces gynogenetically, relying on the sperm of males of the sexual "host," C. auratus subspp. Theoretically, frequency-dependent mating preference of males to conspecific females can lead to the coexistence of asexual and sexual fish, if all else is equal. Our specific questions are whether males prefer conspecific females over asexual females and whether individuals show dominance hierarchies that potentially cause frequency-dependent mating preference. In an individual choice experiment, a tank was partitioned into three compartments with the middle one for a single male and the two outer ones for a sexual and an asexual female. The males of C. auratus bürgeri demonstrated a significant preference for ovulated conspecific females over ovulated asexual females. In contrast, in a group mating experiment, a single experimental tank included two males, a sexual female, and an asexual female together, and males chased and mated with both asexual and sexual females equally. Male mate preference was weak in group mating, which is typical in natural populations. Males and females of crucian carp showed no apparent agonistic behavior to each other in the group mating experiment. This is different from other gynogenetic complexes with the dominance hierarchy of males showing strong frequency-dependent mating preference (e.g., Poeciliopsis). We conclude that male mate preference is unlikely to be a strong frequency-dependent force maintaining the coexistence of asexual–sexual complexes of Japanese crucian carp. Received in revised form: 5 February 2001 Electronic Publication     

Chromosome synapsis and recombination during meiotic division in gynogenetic triploid ginbuna,Carassius auratus langsdorfii

Electron microscope examinations of primary oocytes of gynogenetic triploid ginbuna (Carassius auratus langsdorfii) and diploid gengoroubuna (Carassius auratus cuvieri) were carried out. Typical synaptonemal complexes were observed in both subspecies. In addition, clear differences in PGM-A electrophoretic patterns between a female parent ginbuna and her offspring were detected. It was concluded that synapsis and recombination occur between at least some homologous chromosomes in triploid ginbuna.     

Composition of leucocytes of the head kidney of the crucian carp (<Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>, Cypriniformes: Cyprinidae) as affected by invasion of cestode <Emphasis Type="Italic">Digramma interrupta</Emphasis> (Cestoda; Pseudophyllidea)

The composition of leucocytes of the head kidney is studied in the crucian carps (Carassius auratus) either contaminated or uncontaminated with Digramma interrupta. The composition of leucocytes in the pronephros of the crucian carp from Lake Baikal basin has a lymphoid character. Compared to the crucian carp from the European part of Russia, in the fish from Baikal the granulocytopoetic processes are more pronounced. This is proved by the high content of young forms of granulocytes. In the fish infected with digramma, the immune suppression of proliferation of blasts and young forms of eosinophils was revealed. On the other hand, the inflammatory and humoral specific immune reactions are enhanced. Partial suppression of the immune response of C. auratus to invasion by D. interrupta facilitates development of the parasite.     

Studies on resistance characteristic and cDNA sequence conservation of transferrin from crucian carp, Carassius auratus

Transferrin (Tf) is a kind of non-heme β-globulin with two iron ions (Fe³⁺)-binding sites. To prove Tf’s physiological functions, Fe³⁺-proteins, serum iron contents, and total iron-binding capabilities were tested for Tfs of crucian carps (Carassius auratus) and sliver carps (Hypophthalmichthys molitrix). The above results demonstrated that sliver carps shared 1/3 Tf alleles with crucian carps; Tf of crucian carps had stronger Fe³⁺-binding ability and transportation ability in plasma than that of sliver carps. In addition, the results of oxygen consumption experiments indicated that crucian carps had the higher oxygen utility rate than sliver carps. For acute hypoxia exposure assay, normoxic gas mixture, hypoxic gas mixture A, and hypoxic gas mixture B were used to induce oxygen-regulated gene expression of crucian carps in acute hypoxia. The results of quantitative real-time PCR revealed that mRNA levels of Tf gene, Tfr gene and ATPase gene were down-regulated in acute hypoxia but mRNA level of LDHa gene was up-regulated in acute hypoxia. The results of crucian carp Tf-cDNA sequence analysis showed that cDNA regions of two Fe³⁺-binding sites were T₇₄₇–T₁₀₂₆ and T₁₇₃₇–A₁₈₈₄ based on the principle of bioinformatics. The sequence conservation of two Fe³⁺-binding sites was higher than that of the other five regions, which were confirmed according to the subregion model of Tf-cDNA sequence.     

Toward a preliminary assessment of the diversity and origin of Cyprinid fish genus Carassius in Iran

Introduction of non-native fish species has been criticized as one of the major threats to freshwater ecosystems. During recent years, the cyprinid fish genus Carassius has been the subject of various studies in terms of introduction biology and ecology. The taxonomy and identification of described species in the genus Carassius is still ambiguous. In order to increase the knowledge of introduced species of the genus in Iran, molecular approaches were employed to identify species based on the mitochondrial gene, cytochrome b (Cyt b). In total, 417 specimens across Eurasia and some other parts of the world have been analyzed. Afterwards, investigation of the possible origin and pathways of introduction was done using analysis of haplotype networks based on the Cyt b gene. The results revealed that there are three introduced species i.e., Carassius auratus, Carassius gibelio and Carassius langsdorfii, in inland waters of Iran. Carassius langsdorfii was recorded from Iran and western Asia for the first time. Analysis of haplotype network showed various potential sources of introduction for each species in Iran and regions beyond their native distribution ranges.     

A molecule in teleost fish, related with human MHC-encoded G6F, has a cytoplasmic tail with ITAM and marks the surface of thrombocytes and in some fishes also of erythrocytes

In teleost fish, a novel gene G6F-like was identified, encoding a type I transmembrane molecule with four extracellular Ig-like domains and a cytoplasmic tail with putative tyrosine phosphorylation motifs including YxN and an immunoreceptor tyrosine-based activation motif (ITAM). G6F-like maps to a teleost genomic region where stretches corresponding to human chromosomes 6p (with the MHC), 12p (with CD4 and LAG-3), and 19q are tightly linked. This genomic organization resembles the ancestral “Ur-MHC” proposed for the jawed vertebrate ancestor. The deduced G6F-like molecule shows sequence similarity with members of the CD4/LAG-3 family and with the human major histocompatibility complex-encoded thrombocyte marker G6F. Despite some differences in molecular organization, teleost G6F-like and tetrapod G6F seem orthologous as they map to similar genomic location, share typical motifs in transmembrane and cytoplasmic regions, and are both expressed by thrombocytes/platelets. In the crucian carps goldfish (Carassius auratus auratus) and ginbuna (Carassius auratus langsdorfii), G6F-like was found expressed not only by thrombocytes but also by erythrocytes, supporting that erythroid and thromboid cells in teleost fish form a hematopoietic lineage like they do in mammals. The ITAM-bearing of G6F-like suggests that the molecule plays an important role in cell activation, and G6F-like expression by erythrocytes suggests that these cells have functional overlap potential with thrombocytes.     

Phylogenetic relationships of silver crusian carp Carassius auratus gibelio, C. auratus cuvieri, crucian carp Carassius carassius, and common carp Cyprinus carpio as inferred from mitochondrial DNA variation

PCR-RFLP analysis of the ND3/ND4L/ND4 and 12S/16S rRNA regions and nucleotide sequence variation of the cytochrome b gene were used to study the mtDNA divergence in species of the family Cyprinidae, to examine the phylogenetic relationships of the species, and to identify their taxonomic status. The results indicated that an ancestral form diverged into silver crucian carp and crucian carp after its separation from the common carp lineage. The divergence of continental Carassius auratus gibelio and Japanese C. auratus cuvieri occurred more recently. Two well distinguishable mtDNA phylogroups, suggesting divergent evolution, were observed in continental C. auratus gibelio populations. The divergence was possibly related to the formation of two silver crucian carp groups with different types of reproduction, triploid gynogenetic and diploid gonochoric. At the same time, the results supported the high probability of current genetic exchange between the forms. In view of these findings and high morphological similarity of the two forms, they were not considered to be separate species.     

Relationships of morphological groups in the northern flicker superspecies complex (Colaptes auratus & C. chrysoides)

Many species complexes have diversified rapidly and recently, resulting in morphologically diverse populations; however, the rapid pace of diversification often prevents identification of clear phylogeographic structure. Recently, the use of large genomic and reduced-representation genomic datasets has improved resolution of the evolutionary histories in such species and allowed identification of lineages on distinct evolutionary trajectories. The northern flicker (Colaptes auratus) and gilded flicker (Colaptes chrysoides) form a polytypic superspecies group with a complex taxonomic history. The superspecies group includes up to 13 described subspecies, which represent slight geographic variation among five main morphological groups: red-shafted flickers of western North America (cafer group), yellow-shafted flickers of eastern North America (auratus group), Cuban flickers of the Caribbean (chrysocaulosus group), gilded flickers of the U.S. south-west and Mexican north-west (chrysoides group), and Guatemalan flickers of Central America (mexicanoides group). These groups are largely differentiable by variation in feather shaft colour, malar colour, throat colour, crown colour, and back barring. Here, using mitochondrial DNA (mtDNA) and hundreds of single nucleotide polymorphisms (SNPs), we characterized the genetic relationships and genomic distinctiveness of the five morphological groups. We found the mexicanoides group to be the most genetically distinct in both mtDNA (～1.4% sequence divergence) and large SNP panels. The chrysocaulosus group is differentiated by a single basepair mutation in a small mtDNA fragment. In both mtDNA and SNP panels, there is little genetic distinctiveness between auratus, cafer, and chrysoides morphological groups, with evidence of admixture and a lack of fixed differences.     

Morphological and genetic analysis of fish of a Carassius complex (Cyprinidae) in Lake Kasumigaura with reference to the taxonomic status of two all-female triploid morphs

The classification of the Carassius complex (Cyprinidae) including all-female triploids, called ginbuna in Japanese, is so confused that three sympatric morphs of crucian carp in Lake Kasumigaura are categorized into two different subspecies within a species. We examined them in order to explain the coexistence of more than one subspecies and determine the founder of the triploid lineages in the crucian carp fauna in the lake. Principal component analysis proved that the three sympatric morphs had a morphometric basis distinguishable from each other. Ploidy was determined by flow cytometry which showed triploids in two morphs and diploids in the other morph. Stepwise discriminant analysis using only meristic characteristics could separate the diploids from the triploids. Phylogenetic analysis using mitochondrial DNA inferred two lineages in which one was composed of a triploid morph and the other was a diploid–triploid mixture. Disagreement between the taxonomic status and the phylogenetic status is explicable by assuming that the triploids in the Carassius complex had independent origins leading to the different subspecies. C. auratus langsdorfii appears to show genetic complexities that traditional taxonomic classification can not unravel.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 351–357.     

Diagnostic features and biometry of head bones for identifying Carassius species in faecal and archaeological remains

The identification of fish species from head bone remains is employed in various sciences, including archaeology, paleontology and field ecology, with the estimation of fish size from biometric relationships being useful in the assessment of predation pressure exerted by increasing numbers of piscivorous species (e.g. Eurasian otter Lutra lutra and great cormorant Phalacrocorax carbo). This is particularly relevant for crucian carp, Carassius carassius, which is in decline in Europe due to changes in land use and to increasing numbers of non‐native Carassius species (i.e. goldfish C. auratus, gibel carp C. gibelio), which hybridize with C. carassius. However, diagnostic keys and biometric relationships are lacking for C. carassius and its most common hybrids, crosses with C. auratus. The present paper addresses this gap in knowledge, providing diagnostic keys and biometric relationships for the head bones of all Carassius species found in Europe as well as for C. carassius × C. auratus hybrids. All bone size to body length relationships were statistically significant. Similarly, all bone size to body weight relationships were significant for C. carassius, C. auratus, and C. gibelio, but none were significant for C. carassius × C. auratus hybrids. Diagnostic structures were found to distinguish easily between the Carassius species and hybrids, which will assist in determining the identity and sizes of prey found in faecal and archaeological remains.     

不同捕食者暴露对鲫幼鱼行为、运动和形态特征的影响

自然界中鱼类的捕食者种类繁多,为验证猎物鱼针对不同捕食者是否会做出相异的反捕食策略选择,选取鲫（Carassius auratus）幼鱼为猎物鱼,乌鳢（Channa argus）和胡子鲶（Clarias fuscus）为捕食者,将鲫幼鱼分别暴露于空白（对照）、乌鳢、胡子鲶和双捕食者（同时存在乌鳢和胡子鲶）环境中持续2个月,随后考查不同组鲫幼鱼的生长、行为特征、形态和运动能力等指标之间的差异。结果表明：经过2个月的捕食胁迫处理,鲫幼鱼的自发运动、隐蔽场所利用率和勇敢性等行为指标在4个组之间均未表现出显著性差异。相比对照组,乌鳢、胡子鲶和双捕食者暴露组鲫幼鱼的快速启动反应时滞显著缩短（P<0.05）;但所有处理组中仅乌鳢暴露组鲫幼鱼的快速启动120ms移动距离（S_120ms）和体高（BD）显著高于对照组（P<0.05）。另外鲫幼鱼的临界游泳速度（U_crit）和活跃代谢率（M_O2active）在各组之间也未表现出显著差异。乌鳢暴露组鲫幼鱼快速启动能力的提升可能与形态上体高的提高有关;另外相比临界游泳能力,快速启动能力可能在鱼类逃避捕食者过程中更为关键;鲫幼鱼表现出对乌鳢的反应最为明显,可能与乌鳢口裂较大,给鲫幼鱼带来的威胁更大有关。整体而言,应对捕食胁迫时,鲫幼鱼仅表现出形态和快速启动能力的反捕食响应,其行为特征的保守性可能是对生长作出的妥协,这对于鱼类适应生境中长期存在的捕食胁迫十分关键。     

Evidence for Introgressive Hybridization of Captive Markhor (Capra falconeri) with Domestic Goat: Cautions for Reintroduction

Markhors (Capra falconeri) are among the most endangered mammal species, and several conservation measures, including ex situ breeding, are implemented to prevent their extinction. We studied sequence diversity and differentiation of the first hypervariable segment of the mitochondrial DNA control region among C. f. heptneri and C. f. megaceros kept in four zoos in relationship to lineages of other wild and domestic goats, to assess for the first time the level of molecular distinctness and variability among those subspecies, and to check for possible introgression by related Capra taxa, such as domestic goats. Levels of differentiation between some Capra falconeri lineages and modern domestic goats were similar to levels between other wild goat species (i.e., Capra aegagrus, Capra ibex) and domestic goats. Among pure markhor lineages, paraphyly was observed for C. f. heptneri, suggesting occurrence of shared ancestral polymorphism among markhor subspecies and/or ancient or recent gene exchange between subspecies. Interestingly, 35.7% of all studied markhors from three zoos are introgressed by the domestic goat. Furthermore, despite relatively small breeding group sizes, markhors have maintained a relatively high proportion of mtDNA variation within zoo groups. In any case, the existence of markhors introgressed with domestic goat DNA in zoos should be considered when selecting markhors for ex situ breeding programs with the aim of building up a stock for later reintroduction into the wild.     

A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio)

1. Releases of non‐native fish into the wild is an increasing problem posing considerable ecological and genetic threats through direct competition and hybridisation. 2. We employed six microsatellite markers to identify first generation hybrids and backcrosses between native crucian carp (Carassius carassius) and introduced goldfish (C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites across Europe. 3. Natural hybridisation between goldfish and crucian carp occurs frequently, although hybrids between all other species pairs were observed. Only 62% of British crucian carp populations (n = 21) consisted exclusively of pure crucian carp. In some populations hybrids were so frequent, that no pure crucian carp were caught, indicating a high competitive ability of hybrids. 4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low frequency in goldfish × crucian carp hybrids and goldfish × common carp hybrids. Furthermore, some local populations had high frequencies of backcrosses, raising the opportunity for introgression. 5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were genetically closely related to goldfish, whereas all individuals identified from British populations proved to be crucian carp × goldfish hybrids. 6. Our study suggests that the release of closely related exotic cyprinids not only poses a threat to the genetic integrity and associated local adaptations of native species, but may also contribute to shifts in community structure through competitive interactions.     

A laboratory study of the effects of shelter availability and invasive crayfish on the growth of native stream fish

1. The alteration of stream habitats by urbanisation reduces the availability of shelter. Reduced shelter availability may increase both predation risks and metabolic costs, negatively affecting the growth performance of stream fish. Although urbanised streams often allow the establishment of invasive species, the additive or interactive effects of shelter availability and invasive species are rarely explored. The invasive red swamp crayfish (Procambarus clarkii) is a strong shelter competitor and predator in streams. 2. We conducted a stream‐channel experiment to investigate how shelter availability and this invasive crayfish species affect the growth performance (measured as the loss in body mass under starved conditions) of two native benthic species, Japanese crucian carp (Carassius auratus complex) and an endangered cobitid fish (Lefua echigonia). 3. We found that both the absence of shelter and the presence of crayfish increased body mass loss in the crucian carp. However, the growth performance of the crucian carp was improved by the presence of shelter except in the presence of crayfish, in which case the shelter available did not ameliorate the negative effect of the crayfish on growth performance (i.e. an interactive effect). This result suggests that crayfish may affect the growth performance of the crucian carp through chemical cues as well as through shelter occupancy. 4. In contrast, shelter availability is the primary factor affecting the body mass loss of the cobitid fish. However, the growth performance of these fish was unaffected by the presence of crayfish. The change in the growth performance of the cobitid fish in response to the absence of shelter was greater than that of the crucian carp. This finding suggests that the cobitid fish may be more vulnerable than the crucian carp to a reduction in shelter availability. 5. Our study demonstrates that reduced shelter availability and/or crayfish invasion can have significant, indirect negative effects on the growth performance of native fish, but whether those effects are interactive might vary depending on the species. Our findings have clear implications for stream restoration and habitat assessment.     

Genetic relationships among the Japanese and Korean striated spined loach complex (Cobitidae: Cobitis) and their phylogenetic positions

The striata complex, a group of spined loaches included in the genus Cobitis and characterized by a striped coloration pattern on the lateral midline, is distributed in rivers in northeastern Asia to western Japan. The complex comprises 2 continental species (Cobitis tetralineata and Cobitis lutheri) and 3 Japanese races of species rank (large race, middle race, and small race), the small race further comprising 6 local forms of subspecific rank (Tokai form, Biwa form, Yodo form, Sanyo form, San-in form, and Kyushu form). Previous karyological studies have revealed that the large race is an allotetraploid, the others being diploid. In this study, mitochondrial (mt) DNA analyses were conducted for 30 diploid populations of the Cobitis striata complex from Japan and Korea to examine: (1) their phylogenetic relationships and the position of the complex among the major lineages of Cobitis; and (2) the genetic relationships among the Japanese and Korean populations. The results, based on cytochrome b sequences (724 base pairs) analyzed with those of the main lineage of European and Japanese Cobitis, indicated that the striata complex should be considered as a monophyletic group, which evolved in northeastern Asia. Initially considered as a subspecies of Cobitis taenia, widely distributed from Europe to Asia, the striata complex does not have a sister-relationship with the former. Although the Korean species C. tetralineata was formerly believed to be closely related to the middle race in Japan, and a second continental species, C. lutheri, closely related to the Kyushu or San-in forms of the small race in Japan, the trees resulting from the present study revealed that the two Korean species were clustered with each other and separated from all Japanese races.     

Growth and potential photosynthesis of cyanobacteria are stimulated by viable gut passage in crucian carp

Growth and potential photosynthetic activity of phytoplankton passed through intestine of crucian carp (Carassius auratus) from a small Siberian reservoir were compared with those of phytoplankton taken the directly from the reservoir. The dominant phytoplankton species in the reservoir, Microcystis aeruginosa, showed a significant increase of growth after the passage. Subdominant Planktothrix agardhii also showed an increase in growth rate, while subdominants Anabaena flos-aquae and Aphanizomenon flos-aquae were not stimulated by the gut passage.     

Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch), and cross‐amplification in the closely related species crucian carp,Carassius auratus auratus (Linnaeus)

We report the development of 20 microsatellite markers for Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch). Nineteen out of 20 showed polymorphism with alleles ranging from two to 14. These loci were screened to amplify the closely related species crucian carp, Carassius auratus auratus (Linnaeus) and all of them can amplify DNA with the size similar to the former. The origin of silver crucian carp is in issue and the population genetic structure is still unclear. Microsatellite markers isolated from the silver crucian carp and their utility in the crucian carp will be useful for these researches.     

Morphological characterization and phylogenetic relationships of Indochinese box turtles—The Cuora galbinifrons complex

The members of the Indochinese box turtle complex, namely Cuora galbinifrons, Cuora bourreti, and Cuora picturata, rank the most critically endangered turtle species on earth after more than three decades of over‐harvesting for food, traditional Chinese medicine, and pet markets. Despite advances in molecular biology, species boundaries and phylogenetic relationships, the status of the C. galbinifrons complex remains unresolved due to the small number of specimens observed and collected in the field. In this study, we present analyses of morphologic characters as well as mitochondrial and nuclear DNA data to reconstruct the species boundaries and systematic relationships within the C. galbinifrons complex. Based on principal component analysis (PCA) and statistical analysis, we found that phenotypic traits partially overlapped among galbinifrons, bourreti, and picturata, and that galbinifrons and bourreti might be only subspecifically distinct. Moreover, we used the mitochondrial genome, COI, and nuclear gene Rag1 under the maximum likelihood criteria and Bayesian inference criteria to elucidate whether C. galbinifrons could be divided into three separate species or subspecies. We found strong support for a sister relationship between picturata and the other two species, and consequently, we recommend maintaining picturata as a full species, and classifying bourreti and galbinifrons as subspecies of C. galbinifrons. These findings provide evidence for a better understanding of the evolutionary histories of these critically endangered turtles.