Comparative studies of the population genetic structure of the Brachionus calyciflorus species complex from four inland lakes in Wuhu,China

To study how the population genetic structure in zooplankton respond to environmental conditions, using comparative limnology, the genetic diversity and genetic differentiation of the B. calyciflorus complex collected from four inland lakes in Wuhu City, China were investigated based on the 16S rRNA gene and nuDNA ITS sequences. The results displayed a high genetic diversity, and the nucleotide diversity of the 16S rRNA gene was higher than that of the ITS sequence. The phylogenetic analyses grouped the four populations into two cryptic species (Bc-JT and Bc-FL) with strong support. The two cryptic species were found in lakes with different trophic levels, demonstrating significant ecological specialization. The origins of clone TW12 were not consistent in the phylogenetic trees between two genetic markers, which might be attributed to the effects of male-mediated gene flow on the phylogenetic relationships of rotifers. The nucleotide diversity of the cryptic species Bc-JT was higher than that of Bc-FL, indicating that eutrophication might decrease the genetic diversity of cryptic species. The total phosphorus concentration in water bodies might be the most important factor affecting the genetic diversity of species.     

Molecular phylogeny and phylogeography of the Cuban cave-fishes of the genus Lucifuga: evidence for cryptic allopatric diversity

Underground environments are increasingly recognized as reservoirs of faunal diversity. Extreme environmental conditions and limited dispersal ability of underground organisms have been acknowledged as important factors promoting divergence between species and conspecific populations. However, in many instances, there is no correlation between genetic divergence and morphological differentiation. Lucifuga Poey is a stygobiotic fish genus that lives in Cuban and Bahamian caves. In Cuba, it offers a unique opportunity to study the influence of habitat fragmentation on the genetic divergence of stygobiotic species and populations. The genus includes four species and one morphological variant that have contrasting geographical distributions. In this study, we first performed a molecular phylogenetic analysis of the Lucifuga Cuban species using mitochondrial and nuclear markers. The mitochondrial phylogeny revealed three deeply divergent clades that were supported by nuclear and morphological characters. Within two of these main clades, we identified five lineages that are candidate cryptic species and a taxonomical synonymy between Lucifuga subterranea and Lucifuga teresinarum. Secondly, phylogeographic analysis using a fragment of the cytochrome b gene was performed for Lucifuga dentata, the most widely distributed species. We found strong geographical organization of the haplotype clades at different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow. At a larger temporal scale, these processes could also explain the diversification and the distribution of the different species.     

Contrasting patterns of population structure and demographic history in cryptic species of Bostrychia intricata (Rhodomelaceae,Rhodophyta) from New Zealand

Spatial patterns of genetic diversity provide insight into the demography and history of species. Morphologically similar but genetically distinct “cryptic” species are increasingly being recognized in marine organisms through molecular analyses. Such species are, on closer inspection, often discovered to display contrasting life histories or occasionally minor morphological differences; molecular tools can thus be useful indicators of diversity. Bostrychia intricata, a marine red alga, is widely distributed throughout the Southern Hemisphere and comprises many cryptic species. We used mitochondrial cytochrome c oxidase I gene sequences to assess the genetic variation, population genetic structure, and demographic history of B. intricata in New Zealand. Our results supported the existence of three cryptic species of B. intricata (N2, N4, and N5) in New Zealand. Cryptic species N4, which was found throughout New Zealand, showed a higher genetic diversity and wider distribution than the other two species, which were only found in the North Island and northern South Island. Our analyses showed low to moderate genetic differentiation among eastern North Island populations for cryptic species N2, but high differentiation among North and South Island populations for N4, suggesting different population structure between these cryptic species. Data also indicated that N2 has recently undergone population expansion, probably since the Last Glacial Maximum (LGM), while the higher genetic diversity in N4 populations suggests persistence in situ through the LGM. The contrasting population structures and inferred demographic histories of these species highlight that life history can vary greatly even among morphologically indistinguishable taxa.     

Unexpected cryptic species diversity in the widespread coral Seriatopora hystrix masks spatial‐genetic patterns of connectivity

Mounting evidence of cryptic species in a wide range of taxa highlights the need for careful analyses of population genetic data sets to unravel within‐species diversity from potential interspecies relationships. Here, we use microsatellite loci and hierarchical clustering analysis to investigate cryptic diversity in sympatric and allopatric (separated by 450 km) populations of the widespread coral Seriatopora hystrix on the Great Barrier Reef. Structure analyses delimited unique genetic clusters that were confirmed by phylogenetic and extensive population‐level analyses. Each of four sympatric yet distinct genetic clusters detected within S. hystrix demonstrated greater genetic cohesion across regional scales than between genetic clusters within regions (<10 km). Moreover, the magnitude of genetic differentiation between different clusters (>0.620 G”_ST) was similar to the difference between S. hystrix clusters and the congener S. caliendrum (mean G”_ST 0.720). Multiple lines of evidence, including differences in habitat specificity, mitochondrial identity, Symbiodinium associations and morphology, corroborate the nuclear genetic evidence that these distinct clusters constitute different species. Hierarchical clustering analysis combined with more traditional population genetic methods provides a powerful approach for delimiting species and should be regularly applied to ensure that ecological and evolutionary patterns interpreted for single species are not confounded by the presence of cryptic species.     

Reproductive isolation and patterns of genetic differentiation in a cryptic butterfly species complex

Molecular studies of natural populations are often designed to detect and categorize hidden layers of cryptic diversity, and an emerging pattern suggests that cryptic species are more common and more widely distributed than previously thought. However, these studies are often decoupled from ecological and behavioural studies of species divergence. Thus, the mechanisms by which the cryptic diversity is distributed and maintained across large spatial scales are often unknown. In 1988, it was discovered that the common Eurasian Wood White butterfly consisted of two species (Leptidea sinapis and Leptidea reali), and the pair became an emerging model for the study of speciation and chromosomal evolution. In 2011, the existence of a third cryptic species (Leptidea juvernica) was proposed. This unexpected discovery raises questions about the mechanisms preventing gene flow and about the potential existence of additional species hidden in the complex. Here, we compare patterns of genetic divergence across western Eurasia in an extensive data set of mitochondrial and nuclear DNA sequences with behavioural data on inter‐ and intraspecific reproductive isolation in courtship experiments. We show that three species exist in accordance with both the phylogenetic and biological species concepts and that additional hidden diversity is unlikely to occur in Europe. The Leptidea species are now the best studied cryptic complex of butterflies in Europe and a promising model system for understanding the formation of cryptic species and the roles of local processes, colonization patterns and heterospecific interactions for ecological and evolutionary divergence.     

Genetic diversity and biogeography of red turpentine beetle Dendroctonus valens in its native and invasive regions

Sequences of 479 bp region of the mitochondrial COI gene were applied to detect population genetic diversity and structure of Dendroctonus valens populations. By comparing the genetic diversity between native and invasive populations, it was shown that the genetic diversity of Chinese populations was obviously lower than that of native populations with both indices of haplotype diversity and Nei's genetic diversity, suggesting genetic bottleneck occurred in the invasive process of D. valens , and was then followed by a relatively quick population buildup. According to phylogenetic analyses of haplotypes, we suggested that the origin of the Chinese population was from California, USA. Phylogenetic and network analysis of native populations of D. valens revealed strong genetic structure at two distinct spatial and temporal scales in North America. The main cause resulting in current biogeographic pattern was supposedly due to recycled glacial events. Meanwhile, a cryptic species might exist in the Mexican and Guatemalan populations.     

First evidence of cryptic species diversity and significant population structure in a widespread freshwater nematode morphospecies (Tobrilus gracilis)

Free‐living nematodes are ubiquitous and highly abundant in terrestrial and aquatic environments, where they sustain ecosystem functioning by mineralization processes and nutrient cycling. Nevertheless, very little is known about their true diversity and intraspecific population structure. Recent molecular studies on marine nematodes indicated cryptic diversity and strong genetic differentiation of distinct populations, but for freshwater nematode species, analogous studies are lacking. Here, we present the first extensive molecular study exploring cryptic species diversity and genetic population structure of a widespread freshwater nematode morphospecies, Tobrilus gracilis, from nine postglacially formed European lakes. Taxonomic species status of individuals, analysed for fragments of the mitochondrial COI gene and for the large (LSU) and small (SSU) ribosomal subunits, were determined by morphological characteristics. Mitochondrial and nuclear markers strongly supported the existence of three distinct genetic lineages (Tg I–III) within Tobrilus gracilis, suggesting that this morphospecies indeed represents a complex of highly differentiated biological species. High genetic diversity was also observed at the population level. Across the nine lakes, 19 mitochondrial, and seven (LSU) and four (SSU) nuclear haplotypes were determined. A phylogeographical analysis revealed remarkable genetic differentiation even among neighbouring lake populations for one cryptic lineage. Priority and persistent founder effects are possible explanations for the observed population structure in the postglacially colonized lakes, but ask for future studies providing direct estimates of freshwater nematode dispersal rates. Our study suggests therefore that overall diversity of limnetic nematodes has been so far drastically underestimated and challenges the assumed ubiquitous distribution of other, single freshwater nematode morphospecies.     

In and out of refugia: historical patterns of diversity and demography in the North American Caesar's mushroom species complex

Some of the effects of past climate dynamics on plant and animal diversity make‐up have been relatively well studied, but to less extent in fungi. Pleistocene refugia are thought to harbour high biological diversity (i.e. phylogenetic lineages and genetic diversity), mainly as a product of increased reproductive isolation and allele conservation. In addition, high extinction rates and genetic erosion are expected in previously glaciated regions. Some of the consequences of past climate dynamics might involve changes in range and population size that can result in divergence and incipient or cryptic speciation. Many of these dynamic processes and patterns can be inferred through phylogenetic and coalescent methods. In this study, we first delimit species within a group of closely related edible ectomycorrhizal Amanita from North America (the American Caesar's mushrooms species complex) using multilocus coalescent‐based approaches; and then address questions related to effects of Pleistocene climate change on the diversity and genetics of the group. Our study includes extensive geographical sampling throughout the distribution range, and DNA sequences from three nuclear protein‐coding genes. Results reveal cryptic diversity and high speciation rates in refugia. Population sizes and expansions seem to be larger at midrange latitudes (Mexican highlands and SE USA). Range shifts are proportional to population size expansions, which were overall more common during the Pleistocene. This study documents responses to past climate change in fungi and also highlights the applicability of the multispecies coalescent in comparative phylogeographical analyses and diversity assessments that include ancestral species.     

Plitvice Lakes National park harbors ancient,yet endangered diversity of trout (genus Salmo)

National park Plitvice Lakes is the oldest, the largest and the most visited national park in Croatia, well known for its stunning karstic watersheds that supply Plitvice Lakes and exceptionally valuable biodiversity. Lack of knowledge on the taxonomic status, population structure and viability of trout in the Plitvice Lakes National park, as well as the possibility of existence of cryptic diversity, motivated this study which aimed to determine: taxonomic status and phylogenetic relationship of Plitvice Lakes trout with other Salmo species, genetic structure and diversity of their populations, as well as future viability of populations. 150 samples of trout were obtained from 17 localities. Two mitochondrial gene markers, gene for cytochrome b and mitochondrial control region were used in phylogenetic reconstruction, as well as in population genetic investigation. Intraspecific and intrapopulation genetic diversity was described by calculating several measures of genetic polymorphism, while gene flow among populations was estimated as the migration rates and as the number of immigrants per generation. Phylogenetic trees revealed that Plitvice Lakes watershed is inhabited by two evolutionary independent units of the genus Salmo, representing two species, Salmo labrax Pallas, 1814 and Salmo trutta Linnaeus, 1758. Possibly ‘pure’ populations of both species were found alongside several admixed ones in which both species are present. Our data has shown low intraspecific and intrapopulational diversities for Plitvice Lakes trout and restricted gene flow. Small effective sizes of populations and weak interconnections between them, which are most likely a consequence of fragmentation and habitat degradation, accentuating the need for urgent conservation measures.     

Multilocus phylogeography of the sea snake Hydrophis curtus reveals historical vicariance and cryptic lineage diversity

The Indo‐Australian archipelago (IAA) supports the world's highest diversity of marine fish, invertebrates and reptiles. Many of the marine fish and invertebrates show congruent phylogeographic patterns, supporting a view that the region's complex geo‐climatic history has played an important role in generating its exceptional biodiversity. Here, we examine population genetic structure of the viviparous sea snake, Hydrophis curtus, to assess how past and present barriers to gene flow in the IAA have contributed to genetic and species diversity in a fully marine reptile. Mitochondrial and anonymous nuclear sequences and ten microsatellite loci were used to identify patterns of historical genetic structure and population expansion, reconstruct dated genealogies and assess levels of recent gene flow. These markers revealed strong concordant geographic structure within H. curtus with a prominent genetic break between populations broadly distributed in the Indian Ocean and the West Pacific. These populations were estimated to have diverged in the late Pliocene or early Pleistocene, and microsatellite admixture analyses suggested limited recent gene flow between them despite the current lack of barriers to dispersal, indicating possible cryptic species. Subsequent divergence in the mid–late Pleistocene was detected within the West Pacific clade among the populations in the Phuket‐Thailand region, South‐East Asia and Australia, and two of these populations also showed genetic signals of recent range expansions. Our results show that climatic fluctuations during the Plio‐Pleistocene generated high levels of cryptic genetic diversity in H. curtus, and add to similar findings for diverse other marine groups in the IAA.     

Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda,Leptosomatidae)

Derycke, S., De Ley, P., De Ley, I.T., Holovachov, O., Rigaux, A. & Moens, T. (2010). Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae).—Zoologica Scripta, 39, 276–289. Recent taxonomic and population genetic studies have revealed the presence of substantial cryptic diversity through sequence analysis of nematode morphospecies classified in different major clades. Correct interpretations of intra‐ and interspecific genetic variation require certainty about the conspecificity of the sequenced specimens, which in turn must depend on appropriate protocols with built‐in verifiability procedures. In this study, we performed a population genetic study in the free‐living marine nematode Thoracostoma trachygaster, a member of one of the earliest major clades to diverge in nematode phylogeny. We collected 367 nematodes from 11 populations located in the Californian Bight, all of which were video captured before DNA extraction to document and verify their individual morphology. Sequences for the cytochrome c oxidase subunit 1 (COI), D2D3 and 18S genes showed eight deeply divergent clades, and using a reverse taxonomy approach, six of these clades proved to be other morphospecies than T. trachygaster. Phylogenetic analyses of COI, internal transcribed spacer and D2D3 showed evidence for two sympatrically distributed cryptic species within the morphospecies T. trachygaster. Population genetic analyses of the most widespread cryptic species showed a moderate genetic structuring (Φ_ST = 0.28), and 18% of this genetic variation was caused by differences between populations north and south of Point Conception. Within the southern Californian Bight, some genetic differentiation could be attributed to differences between populations north and south of Malibu, supporting the idea of a barrier to gene flow near Los Angeles region. The results for T. trachygaster support the contention that species diversity within free‐living nematodes is underestimated, and that dispersal of marine nematodes from tidal environments associated with kelp holdfasts is substantial at scales of a few 100 km.     

Low genetic diversity in the vulnerable Goitred Gazelle,Gazella subgutturosa (Cetartiodactyla: Bovidae), in Iran: potential genetic consequence of recent population declines

The Goitered Gazelle, Gazella subgutturosa, is the most widespread gazelle species in the Middle East and central Asia inhabiting desert and semi-desert habitats. Today it is threatened and its geographic range and population size have experienced significant decline in the last decades. In Iran, the remnant populations are confined to fragmented habitats. We aimed to characterise genetic diversity and phylogenetic status of the populations of Goitered Gazelle in Central Iran and to evaluate the potential effect of a historic population bottleneck on the genetic variation of today’s population. We used noninvasive sampling to uncover structure and level of genetic variation in a fragment of the cytochrome-b gene from 170 samples. Genealogical analyses were performed using HKY+I model and phylogenetic trees reconstructed using Bayesian inference and maximum likelihood. We found extremely low levels of genetic variation, with altogether only five haplotypes in samples from different populations. Overall haplotype diversity was 0.081 and nucleotide diversity 0.0003. The mean observed mismatch between any two sequences was 0.093 with the largest peak for small numbers. The mismatch distribution fit the model of population expansion and suggested that gazelles had experienced a sudden expansion. An unrooted median-joining network analysis of mtDNA haplotypes showed a star-like structure which few mutations steps separating the haplotypes from other regions. Our findings strengthen the urgency of preserving the species’ genetic diversity to prevent local extinction.     

Phylogeography and sexual macrocyst formation in the social amoeba <Emphasis Type="Italic">Dictyostelium giganteum</Emphasis>

Background  

Microorganisms are ubiquitous, yet we are only beginning to understand their diversity and population structure. Social amoebae (Dictyostelia) are a diverse group of unicellular eukaryotic microbes that display a unique social behaviour upon starvation in which cells congregate and then some die to help others survive and disperse. The genetic relationships among co-occurring cells have a major influence on the evolution of social traits and recent population genetic analysis found extensive genetic variation and possible cryptic speciation in one dictyostelid species (Dictyostelium purpureum). To further characterize the interplay among genetic variation, species boundaries, social behaviour, and reproductive isolation in the Dictyostelia, we conducted phylogenetic analyses and mating experiments with the geographically widespread social amoeba Dictyostelium giganteum.     

Ecological Differentiation of Cryptic Species within an Asexual Protist Morphospecies: A Case Study of Filamentous Green Alga Klebsormidium (Streptophyta)

Taxa of microbial eukaryotes defined on morphological basis display a large degree of genetic diversity, implying the existence of numerous cryptic species. However, it has been postulated that genetic diversity merely mirrors accumulation of neutral mutations. As a case taxon to study cryptic diversity in protists, we used a widely distributed filamentous genus, Klebsormidium, specifically the lineage E (K. flaccidum/K. nitens complex) containing a number of morphologically similar strains. Fourteen clades were recognized in the phylogenetic analysis based on a concatenated ITS rDNA + rbcL data set of more than 70 strains. The results of inferred character evolution indicated the existence of phylogenetic signal in at least two phenotypic characters (production of hydro‐repellent filaments and morphology of zoosporangia). Moreover, the lineages recovered exhibited strong ecological preferences to one of the three habitat types: natural subaerial substrata, artificial subaerial substrata, and aquatic habitats. We interpret these results as evidence of existence of a high number of cryptic species within the single morphospecies. We consider that the permanent existence of genetically and ecologically well‐defined cryptic species is enabled by the mechanism of selective sweep.     

Integrative species delimitation reveals cryptic diversity in the southern Appalachian Antrodiaetus unicolor (Araneae: Antrodiaetidae) species complex

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth's quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, the Antrodiaetus unicolor species complex containing two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and use multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include structure , principal components analysis and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large‐scale ecological data for niche‐based distribution modelling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic‐scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.     

BIODIVERSITY RESEARCH: Genetic diversity in two introduced biofouling amphipods (Ampithoe valida & Jassa marmorata) along the Pacific North American coast: investigation into molecular identification and cryptic diversity

Aim We investigated patterns of genetic diversity among invasive populations of Ampithoe valida and Jassa marmorata from the Pacific North American coast to assess the accuracy of morphological identification and determine whether or not cryptic diversity and multiple introductions contribute to the contemporary distribution of these species in the region. Location Native range: Atlantic North American coast; Invaded range: Pacific North American coast. Methods We assessed indices of genetic diversity based on DNA sequence data from the mitochondrial cytochrome c oxidase subunit I (COI) gene, determined the distribution of COI haplotypes among populations in both the invasive and putative native ranges of A. valida and J. marmorata and reconstructed phylogenetic relationships among COI haplotypes using both maximum parsimony and Bayesian approaches. Results Phylogenetic inference indicates that inaccurate species‐level identifications by morphological criteria are common among Jassa specimens. In addition, our data reveal the presence of three well supported but previously unrecognized clades of A. valida among specimens in the north‐eastern Pacific. Different species of Jassa and different genetic lineages of Ampithoe exhibit striking disparity in geographic distribution across the region as well as substantial differences in genetic diversity indices. Main conclusions Molecular genetic methods greatly improve the accuracy and resolution of identifications for invasive benthic marine amphipods at the species level and below. Our data suggest that multiple cryptic introductions of Ampithoe have occurred in the north‐eastern Pacific and highlight uncertainty regarding the origin and invasion histories of both Jassa and Ampithoe species. Additional morphological and genetic analyses are necessary to clarify the taxonomy and native biogeography of both amphipod genera.     

Symbiodinium population genetics: testing for species boundaries and analysing samples with mixed genotypes

Population genetic markers are increasingly being used to study the diversity, ecology and evolution of Symbiodinium, a group of eukaryotic microbes that are often mutualistic with reef‐building corals. Population genetic markers can resolve individual clones, or strains, from samples of host tissue; however, samples may comprise different species that may confound interpretations of gene flow and genetic structure. Here, we propose a method for resolving species from population genetic data using tests for genetic recombination. Assigning individuals to genetically recombining populations prior to further analyses avoids critical errors in the interpretation of gene flow and dispersal. To demonstrate the effectiveness of the approach, we first apply this method to a simulated data set. We then use the method to resolve two species of host generalist Symbiodinium that commonly co‐occur in reef‐building corals collected from Indo‐West Pacific reefs. We demonstrate that the method is robust even when some hosts contain genotypes from two distinct species. Finally, we examine population genetic data sets from two recently published papers in Molecular Ecology. We show that each strongly supports a two species interpretation, which significantly changes the original conclusions presented in these studies. When combined with available phylogenetic and ecological evidence, the use of population genetic data offers a robust method for unambiguously delimiting morphologically cryptic species.     

Population structuring in the monogonont rotifer Synchaeta pectinata: high genetic divergence on a small geographical scale

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Integrating multiple analytical approaches to spatially delineate and characterize genetic population structure: an application to boreal caribou (<Emphasis Type="Italic">Rangifer tarandus caribou</Emphasis>) in central Canada

Individual-based clustering (IBC) methods have become increasingly popular for the characterization and delineation of genetic population units for numerous species. These methods delineate populations based on the genetic assumptions of a breeding unit which may provide a better representation of the behaviour of the species. The increasing use of IBC has resulted in the development of several analytical models all of which vary in their theoretical assumptions to infer genetic population structure. In this paper, we report a comparative strategy utilizing three IBC methods to characterize the spatial genetic structure of the boreal population of woodland caribou (Rangifer tarandus caribou) in central Canada. In addition, we implement both tests for isolation-by-distance (IBD) and frequency-based assignment tests to validate the consensus genetic clusters as defined by IBC. We also compare indirect metrics of genetic diversity and gene flow using both a priori defined herds and the IBC defined populations. Although our results show some concordance between both pre-defined herds and IBC derived genetic clusters, the IBC analyses identified a cluster that was cryptic to observation-based caribou herds and found no difference between several adjacent herds. By comparing multiple IBC methods and integrating both IBD and indirect genetic diversity metrics a posteriori, our strategy provides an effective means to delineate wildlife population structure and accurately assess genetic diversity and connectivity.     

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.