Contrasting Gene Decay in Subterranean Vertebrates: Insights from Cavefishes and Fossorial Mammals.

Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxes functional constraints. Subterranean vertebrates are outstanding models to analyze this process, and gene decay can serve as a readout. We sought to understand some general principles on the extent and tempo of the decay of genes involved in vision, circadian clock, and pigmentation in cavefishes. The analysis of the genomes of two Cuban species belonging to the genus Lucifuga provided evidence for the largest loss of eye-specific genes and nonvisual opsin genes reported so far in cavefishes. Comparisons with a recently evolved cave population of Astyanax mexicanus and three species belonging to the Chinese tetraploid genus Sinocyclocheilus revealed the combined effects of the level of eye regression, time, and genome ploidy on eye-specific gene pseudogenization. The limited extent of gene decay in all these cavefishes and the very small number of loss-of-function mutations per pseudogene suggest that their eye degeneration may not be very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several vision genes carrying many loss-of-function mutations in ancient fossorial mammals, further suggesting that blind fishes cannot thrive more than a few million years in cave ecosystems.

Phylogeographic evidence that the distribution of cryptic euryhaline species in the Gambusia punctata species group in Cuba was shaped by the archipelago geological history.

The main drivers of diversification of freshwater fishes in Cuba are not yet well understood. For example, salt tolerance was thought as the main factor involved in the diversification of Gambusia punctata species group in this archipelago. However, evidence from a recent DNA barcoding survey suggested the presence of cryptic species and no correlation between species delimitation and level of salinity. In this study, we analyzed the cryptic diversification of G. punctata species group in Cuba, based on a comprehensive sampling of its distribution and including habitats with different salinity levels. We evaluated the patterns of molecular divergence of the samples by sequencing a set of mitochondrial DNA (mtDNA) regions and genotyping nine nuclear microsatellite loci. We also used cytochrome b gene (cytb) partial sequences and these microsatellite loci to analyze population structure inside putative species. Five mtDNA well-differentiated haplogroups were found, four of them also identified by the analysis of the microsatellite polymorphism which corresponds to two already recognized species, G. punctata, and G. rhizophorae, and three putative new species. The extent of hybrid zones between these groups is also described. In each group, populations inhabiting environments with contrasting salinity levels were identified, indicating a generalized trait not specific to G. rhizophorae. The geographic distribution of the groups suggested a strong association with major relict territories of the Cuban Archipelago that was periodically joined or split-up by changes in seawater levels and land uplifts. Salinity tolerance might have facilitated sporadic and long-distance oversea dispersal but did not prevent speciation in the Cuban archipelago.

Phylogeography of Cuban Rivulus: evidence for allopatric speciation and secondary dispersal across a marine barrier.

The genus Rivulus is currently comprised of two species, R. cylindraceus and R. insulaepinorum, which are endemic to Cuba. However, the taxonomic status of the latter species remains dubious because of the poor quality of the original description. In addition, a recent barcoding survey suggests that the two species may be conspecific. The aim of this study was to test the hypothesis that the two species represent a single evolutionary clade. To delimit the species and their evolutionary history, we used a combination of molecular phylogenetic analyses, with both mitochondrial and nuclear sequences, tests of phylogeographic hypotheses, combined with morphological measurements and information on known dispersal barriers and species distribution. None of the data sets support R. insulaepinorum and R. cylindraceus as separate taxa. However, a new species, restricted to the northwestern part of the main island, was identified by phylogenetic analyses, body colour pattern and geographical distribution. The evolutionary distance between the two lineages (cytb, d=15%; CAM-4, d=2.5%) indicates a long period of divergence. Phylogeographic analyses shed light on the dispersal history of R. cylindraceus, which probably originated on the Isla de la Juventud. They also suggest that each lineage had contrasting histories; Rivulus sp. is restricted to a relatively small geographic area whereas R. cylindraceus has dispersed considerably and more than once from its centre of origin, probably facilitated by sea level fluctuations. These results strengthen previous findings, i.e. that the diversity of Cuban freshwater fishes is far from well-known and deserves more in-depth studies, and that vicariance and dispersal events have resulted in a complex biogeographical landscape which has had a significant impact on the freshwater fishes of the Caribbean islands.

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.

A new species of the cave-fish genus Lucifuga (Ophidiiformes, Bythitidae), from eastern Cuba.

Recently, a barcoding study and a molecular phylogenetic analysis of the Cuban species of the cave-fish genus Lucifuga Poey, 1858 revealed the existence of different evolutionary lineages that were previously unknown or passed unnoticed by morphological scrutiny (i.e., cryptic candidate species). In the present study, Lucifuga gibarensis is described as a new species restricted to anchialine caves in the northeastern karst region of the main island. The species was earlier described as a variety of Lucifuga dentata, but since the name was introduced as a variety after 1960, it is deemed to be infrasubspecific and unavailable according to the International Code of Zoological Nomenclature Art. 15.2. The new species differs from L. dentata by pigmented eyes vs. eyes absent and lack of palatine teeth vs. present. Lucifuga gibarensis seems to be most similar to the Bahamian species L. lucayana by showing pigmented eyes, 13 or 14 precaudal vertebrae and ten caudal fin rays. However, differs from it by a larger size of the pigmented eye (1.1-1.9 vs. 0.9-1.0% SL) and number of posterior lateral line neuromasts (30-33 vs. 34-35).

The complete mitochondrial DNA of the Cuban gar (Atractosteus tristoechus).

The Cuban gar (Atractosteus tristoechus) is an endemic lepisosteid living in Cuba. Among gars, this species is one of the most threatened and has the smallest natural distribution range. Lepisosteids are air-breathing fishes belonging to the Holostean, a basal non-teleost clade of actinopterygians. Recent studies have indicated that these fishes could be a 'bridge between tetrapods and teleost biomedical models'. Herein, we sequenced and assembled the first complete mitochondrial genome of A. tristoechus. The total length of the mt genome is 16,290 bp, containing the typical 13 protein-coding genes, two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a 537 bp length control region.

Phylogeny and biogeography of the scaleless scale worm Pisione (Sigalionidae, Annelida).

Pisione is a scaleless group of small scale worms inhabiting sandy bottoms in shallow marine waters. This group was once considered rare, but now 45 described species can be characterized, among others, by their paired, segmental copulatory organs (one to multiple external pairs), which display a complexity of various accessory structures. The evolutionary significance of these unique organs was suggested in the late 1960s, but has been heavily debated since the late 1990s and remains controversial. In the present paper, we study the internal relationships within Pisione, employing combined phylogenetic analyses of both molecular and morphological data from 16 terminals of Pisione, as well as two terminals of Pisionidens, and eight additional scale worms as outgroups. Our taxon sampling covers all geographical areas where the genus has been reported, as well as most of their morphological and copulatory variability, including representatives of the "africana," "remota," "crassa," and "papuensis" groups, established previously by Yamanishi. We hereby provide a first insight into the relationships of the genus, testing previously proposed hypotheses on the evolutionary significance of male copulatory structures within Pisione, while attempting to understand patterns of distribution. The phylogenetic analyses using maximum likelihood and Bayesian methods consistently recovered two large clades spanning the East Atlantic (including the Mediterranean) and the Indo-Pacific-West Atlantic, respectively. Character optimization on our trees revealed a high degree of homoplasy in both non-reproductive and sexual characters of Pisione, with buccal acicula found to be the sole apomorphy among the morphological features assessed herein, with none defining the biogeographical subclades within. Overall, our comparative analyses highlight the high degree of morphological variation in this widely distributed genus, rejecting previous assertions of an increasing number and complexity of copulatory structures across the genus.

Go West: A One Way Stepping-Stone Dispersion Model for the Cavefish Lucifuga dentata in Western Cuba.

Consistent with the limited dispersal capacity of most troglobitic animals, almost all Lucifuga cavefish species have very narrow geographic distribution in Cuba. However, one species, L. dentata, has a wide but disjointed distribution over 300 km in the west of the island. In order to estimate the relative role of vicariance and dispersal in the unexpected L. dentata distribution, we obtained partial sequences of the mitochondrial DNA (mtDNA) cytochrome b (cytb) gene and control region (CR), and then applied Approximate Bayesian Computation (ABC), based on the identification of five genetic and geographic congruent groups of populations. The process that best explains the distribution of genetic diversity in this species is sequential range expansion from east Matanzas to the western Pinar del Río provinces, followed by isolation of groups of populations. We found relative high haplotype diversity and low nucleotide diversity in all but the Havana group, which has high values for both diversity parameters, suggesting that this group has been demographically stable over time. For two groups of populations (Cayuco and Bolondrón), the mismatch distribution analyses suggests past demographic expansion. In the case of the Cayuco region, the star like relationships of haplotypes in the network suggests a recent founding event, congruent with other evidence indicating that this is the most recently colonized region. Over all, the results suggest that a combination of habitat availability, temporal interconnections, and possibly the biological properties of this species, may have enabled its dispersal and range expansion compared to other species of the genus, which are more geographically restricted.

Genetic multipartitions based on D-Loop sequences and chromosomal patterns in brown chromis, Chromis multilineata (Pomacentridae), in the Western Atlantic.

Connectivity levels among Brazilian reef fish fauna populations have attracted growing interest, mainly between mainland shores and oceanic islands. The Pomacentridae, whose phylogeographic patterns are largely unknown in the Atlantic, are a family of dominant fish in reef regions. We present data on the variability and population structure of damselfish Chromis multilineata in different areas along the northeast coast of Brazil and in the waters around the oceanic islands of Fernando de Noronha (FNA) and Saint Peter and Saint Paul Archipelago (SPSPA) through analysis of the HVR1 mtDNA sequence of the control region. The remote SPSPA exhibits the highest level of genetic divergence among populations. Conventional and molecular cytogenetic analysis showed similar karyotype patterns (2n=48 acrocentrics) between these insular areas. Our estimates reveal three genetically different population groups of C. multilineata on the Brazilian coast. The level of genetic structure is higher than previous data suggested, indicating complex panel of interactions between the oceanic island and coastal populations of Brazil.

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.

Spatiotemporal genetic differentiation of Cuban natural populations of the pink shrimp Farfantepenaeus notialis.

We analyzed the spatiotemporal genetic structure of Farfantepenaeus notialis populations using five microsatellites loci in order to understand the influence of natural events such as hurricanes on the genetic drift/migration balance as the main cause for the variation of allele frequencies over time. The results were compared with the previous ones obtained from allozymes and mtDNA. High and stable genetic diversity levels (He=0.879+/-0.0015) were found over eight years for the populations that inhabit the south Cuban platform, however significant changes of allele frequencies were detected over time. The F(ST) estimates, albeit low, revealed significant differences among populations inside the Ana Maria Gulf for 1995 but not for the 1999 and 2003 samples. The F(ST), AMOVA and the genetic distance analysis revealed the instability of the genetic structure over time in accordance with allozymes results. The correspondence of the microsatellite results with those obtained from allozymes confirm the effects of migration enhanced by natural events as the main cause of the temporal variation of allele frequencies. The genetic drift effect was discarded through the evaluation of Ne and the M ratio, while natural selection effects were rejected because of the lowest probability of microsatellite loci being under selective pressures. The microsatellite data are also consistent with the results obtained with mtDNA in detecting significant and persistent genetic differences between the Gulfs of Ana María and Batabanó for the years 1995 and 2003.

Temporal variation of the population structure and genetic diversity of Farfantepenaeus notialis assessed by allozyme loci.

Population genetic studies carried out on penaeid shrimps have disclosed different patterns of population subdivision, revealing new aspects of shrimp biology as well as the effects of historical contingency molding those patterns. However, the stability of observed allele frequencies over time still remains untested. The objective of this article is to show the analysis of the temporal variation of allozymes in a shrimp species inhabiting Cuba which proves that the genetic structure of this species could significantly change in time. The study involves four populations of Farfantepenaeus notialis sampled in a period of 8 years. The significant statistics obtained from partitions observed in 1995 were not detected in 2003 (as suggested by AMOVA and F(ST)), whereas temporal genetic differentiation and heterozygosity became highly significant. The results strongly suggest that the effect of migrations could be the cause for the loss of F. notialis genetic structure in 2003. It is therefore imperative to call attention on the vulnerability of these populations when facing unstable environmental and habitat conditions.