Genomic Signatures Associated with Transitions to Viviparity in Cyprinodontiformes.

The transition from oviparity to viviparity has occurred independently over 150 times across vertebrates, presenting one of the most compelling cases of phenotypic convergence. However, whether the repeated, independent evolution of viviparity is driven by redeployment of similar genetic mechanisms and whether these leave a common signature in genomic divergence remains largely unknown. Although recent investigations into the evolution of viviparity have demonstrated striking similarity among the genes and molecular pathways involved across disparate vertebrate groups, quantitative tests for genome-wide convergent have provided ambivalent answers. Here, we investigate the potential role of molecular convergence during independent transitions to viviparity across an order of ray-finned freshwater fish (Cyprinodontiformes). We assembled de novo genomes and utilized publicly available genomes of viviparous and oviparous species to test for molecular convergence across both coding and noncoding regions. We found no evidence for an excess of molecular convergence in amino acid substitutions and in rates of sequence divergence, implying independent genetic changes are associated with these transitions. However, both statistical power and biological confounds could constrain our ability to detect significant correlated evolution. We therefore identified candidate genes with potential signatures of molecular convergence in viviparous Cyprinodontiformes lineages. Motif enrichment and gene ontology analyses suggest transcriptional changes associated with early morphogenesis, brain development, and immunity occurred alongside the evolution of viviparity. Overall, however, our findings indicate that independent transitions to viviparity in these fish are not strongly associated with an excess of molecular convergence, but a few genes show convincing evidence of convergent evolution.

Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation.

Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.

Congenital feeding response to a novel prey in a Mexican gartersnake.

In this study, we explored chemosensory, ingestive and prey-catching responses of neonate Mexican Black-bellied Gartersnakes (Thamnophis melanogaster) to crayfish (Cambarellus montezumae). By comparing snakes from a recently discovered crayfish-eating population and a typical non-crayfish-eating population, we asked which behavioral components change as a species enlarges its feeding niche. In the crayfish-eating population chemosensory responsiveness to crayfish was not enhanced but its heritability was higher. Neonates of both populations showed similar preference for freshly-molted versus unmolted crayfish, and whereas the tendency to ingest both crayfish stages remained stable between ages 15 and 90 days in the non-crayfish-eating population, in the crayfish-eating population it actually decreased. Techniques to catch and manipulate molted crayfish were similar in the two populations. We discuss the possibility that there is no increase in the behavioral response to eat crayfish by the neonates of the crayfish-eating populations, possibly due to the absence of ecological and spatial isolation between the two T. melanogaster populations. The crayfish ingestion in some population of T. melanogaster can be explained by environmental differences between populations, or by recent origin of crayfish ingestion in T. melanogaster.

Sexually selected sexual selection: Can evolutionary retribution explain female ornamental colour?

By preferring mates with increasingly costly ornaments or courtship displays, females cause an escalation of male reproductive costs. Such increased costs should promote male selectivity based on fecundity-linked female attributes, leading to female ornamentation in species with traditional sex roles. Consequently, female ornamentation should evolve more frequently in taxa where male reproduction is costly than in comparable taxa where it is cheaper. We assessed the prevalence of female ornamental colouration in two clades of viviparous cyprinodontid fish: the Goodeinae, where stringent female choice imposes male mating costs, and the Poeciliinae, whose males can circumvent female mate choice. We found that although in the Poeciliinae female ornamental colour is a correlated, but paler version of male coloration, females of the Goodeinae often display vivid ornamental colours that are distinct from those of males. Thus, male and female ornaments are not (phylo)genetically correlated in the Goodeinae. Furthermore, phylogenetic signal on male and female colour is clearly detectable in the Poeciliinae, but absent in the Goodeinae, suggesting that ornamental colour of males and females in the latter may be the consequence of selection. Given that enforceable female choice has promoted male ornaments, we propose that evolutionary retribution has promoted distinct female ornaments in the Goodeinae.

Morphological convergence in a Mexican garter snake associated with the ingestion of a novel prey.

Morphological convergence is expected when organisms which differ in phenotype experience similar functional demands, which lead to similar associations between resource utilization and performance. To consume prey with hard exoskeletons, snakes require either specialized head morphology, or to deal with them when they are vulnerable, for example, during molting. Such attributes may in turn reduce the efficiency with which they prey on soft-bodied, slippery animals such as fish. Snakes which consume a range of prey may present intermediate morphology, such as that of Thamnophiine (Natricinae), which may be classified morphometrically across the soft-hard prey dietary boundary. In this study, we compared the dentition and head structure of populations of Thamnophis melanogaster that have entered the arthropod-crustacean (crayfish)-eating niche and those that have not, and tested for convergence between the former and two distantly related crayfish specialists of the genus Regina (R. septemvittata and R. grahamii). As a control, we included the congener T. eques. Multivariate analysis of jaw length, head length, head width, and number of maxillary teeth yielded three significant canonical variables that together explained 98.8% of the variance in the size-corrected morphological data. The first canonical variable significantly discriminated between the three species. The results show that head dimensions and number of teeth of the two Regina species are more similar to those of crayfish-eating T. melanogaster than to non-crayfish-eating snakes or of T. eques. It is unclear how particular head proportions or teeth number facilitates capture of crayfish, but our results and the rarity of soft crayfish ingestion by T. melanogaster may reflect the novelty of this niche expansion, and are consistent with the hypothesis that some populations of T. melanogaster have converged in their head morphology with the two soft crayfish-eating Regina species, although we cannot rule out the possibility of a morphological pre-adaptation to ingest crayfish.

Asymmetric paternal effect on offspring size linked to parent-of-origin expression of an insulin-like growth factor.

Sexual reproduction brings together reproductive partners whose long-term interests often differ, raising the possibility of conflict over their reproductive investment. Males that enhance maternal investment in their offspring gain fitness benefits, even if this compromises future reproductive investment by iteroparous females. When the conflict occurs at a genomic level, it may be uncovered by crossing divergent populations, as a mismatch in the coevolved patterns of paternal manipulation and maternal resistance may generate asymmetric embryonic growth. We report such an asymmetry in reciprocal crosses between populations of the fish Girardinichthys multiradiatus. We also show that a fragment of a gene which can influence embryonic growth (Insulin-Like Growth Factor 2; igf2) exhibits a parent-of-origin methylation pattern, where the maternally inherited igf2 allele has much more 5' cytosine methylation than the paternally inherited allele. Our findings suggest that male manipulation of maternal investment may have evolved in fish, while the parent-of-origin methylation pattern appears to be a potential candidate mechanism modulating this antagonistic coevolution process. However, disruption of other coadaptive processes cannot be ruled out, as these can lead to similar effects as conflict.

Pre- and post-experimental manipulation assessments confirm the increase in number of birds due to the addition of nest boxes.

Secondary cavity nesting (SCN) birds breed in holes that they do not excavate themselves. This is possible where there are large trees whose size and age permit the digging of holes by primary excavators and only rarely happens in forest plantations, where we expected a deficit of both breeding holes and SCN species. We assessed whether the availability of tree cavities influenced the number of SCNs in two temperate forest types, and evaluated the change in number of SCNs after adding nest boxes. First, we counted all cavities within each of our 25-m radius sampling points in mature and young forest plots during 2009. We then added nest boxes at standardised locations during 2010 and 2011 and conducted fortnightly bird counts (January-October 2009-2011). In 2011 we added two extra plots of each forest type, where we also conducted bird counts. Prior to adding nest boxes, counts revealed more SCNs in mature than in young forest. Following the addition of nest boxes, the number of SCNs increased significantly in the points with nest boxes in both types of forest. Counts in 2011 confirmed the increase in number of birds due to the addition of nest boxes. Given the likely benefits associated with a richer bird community we propose that, as is routinely done in some countries, forest management programs preserve old tree stumps and add nest boxes to forest plantations in order to increase bird numbers and bird community diversity.

Spatial and temporal variation in superfoetation and related life history traits of two viviparous fishes: Poeciliopsis gracilis and P. infans.

Superfoetation is the ability of females to simultaneously bear multiple broods of embryos at different developmental stages. Most studies on the phylogenetic distribution of superfoetation and on the factors that potentially promote superfoetation ignore variation within species. Here, we studied 11 populations of two species of viviparous fishes of the family Poeciliidae (Poeciliopsis gracilis and Poeciliopsis infans) and document wide variation in superfoetation and in three related life history traits: brood size, individual embryo mass and total reproductive allotment. We found significant differences in the average number of simultaneous broods among populations of P. gracilis but not among populations of P. infans. In addition, we found even greater variation between months within populations for both species, although no specific pattern of temporal variation was evident. Instead of the expected consistency of seasonal differences in superfoetation across populations, we found that large variation among months within seasons and the amount and direction of this monthly variation differed widely between populations. Our results emphasize the importance of including intraspecific variation in superfoetation and other life history traits in studies that aimed at finding general explanations of life history trait evolution.

Geographic variation in the advertisement calls of Hyla eximia and its possible explanations.

Populations of species occupying large geographic ranges are often phenotypically diverse as a consequence of variation in selective pressures and drift. This applies to attributes involved in mate choice, particularly when both geographic range and breeding biology overlap between related species. This condition may lead to interference of mating signals, which would in turn promote reproductive character displacement (RCD). We investigated whether variation in the advertisement call of the mountain treefrog (Hyla eximia) is linked to geographic distribution with respect to major Mexican river basins (Panuco, Lerma, Balsas and Magdalena), or to coexistence with its sister (the canyon treefrog, Hyla arenicolor) or another related species (the dwarf treefrog, Tlalocohyla smithii). We also evaluated whether call divergence across the main river basins could be linked to genetic structure. We found that the multidimensional acoustic space of calls from two basins where H. eximia currently interacts with T. smithii, was different from the acoustic space of calls from H. eximia elsewhere. Individuals from these two basins were also distinguishable from the rest by both the phylogeny inferred from mitochondrial sequences, and the genetic structure inferred from nuclear markers. The discordant divergence of H. eximia advertisement calls in the two separate basins where its geographic range overlaps that of T. smithii can be interpreted as the result of two independent events of RCD, presumably as a consequence of acoustic interference in the breeding choruses, although more data are required to evaluate this possibility.

Female vibration discourages male courtship behaviour in the Amarillo fish (Girardinichthys multiradiatus).

Amarillo fish females (Girardinichthys multiradiatus) vibrate when conspecific males approach them; the reason behind this behaviour is unclear. Hypotheses are that females vibrate either to avoid aggression from males or to court them. We prevented females from vibrating by temporarily blocking their lateral line organs and eyes, on the assumption that they rely on these senses to detect approaching males. Females with the lateral line organs obstructed vibrated less frequently than females with the lateral line intact, indicating that the mechanosensory lateral line system is necessary for perceiving approaching males. Males displayed more courtship behaviour to sighted females with the lateral line organs obstructed than to sighted females with the lateral line intact. A general tendency indicated that the less the females vibrated the more the males courted them. These findings indicate that female vibration discourages male courtship behaviour.

What do we need to know about speciation?

Speciation has been a major focus of evolutionary biology research in recent years, with many important advances. However, some of the traditional organising principles of the subject area no longer provide a satisfactory framework, such as the classification of speciation mechanisms by geographical context into allopatric, parapatric and sympatry classes. Therefore, we have asked where speciation research should be directed in the coming years. Here, we present a distillation of questions about the mechanisms of speciation, the genetic basis of speciation and the relationship between speciation and diversity. Our list of topics is not exhaustive; rather we aim to promote discussion on research priorities and on the common themes that underlie disparate speciation processes.

Effects of methyl parathion exposure on development and reproduction in the viviparous fish Girardinichthys multiradiatus.

Methyl parathion (MeP), an insecticide commonly used in Mexico, has been detected in water bodies adjacent to agricultural crops and is accumulated in the tissue of the amarillo (Girardinichthys multiradiatus), a viviparous fish unique to Central Mexico. Evidence of MeP accumulation in a G. multiradiatus population prompted assessment of the consequences of exposure to MeP during the gestation period and adult life. We exposed adult fish to MeP in the diet at concentrations ranging from 0.001 to 0.165 microg/g dry weight of food. Fish accumulated MeP at concentrations from 0.21 to 5.87 microg/g of fish and exhibited a significant reduction in weight and growth rate. We also evaluated the effects on broods of pregnant females exposed to 0.005, 0.01, and 0.1 microg/g dry weight of food. Although these pregnant females were not affected, their offspring showed an increase in spinal cord malformation and a reduction in survival to adulthood. Finally, we sampled seven localities inhabited by G. multiradiatus; this revealed the presence of MeP in two water bodies at concentrations of 0.0036 and 0.037 microg/mL respectively. Our results suggest that low MeP concentrations affect immature fish with possible consequences later in life.

Exposure to pesticides impairs the expression of fish ornaments reducing the availability of attractive males.

Ornament magnitude often reflects a local balance between sexual selection and other sources of natural selection opposing their elaboration. Human activity may disrupt this balance if it modifies the costs of producing, maintaining or displaying the ornaments. When costs are increased, a shortage of acceptable partners may ensue, with consequences commensurate with how stringent (and effective) the process of mate choice is. Here, we show that the expression of ornaments in the viviparous amarillo fish (Girardinichthys multiradiatus) is influenced by embryonic exposure to low concentrations of an organophosphorus insecticide. Male ornamental fin size, dimorphic yellow coloration and display rates were all compromised in exposed fish, but unaffected in their paternal half-sibling controls and in their sisters (morphology and colour). Exposure resulted in smaller fish of both sexes, thus the differential effect by sex was restricted to attributes such as fin size only above the naturally selected magnitude shown by females. Father phenotype predicted offspring morphology of controls, but not of exposed males, which were discriminated against by both control and exposed females. Since stringent female mate choice can result in females refusing to mate with suboptimal mates, this sub-lethal developmental effect can reduce the effective population size of amarillo fish populations.

Heterospecific harassment of native endangered fishes by invasive guppies in Mexico.

Persistent courtship by male Trinidadian guppies (Poecilia reticulata) is costly for conspecific females. Since male guppies are known to attempt matings with other poeciliid females, we asked whether persistent courtship is also directed towards morphologically similar but phylogenetically distant females encountered following invasion. Skiffia bilineata is one of several endangered viviparous goodeids from Central México, whose remaining habitats are increasingly shared with invasive guppies. Experiments in which guppy sex ratios were manipulated to vary the proportion of heterospecific to conspecific females showed that male guppies courted and attempted forced copulations with S. bilineata females even when females of their own species were in excess. This behaviour places an additional, and previously unrecognized, burden on a group of endemic Mexican fishes already in risk of extinction.

Phenotypic differentiation and pre-mating isolation between allopatric populations of Girardinichthys multiradiatus.

Sexual selection may lead to reproductive isolation between populations through divergence in female mate choice, and population differentiation driven by female mate choice is expected to produce pre- but not post-mating isolation. We tested these hypotheses by looking at whether allopatric populations of the Amarillo (Girardinichthys multiradiatus), a sexually dimorphic viviparous fish with effective female choice, (i) have undergone phenotypical differentiation that may be attributed to divergence in female mate choice, and (ii) are already separated by pre- and/or post-mating reproductive barriers. We found substantial divergence in morphological traits which are the target of female mate choice, and in male courtship behaviour. Strong female preferences for homogametic males indicate substantial and symmetric pre-copulatory isolation, but the few successful heterogametic crosses produced in confinement yielded litters of the same size as those produced in homogametic matings, suggesting that post-copulatory isolation between populations is non-existent or weak. It appears that the studied populations have undergone incipient speciation with a pattern that is consistent with speciation driven by sexual selection, yet further work should assess whether divergence in female preferences has promoted male phenotypic differentiation or whether variation in male attributes has driven divergence in female preferences.

Molecular phylogeny of the livebearing Goodeidae (Cyprinodontiformes).

The Goodeinae is a speciose group of viviparous freshwater fishes endemic to the Mesa Central of Mexico. The current taxonomy of the group is based on morphology associated with viviparity and several of the groupings are questionable. We sequenced part of the mitochondrial cytochrome c oxidase subunit I (COI) gene (627bp) and control region (approximately 430bp aligned) of representatives of 36 species (all genera) of goodeid fishes in order to establish phylogenetic relationships among the taxa. Findings support the monophyly of the Goodeidae, the sister-group relationship of the Empetrichthyinae and Goodeinae, and the relationship of Profundulus to the Goodeidae. All goodeine genera but Xenotoca were recovered as monophyletic. Many of the higher-level relationships within the group contradict the findings of previous studies based upon morphology. The rate of molecular change in COI (0.9% per Myr), calibrated with the fossil record and geological data, suggests an approximate age for the Goodeidae of 16.5Myr. The majority of divergence within the Goodeinae appears to have occurred during the Miocene, with subsequent cladogenesis in the Pliocene and Pleistocene. Most recent speciation appears allopatric. River piracy, particularly involving the Rio Ameca basin, has played a significant role in the diversification of the Goodeinae.