Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis.

BACKGROUND: In recent years, the relaxin family of signaling molecules has been shown to play diverse roles in mammalian physiology, but little is known about its diversity or physiology in teleosts, an infraclass of the bony fishes comprising approximately 50% of all extant vertebrates. In this paper, 32 relaxin family sequences were obtained by searching genomic and cDNA databases from eight teleost species; phylogenetic, molecular evolutionary, and syntenic data analyses were conducted to understand the relationship and differential patterns of evolution of relaxin family genes in teleosts compared with mammals. Additionally, real-time quantitative PCR was used to confirm and assess the tissues of expression of five relaxin family genes in Danio rerio and in situ hybridization used to assess the site-specific expression of the insulin 3-like gene in D. rerio testis. RESULTS: Up to six relaxin family genes were identified in each teleost species. Comparative syntenic mapping revealed that fish possess two paralogous copies of human RLN3, which we call rln3a and rln3b, an orthologue of human RLN2, rln, two paralogous copies of human INSL5, insl5a and insl5b, and an orthologue of human INSL3, insl3. Molecular evolutionary analyses indicated that: rln3a, rln3b and rln are under strong evolutionary constraint, that insl3 has been subject to moderate rates of sequence evolution with two amino acids in insl3/INSL3 showing evidence of positively selection, and that insl5b exhibits a higher rate of sequence evolution than its paralogue insl5a suggesting that it may have been neo-functionalized after the teleost whole genome duplication. Quantitative PCR analyses in D. rerio indicated that rln3a and rln3b are expressed in brain, insl3 is highly expressed in gonads, and that there was low expression of both insl5 genes in adult zebrafish. Finally, in situ hybridization of insl3 in D. rerio testes showed highly specific hybridization to interstitial Leydig cells. CONCLUSIONS: Contrary to previous studies, we find convincing evidence that teleosts contain orthologues of four relaxin family peptides. Overall our analyses suggest that in teleosts: 1) rln3 exhibits a similar evolution and expression pattern to mammalian RLN3, 2) insl3 has been subject to positive selection like its mammalian counterpart and shows similar tissue-specific expression in Leydig cells, 3) insl5 genes are highly represented and have a relatively high rate of sequence evolution in teleost genomes, but they exhibited only low levels of expression in adult zebrafish, 4) rln is evolving under very different selective constraints from mammalian RLN. The results presented here should facilitate the development of hypothesis-driven experimental work on the specific roles of relaxin family genes in teleosts.

Relaxin family genes in humans and teleosts.

Functions of the relaxin peptide family have been primarily investigated in mammals, and little attention has been paid to nonmammalian species. In this work, we performed phylogenetic and syntenic data analyses to identify the number and orthologous and paralogous relationships of relaxin family genes in teleosts and humans. Additionally, we performed reverse transcription PCR to determine the expression of three members of the relaxin family in zebrafish brain and gonads. We found evidence that teleosts harbor orthologs of the human INSL5, H2, H3, and INSL3 genes, so-named RLND/RLNE, RLNC, RLN3a/RLN3b, and RLNF in teleosts, respectively. Moreover, the presence of mRNA for RLN3a, RLND, and RLNF in both male and female brain and gonads of zebrafish suggests that all three genes are expressed in these tissues of this teleost. Differential expression and/or regulation of these genes will be explored in future experimental work.

Effect of variation in self-incompatibility on pollen limitation and inbreeding depression in Flourensia cernua (Asteraceae) scrubs of contrasting density.

BACKGROUND AND AIMS: Selection may favour a partial or complete loss of self-incompatibility (SI) if it increases the reproductive output of individuals in the presence of low mate availability. The reproductive output of individuals varying in their strength of SI may also be affected by population density via its affect on the spatial structuring and number of S-alleles in populations. Modifiers increasing levels of self-compatibility can be selected when self-compatible individuals receive reproductive compensation by, for example, increasing seed set and/or when they become associated with high fitness genotypes. METHODS: The effect of variation in the strength of SI and scrub density (low versus high) on seed set, seed germination and inbreeding depression in seed germination (delta(germ)) was investigated in the partially self-incompatible species Flourensia cernua by analysing data from self-, cross- and open-pollinated florets. KEY RESULTS: Examination of 100 plants in both high and low scrub densities revealed that 51% of plants were strongly self-incompatible and 49 % varied from being self-incompatible to self-compatible. Seed set after hand cross-pollination was higher than after open-pollination for self-incompatible, partially self-incompatible and self-compatible plants but was uniformly low for strongly self-incompatible plants. Strongly self-incompatible and self-incompatible plants exhibited lower seed set, seed germination and multiplicative female fitness (floral display x seed set x seed germination) in open-pollinated florets compared with partially self-incompatible and self-compatible plants. Scrub density also had an effect on seed set and inbreeding depression: in low-density scrubs seed set was higher after open-pollination and delta(germ) was lower. CONCLUSIONS: These data suggest that (a) plants suffered outcross pollen limitation, (b) female fitness in partially self-incompatible and self-compatible plants is enhanced by increased mate-compatibility and (c) plants in low-density scrubs received higher quality pollen via open-pollination than plants in high-density scrubs.

Macrophylogenetic analyses of the gain and loss of self-incompatibility in the Asteraceae.

The self-incompatibility (SI) status of 571 taxa from the Asteraceae was identified and the taxa were scored as having SI, partial SI or self-compatibility (SC) as their breeding system. A molecular phylogeny of the internal transcribed spacer (ITS) region was constructed for 211 of these taxa. Macrophylogenetic methods were used to test hypotheses concerning the ancestral state of SI in the Asteraceae, the gain and loss of SI, the irreversibility of the loss of SI and the potential for partial SI or SC to be terminal states. The ancestral breeding system in the family could not be resolved. Both maximum likelihood and parsimony analyses indicated that transitions among all breeding system states provide the best fit to the data and that neither partial SI nor SC is a terminal state. Furthermore, the data indicated that the loss of SI is not irreversible, although breeding system evolution has been more dynamic in some clades than in others. These results are discussed within the context of evidence for the gain and loss of SI, the evolutionary role of partial SI and methodological assumptions of tests of breeding system evolution.

Timing and rate of speciation in Agave (Agavaceae).

The Agave (Agavaceae) are keystone species of semiarid to arid regions where the geographic center of origin is Mexico but whose populations spread from the southwestern U.S. through Central America, the Caribbean, and into northern South America. Our analyses indicate that Agave is a young genus, between 7.8 and 10.1 million years old, and yet it harbors the most species of any genera in the family. Of the eight genera in the family, Agave is paraphyletic with respect to three of them, and these four genera are often grouped into a genus termed Agave sensu lato, which harbors 208 of the 293 recognized species in the Agavaceae. In this article, we examine the phylogenetic limits of Agave sensu lato and present analyses elucidating the origin and rate of speciation in the group. These analyses lead to some new insights into the phylogenetic limits of Agave, indicate an estimated age of the family between 20 and 26 million years and an age of the Agave sensu lato of

Parental effects in a partially self-incompatible herb Campanula rapunculoides L. (Campanulaceae): influence of variation in the strength of self-incompatibility on seed set and progeny performance.

We employ a full reciprocal diallel design between 10 parental plants that differed in their strength of self-incompatibility (SI; strong, intermediate, and weak) to examine parental effects on seed set and 10 components of fitness of progeny performance in Campanula rapunculoides. We perform ANOVAs to separate the influence of the strength of SI and the identity of the maternal and paternal parent on family performance. We calculate the phenotypic and genetic correlations between traits to determine potentially evolutionary constraints. Finally, we employ maximum likelihood methods to estimate the components of quantitative genetic variance, as defined by Cockerham and Weir in their BioModel c. Our most significant finding is that weak SI plants have high outcrossed seed set as maternal parents. We argue that direct or pleiotropic effects of modifiers of SI probably cause this. Second, we find that extranuclear interactions, as defined by the BioModel, have strong effects on seed set and several vegetative and flowering traits. These findings indicate that some maternal plants selectively provision seeds sired by specific paternal donors and that some of this variation appears to be associated with modifiers of the strength of SI. We find other sources of significant quantitative genetic variation for all of the traits we examine and discuss the possible role these play in the evolution of the reproductive system. Taken together, our findings show that variation in the strength of SI may influence levels of quantitative genetic variation that, in turn, can influence the reproductive success of individuals in C. rapunculoides

Effects of inbreeding on male function and self-fertility in the partially self-incompatible herb Campanula rapunculoides (Campanulaceae).

We examined the effect of inbreeding on fitness (through both male and female functions) and changes in self-fertility in the partially self-incompatible species Campanula rapunculoides. Individuals in natural populations of C. rapunculoides varied extensively in their strength of self-incompatibility (SI). We crossed 11 individuals that differed in their strength of SI to generate families with four levels of inbreeding (f = 0.0, 0.25, 0.5, and 0.75). Progeny were scored for three traits related to male fitness and for outcrossed and selfed seed production. Analyses of variance revealed significant inbreeding depression for the three male traits and seed set. Families with strong or weak SI differed in their response to inbreeding. Families with weak SI had lower levels of inbreeding depression for most traits than families with strong SI, but strong SI families had a greater increase in selfed seed set, but not self-fertility, with inbreeding. Finally, we found evidence of a significant linear response to inbreeding for all three male reproductive traits and outcrossed seed, indicating that inbreeding depression was primarily caused by partially or fully recessive deleterious alleles. Variation in genetic load was associated with variation in self-fertility, a finding that suggests an evolutionary role for partial self-fertility in natural populations of C. rapunculoides.

The inheritance of modifiers conferring self-fertility in the partially self-incompatible perennial, Campanula rapunculoides L. (Campanulaceae).

The role of partial self-incompatibility in plant breeding system evolution has received little attention. Here, we examine the genetic basis of modifiers conferring self-fertility in the creeping bellflower, Campanula rapunculoides L. (Campanulaceae), a partially self-incompatible herb. A survey of 35 individuals from two natural populations indicates that 45% of them are strongly self-incompatible, 40% intermediately self-incompatible, and 15% weakly self-incompatible and that some plants show a strong breakdown in self-incompatibility over floral age. We generated 101 F1 families by random crossing among 31 parental plants and estimated the heritability of self-fertility in day 1 and day 4 female-phase flowers, the genetic correlation between day 1 and day 4 self-fertility, and the coefficient of additive genetic variance of self-fertility. We use linear regression and data from additional crosses to examine whether there are significant maternal effects in the expression of self-fertility. We use Fain's test to determine if a major gene influences self-fertility and, finding no evidence, use data from additional crosses on an F2 generation to estimate the mean number and dominance of genes conferring self-fertility. These analyses indicate that the heritability (h2) of self-fertility is 0.24 in day 1 female-phase flowers and 0.44 in day 4 flowers, self-fertility is primarily additive but shows some recessive effects, and self-fertility is estimated to be controlled by four genetic factors. In addition, we have evidence that there may be maternal effects for self-fertility, especially for weakly self-incompatible plants. The significance of these results in the context of mating system evolution is discussed.