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Genetics of Drosophila subobscura populations: VIII. Allozyme loci and their chromosome maps
Abstract
Genetic information on 25 enzyme polymorphisms in Drosophila subobscura, the location of corresponding genes, and cytological and genetic maps of the chromosomes are presented, as well as photographic maps of the salivary gland chromosomes in the third instar larva and white-case early pupal stages. Biochemical markers were used to establish chromosomal homologies among D. subobscura, D. pseudoobscura, D. melanogaster, and D. willistoni.
... Early investigations on its salivary gland nucleus revealed that it has the ancestral Drosophila karyotype of a small dot and five large acrocentric rods, does not show a chromocenter [10] and, especially, shows extraordinary levels of chromosomal polymorphism caused by large, cytologically visible paracentric inversions segregating on all five rods. Elaboration of detailed polytene drawings [11,12] and photomaps [13][14][15] greatly facilitated the study of the inversions, and paved the way for subsequent development of the over 600 linkage [16] and cytologically mapped genetic markers presently available, which cover most of the euchromatic genome [17,18]. ...
... 20,150,897 [110]) identified repetitive sequences, which were re-examined after masking of the repeats; in the third case, BLASTN was used to confirm that the target contig mapped exclusively to one scaffold. Cross-species synteny information obtained in this way was combined with ii) the wealth of available D. subobscura's physical mapping [18,84,111] and genetic linkage [13,112,113] data. Markers' sequences were retrieved from FlyBase 2.0 (release FB2017_02) using gene names and/or annotation symbols provided by the authors. ...
Background
Drosophila subobscura has long been a central model in evolutionary genetics. Presently, its use is hindered by the lack of a reference genome. To bridge this gap, here we used PacBio long-read technology, together with the available wealth of genetic marker information, to assemble and annotate a high-quality nuclear and complete mitochondrial genome for the species. With the obtained assembly, we performed the first synteny analysis of genome structure evolution in the subobscura subgroup.
Results
We generated a highly-contiguous ~ 129 Mb-long nuclear genome, consisting of six pseudochromosomes corresponding to the six chromosomes of a female haploid set, and a complete 15,764 bp-long mitogenome, and provide an account of their numbers and distributions of codifying and repetitive content. All 12 identified paracentric inversion differences in the subobscura subgroup would have originated by chromosomal breakage and repair, with some associated duplications, but no evidence of direct gene disruptions by the breakpoints. Between lineages, inversion fixation rates were 10 times higher in continental D. subobscura than in the two small oceanic-island endemics D. guanche and D. madeirensis. Within D. subobscura, we found contrasting ratios of chromosomal divergence to polymorphism between the A sex chromosome and the autosomes.
Conclusions
We present the first high-quality, long-read sequencing of a D. subobscura genome. Our findings generally support genome structure evolution in this species being driven indirectly, through the inversions’ recombination-suppression effects in maintaining sets of adaptive alleles together in the face of gene flow. The resources developed will serve to further establish the subobscura subgroup as model for comparative genomics and evolutionary indicator of global change.
... In Drosophila, recombination does not occur in males and, in females that are heterozygous for paracentric inversions, single crossovers are suppressed, and double crossovers are unlikely for short inversions (recombination length <20 cM; Navarro, Betr an,Barbadilla, & Ruiz, 1997), whereas for large inversions, they are more likely to affect the central part of the inversion. In contrast, noncrossover gene conversion events are expected to occur uniformly along inversions regardless their size.The inversions of this study should all be considered long [O 3 , the shortest one, is~23 cM long, or 12%(Krimbas, 1992;Loukas, Krimbas, Mavragani-Tsipidou, & Kastritsis, 1979) of the total O chromosome length of 190.7 cM (Pegueroles, Araúz, Pascual, & Mestres, 2010)] and old enough [0.33 AE 0.13 vs. 0.35 AE 0.05 Mya, respectively; average across the rp49 (Ramos-Onsins et al., 1998; Rozas, Segarra, Rib o, & Aguad e, 1999); Acph-1 (Navarro-Sabat e, mosomal differentiation. The position of the Hsp70 locus is, however, well outside the central one-third of the inversions length in all cases, whereby the likelihood of it being affected by double crossovers should be small. ...
Heat-shock (HS) assays to understand the connection between standing inversion variation and evolutionary response to climate change in D. subobscura found that “warm-climate” inversion O3+4 exhibits non-HS levels of Hsp70 protein like those of “cold-climate” OST after HS induction. This was unexpected, as overexpression of Hsp70 can incur multiple fitness costs. To understand the genetic basis of this finding, we have determined the genomic sequence organization of the Hsp70 family in four different inversions, including OST, O3+4, O3+4+8 and O3+4+16, using as outgroups the remainder of the subobscura species subgroup, namely D. madeirensis and D. guanche. We found: i) In all the assayed lines, the Hsp70 family resides in cytological locus 94A, and consists of only two genes, each with four HS elements (HSEs) and three GAGA sites on its promoter. Yet in OST the family is comparatively more compact. ii) The two Hsp70 copies evolve in concert through gene conversion, except in D. guanche. iii) Within D. subobscura, the rate of concerted evolution is strongly structured by inversion, being higher in OST than in O3+4. And iv) In D. guanche the two copies accumulated multiple differences, including a newly evolved “gap-type” HSE2. The absence of concerted evolution in this species may be related to a long-gone-unnoticed observation that it lacks Hsp70 HS response (HSR), perhaps because it has evolved within a narrow thermal range in an oceanic island. Our results point to a previously unrealized link between inversions and concerted evolution, with potentially major implications for understanding genome evolution.
... The chromosomal location in D. subobscura of the sixteen studied Polycomb group (PcG) genes is shown in Fig 1. These genes map at the expected chromosome according to their cytological location in D. melanogaster and the well-established chromosomal homologies between this species and D. subobscura [42]. Only the two tandem paralogs Ph-p and Ph-d are X-linked, all other genes are located in autosomes and Pho maps in the dot-like chromosome (Muller's element F). ...
Polycomb group (PcG) proteins are important regulatory factors that modulate the chromatin state. They form protein complexes that repress gene expression by the introduction of posttranslational histone modifications. The study of PcG proteins divergence in Drosophila revealed signals of coevolution among them and an acceleration of the nonsynonymous evolutionary rate in the lineage ancestral to the obscura group species, mainly in subunits of the Pcl-PRC2 complex. Herein, we have studied the nucleotide polymorphism of PcG genes in a natural population of D. subobscura to detect whether natural selection has also modulated the evolution of these important regulatory genes in a more recent time scale. Results show that most genes are under the action of purifying selection and present a level and pattern of polymorphism consistent with predictions of the neutral model, the exceptions being Su(z)12 and Pho. MK tests indicate an accumulation of adaptive changes in the SU(Z)12 protein during the divergence of D. subobscura and D. guanche. In contrast, the HKA test shows a deficit of polymorphism at Pho. The most likely explanation for this reduced variation is the location of this gene in the dot-like chromosome and would indicate that this chromosome also has null or very low recombination in D. subobscura, as reported in D. melanogaster.
... Subsequent studies supported Muller's original proposal. Included among these studies are comparisons of homologous morphological markers (Sturtevant and Novitsky 1941;Patterson and Stone 1952), enzyme linkage maps (Loukas, et al. 1979), chromosome pairing in interspecific hybrids (Throckmorton 1982;Krimbas and Loukas 1984), banding patterns in polytene chromosomes (Ash burner and Berendes 1978), and in situ hybridization (Whiting et al. 1989). Foster et al. (1981) raised the study ofdipteran chromosomal evolution to a higher phylogenetic level by comparing linkage maps (consisting pri-marily of morphological markers) ofDrosophila. ...
Chromosome number and morphology in mosquitoes is remarkably uniform: virtually all mosquitoes have a diploid chromosome number of six (2N = 6), and their chromosomes are invariably metacentric or submetacentric. Numerical changes obviously have not been important in mosquito chromosomal evolution, and because of the morphological similarity of their chromosomes, it appears that structural changes have played little or no role in mosquito karyotypic evolution. The goal of the present study was to identify the types and relative numbers of chromosomal changes in mosquito evolution and to extend the comparison where possible to the higher diptera. To do this, we compared the enzyme linkage maps of six species of Aedes to each other and to enzyme maps of seven other mosquito species and to Drosophila melanogaster. Our results indicate that Aedes chromosomes have been modified by inversions, most which were paracentric, and by translocations, most which were Robertsonian. Intrageneric comparison of Aedes enzyme maps also revealed groups of linked enzyme loci whose integrity has been maintained throughout Aedes evolution (conserved linkages/syntenies). Intergeneric comparisons of Aedes enzyme maps with those of species in the genera Culex, Anopheles, and Toxorhynchites disclosed conserved associations of enzyme loci between mosquito genera. These findings lead us to postulate that the ancestral mosquito karyotype consisted of six chromosomal elements which, other than being combined in different ways in various mosquito groups, have remained essentially intact during mosquito evolution. Furthermore, the identification of groups of linked enzyme loci common to mosquitoes and to D. melanogaster indicates that linkage group conservation may characterize the karyotypic evolution of all dipteran insects.
... To quantify the amount of recombina- tion, the following technique was devised: males from a strain homokaryotypic for the 03+4 gene arrangement and homozygous for the 1.88 allele ofthe gene codifying the acid phosphatase allozyme (Acph) were crossed with virgin females from the VaiBa strain (Fig. 2). This allozyme locus was chosen because it maps near the tip of the°chromosome and thus is located within the 0 3 +4 inversion (Loukas et al., 1979). Furthermore, the "Varicose" chromosome carries the 1.00 allele ofthis gene. ...
In this work, the process of colonization of North and South America by the species Drosophila subobscura has been studied by analyzing the variability of lethal genes. The genetic structures of a Palearctic natural central population (Bordils, Spain) and a colonizer population from America (Gilroy, California) have been compared. The frequencies of lethal chromosomes and their allelism are 29.007% and 0.0069 in the first population and 14.414% and 0.0526 in the American population. A founder effect is detected after the computation of some population parameters (Ne , h, he and the lethal load). Furthermore, the allelism of lethal chromosomes has revealed a strong association between a lethal gene and the O5 inversion both in Gilroy and in the population of Puerto Montt (Chile). The interpopulation allelism shows that the O5 arrangement from the USA and Chile is the same, confirming that the colonizing processes of North and South America are correlated. The O5 arrangement can also be useful as a genetic marker to trace the origin of the colonization. The frequency of the O5 arrangement in the original population of the colonization could be used to estimate the number of colonizers. This population is still unknown, but taking the extreme values of the frequency of the O5 inversion in natural Palearctic populations (1-15%), the number of colonizers could vary between 9 and 149 individuals.
... In contrast, noncrossover gene conversion events are expected to occur uniformly along inversions regardless their size. The inversions of this study should all be considered long [O 3 , the shortest one, is 23 cM long, or 12% ( Loukas et al. 1979;Krimbas 1992 et al. 1999); Acph-1 ( Navarro-Sabaté et al. 1999); and Fmr1 ( Pegueroles et al. 2013) loci] for recombination to have eroded interchromosomal differentiation. The position of the Hsp70 locus is, however, well outside the central one-third of the inversions length in all cases, whereby the likelihood of it being affected by double crossovers should be small. ...
Numerous studies have confirmed the adaptive value of the rich chromosomal inversion polymorphism in the drosophilid D. subobscura. However, until recently very little was known about the molecular basis behind its maintenance in natural populations. In search of candidate loci, a previous heat shock experiment quantified Hsp70 protein levels in homokaryotypic strains for the OST, O3+4+8 and O3+4 arrangements. Unexpectedly, individuals of the warm climate-associated O3+4 arrangement showed increased levels in absence of thermal stress that did not boost after the heat shock. Unfortunately, by the time this experiment was performed there was very little data available on the molecular organization of the Hsp70IR locus in D. subobscura. The previously mentioned results led to the present thesis work, whose objectives are to locate the Hsp70IR locus in the karyotype and to know the genomic organization, molecular characteristics and gene expression patterns in several representative chromosomal arrangements that comprise the genomic region where the hsp70 gene family is located: O3+4+16+2, O3+4+8, O3+4 and OST. Using the sequence of a clone from an OST line genomic library and contigs from the unassembled genome of D. subobscura, we designed a probe from the hsp70 coding region that enabled us to determine the location of the locus by in situ (ISH) hybridization. Concomitantly, we completed the sequencing of a 9-10 kb region in the Hsp70IR locus in 12 lines isogenic for the aforementioned arrangements and in D. madeirensis and D. guanche to shed light on the evolution of this locus in the last 1. 8 - 2. 8 million years (myr). ISH results showed a single hybridization site in the 94A band in the distal segment (SI) of the O chromosome coincident in the 4 studied karyotypes: O3+4+16+2, O3+4+8, O3+4 and OST. The sequences corresponding to the 12 isogenic lines and to D. madeirensis and D. guanche indicated that in these three species of the subobscura cluster, the Hsp70IR locus consists of two 2. 5 - 3. 0 kb long paralogous copies in divergent orientation separated by a 0. 5 - 1. 4 kb nonduplicated central spacer region. The two copies show a high degree of conservation between the different gene arrangements and species analyzed, while the central spacer region is highly polymorphic. Among the most relevant aspects of polymorphism analyses, we highlight the high degree of conservation in the coding regions (CDSs) and the cis-regulatory elements (CREs) in the proximal promoter of all the analyzed hsp70 genes, which might indicate that these are functional in all studied lines, and that their regulation might be similar. Curiously, at the sequence level, the paralogous 5'-UTR and CDS regions tend to be significantly more similar within the same arrangement and, in some cases, within the same line, probably as a result of ectopic gene conversion. Lastly, we carried out the quantification of basal hsp70 mRNA and protein levels in adult males and females of six lines isogenic for the cold climate-associated OST and six for the warm climate-associated O3+4 arrangements. Basal mRNA quantification results indicate that the two arrangements exhibit similar levels, yet significant differences are observed between males and females of the warm O3+4 arrangement. Regarding the quantification of basal Hsp70 protein levels, these suggest that there are no differences between sexes nor between the two arrangements, but instead we observe a significant interaction between sex and arrangement. Overall, the results for both, mRNA and protein data, indicate that hsp70 expression might be influenced by sex.
The development of genetic maps multiple species of Drosophila to understand the basis for species formation became problematic because visible mutations were not easily compared among species and species-specific linkage groups lacked a standard nomenclature...
The concept of synteny, or conservation of genes on the same chromosome, traces its origins to the early days of Drosophila genetics. This discovery emerged from comparisons of linkage maps from different species of Drosophila with the goal of understanding the process of speciation. H. J. Muller published a landmark article entitled Bearings of the “Drosophila” work on systematics, where he synthesized genetic and physical map data and proposed a model of speciation and chromosomal gene content conservation. These models have withstood the test of time with the advent of molecular genetic analysis from protein to genome level variation. Muller’s ideas provide a framework to begin to answer questions about the evolutionary forces that shape the structure of the genome.
The capacity of the usual tests (chi-square and related tests) to detect gametic disequilibrium between allozyme loci in natural populations of Drosophila has been investigated. We analyzed a large collection of previously reported gametic samples from natural populations involving a variety of loosely linked allozyme loci located along the O chromosome of Drosophila subobscura and the second chromosome of D. melanogaster. It is found that the statistical power of the individual tests to detect the sample disequilibria between allozyme loci is remarkably low, being the average (over pairs of loci) of power estimates close to 0.20 in both species. Moreover, the average minimum disequilibrium (D'min ) that would be required to reject (90% probability) the hypothesis of gametic equilibrium is higher than 0.50 given the observed degree of polymorphism and sample sizes used. This means that statistically significant associations between allozyme loci would rarely be detected by single-sample tests even when much disequilibrium is present in natural populations of Drosophila. However, an alternative approach based on the analysis of disquilibrium for large sets of gametic samples, combining probabilities from single independent tests and assessing significance by a bootstrap procedure, reveals that most of the locus pairs within segment I and II of the O chromosome of D. subobscura and left arm of the second chromosome of D. melanogaster present significant nonrandom associations. Within these chromosomal sections, the observed average absolute value of disquilibrium (D') between loci is around 0.25 (under the more conservative estimation). Also, a positive relationship between the magnitude of disequilibrium and linkage was detected. These findings suggest that weak or moderate values of disequilibrium between loosely linked allozyme loci are more frequent in natural populations of Drosophila than is currently believed.
In 1954, Marvin Wasserman, in his initial cytological analysis of the repleta species group of Drosophila, discovered there were six “mulleri-like” species united and set apart from the others by one fixed inversion common to all six forms and five other inversions shared among the species in various combinations, even though in the homozygous condition. Also of special interest, which was not considered an issue at the time, was the fact that three of the species, when hybridized, showed no inversions in the salivary chromosomes; D. mulleri, D. aldrichi and D. wheeleri were homosequential in banding pattern (Wasserman, 1954).