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Evolution of salivary glue genes in Drosophila species
Jean-Luc Da Lage ORCID: orcid.org/0000-0002-1372-67621,
Gregg W. C. Thomas2,
Magalie Bonneau1 &
Virginie Courtier-Orgogozo3
At the very end of the larval stage Drosophila expectorate a glue secreted by their salivary glands to attach themselves to a substrate while pupariating. The glue is a mixture of apparently unrelated proteins, some of which are highly glycosylated and possess internal repeats. Because species adhere to distinct substrates (i.e. leaves, wood, rotten fruits), glue genes are expected to evolve rapidly.
We used available genome sequences and PCR-sequencing of regions of interest to investigate the glue genes in 20 Drosophila species. We discovered a new gene in addition to the seven glue genes annotated in D. melanogaster. We also identified a phase 1 intron at a conserved position present in five of the eight glue genes of D. melanogaster, suggesting a common origin for those glue genes. A slightly significant rate of gene turnover was inferred. Both the number of repeats and the repeat sequence were found to diverge rapidly, even between closely related species. We also detected high repeat number variation at the intrapopulation level in D. melanogaster.
Most conspicuous signs of accelerated evolution are found in the repeat regions of several glue genes.
Animals interact with their environment (viruses, bacteria, food, chemicals, conspecifics, etc.) in many different ways, particularly through their immune and sensory systems. As animals adapt to new places, the way they interact with their environment is expected to change. Accordingly, the gene families that have been shown to exhibit accelerated rates of gene gain and loss in several animal groups are mostly genes that mediate the interactions with the environment: immune defense, stress response, metabolism, cell signaling, reproduction and chemoreception [1]. Rapid changes in gene copy number can lead to fast phenotypic changes via gene deletion and can provide raw material for genes with new functions via gene duplication [2, 3]. Rapid turnover of genes within a gene family has also been shown to correlate with fast evolution at the sequence level [4, 5].
One particularly interesting environmental interaction occurs in Drosophila. Metamorphosis is a critical stage of fruitfly development [6] during which the animal is vulnerable and motionless. In Drosophilids pupae are generally attached to a substrate until the imago leaves the puparium. It is critical for the pupa to be firmly attached in order not to be moved away by some external event (i.e. rain or wind). Furthermore, for the emerging adult to be able to hold on the external substrate and thus get out of the pupal case, it is necessary for the pupa to adhere to a substrate, whether dry or wet. When the pupal case freely moves and is not attached, adults are unable to hatch and eventually die (J. R. David, personal communication).
Here we focus on the Salivary gland secretion (Sgs) genes, a functional group that mediates the physical interaction of flies in the genus Drosophila with an external substrate during metamorphosis. The Sgs genes encode proteins that make up the glue produced by Drosophila larvae that serves to attach the animal to a surface where it can undergo metamorphosis. In D. melanogaster, the glue is composed of several salivary gland secretion proteins which accumulate in the salivary glands of late third instar larvae [7]. As the puparium forms, the bloated salivary glands release their contents through the mouth. This secretion then hardens within seconds of contact with the air and becomes a glue which firmly attaches the pupa to the substrate.
Pupariation sites of Drosophila species in nature have not been extensively characterized, but a large variety of of pupariation sites have been found. In the wild, D. melanogaster pupae have been found adhered to wood, fixed to grape stalks, attached to the dry parts of various rotten fruits, or adhered to one another on the land beneath grape stalks [8,9,10]. D. mauritiana pupae may be found on the surface of decaying Pandanus fruit, which is hard and lignous (D. Legrand, personal communication). Many Hawaiian Drosophila species pupariate several inches deep in the soil [11]. Some other Drosophila species, such as D. sechellia, D. simulans, and the invasive D. suzukii, appear to pupariate directly within the wet rotten part of fruits (J. David, personal communication, [12]). Given the diversity of pupariation sites, we hypothesized that the different Drosophila species would require distinct types of glue meaning that the Sgs genes might evolve rapidly within the genus.
The glue genes
The glue genes have long been an important model for the regulation of gene expression. In the 1970s and 1980s it was discovered that genes for proteins contained in salivary secretions correlate with the chromosomal location of major puffs. This led to the discovery that, on an acid-urea electrophoresis gel, the salivary glue was resolved into five major bands, numbered from 1 to 5 in order of increasing electrophoretic mobility [13, 14]. Band 2, which was variable and detected in many other tissues, was considered to be a tissue contamination rather than a true glue protein [13]. From this, seven glue genes were eventually identified, and their nucleotide sequences are now well characterized: Sgs1 (band 1, CG3047, 2 L), Sgs3 (band 3, CG11720, 3 L), Sgs4 (band 4, CG12181, X), Sgs5 (band 5, CG7596, 3R), Sgs7 (CG18087, 3 L), and Sgs8 (CG6132, 3 L) and Eig71Ee (also named geneVII I71–7 or gp150, CG7604, 3 L) [15,16,17,18,19,20,21,22,23,24,25]. Eig71Ee, located at position 71E, is not only expressed in salivary glands but also in hemocytes and in the gut, where it appears to be involved in immunity and clotting [26,27,28].
A sixth electrophoretic band migrating slightly slower than the Sgs3 protein was also detected in a few D. melanogaster lines [14, 29, 30]. The nucleotide sequence of the corresponding gene, Sgs6, remains unknown but cytogenetic and genetic mapping indicates that Sgs6 is located in region 71C3–4 and differs from Eig71Ee [21, 26, 30].
The three genes Sgs3, Sgs7 and Sgs8 form a tightly linked cluster on the 3 L chromosomal arm at position 68C [31, 32]. All glue genes were found to start with a signal peptide. The largest glue genes, Sgs1, Sgs3 and Sgs4 and Eig71Ee were shown to harbor numerous internal repeats of amino acid motifs, rich in proline, threonine and serine [16, 23, 27, 33]. Molecular studies showed that the number of internal repeats was variable between strains in Sgs3 [34], and Sgs4 [33]. Additionally, consistent with missing protein bands, a few laboratory strains were inferred to carry loss-of-function mutations in Sgs4 [7, 13, 33, 35], Sgs5 [25] and Sgs6 [14, 29, 30].
In the present study, we characterize the diversity and evolution of the Sgs genes within the Drosophila genus. We inferred loss and gain of glue genes and we investigated repeat number variation and sequence repeat diversity across 19 species and across paralogs.
We used the six Sgs genes and Eig71Ee annotated in D. melanogaster as BLAST queries to identify their putative homologs in 19 other Drosophila species (Table 1). The homologs are summarized in Fig. 1 and Table 2. In D. melanogaster, the glue genes are "extremely highly" or "very highly" expressed in late larval salivary glands according to the RNAseq data in Flybase. But transcript data that would be useful for annotating the genes were not available for all species, probably because the expression window of the glue genes (late third larval instar and only in salivary glands) is narrow [7]. The organization of the Sgs genes was found to be generally conserved across the Drosophila species we investigated (Fig. 1). Proper identification of each ortholog was based on sequence similarity and, when possible, synteny. We describe below our findings for each category of Sgs genes.
Table 1 List of species and databases used in this study
Schematic species tree showing glue gene distribution and the most parsimonious scenario for gene gains and losses. Gains are indicated by "+" and losses by "-". Numbers correspond to the glue gene name (eg. "3" for Sgs3). An inferred distribution of glue genes in the last common ancestor is shown at the bottom. The tree is from Thomas, G.W.C. and Hahn M.W. (2017) https://doi.org/10.6084/m9.figshare.5450602. Pink is for Sgs1, yellow is for Sgs3, dark blue is for Sgs7, light blue is for Sgs8, green is for Sgs4, orange is for Sgs5-5bis, purple is for Eig71Ee. Along with each species is a schematic representation of the organization of the glue gene cluster, with relative position and orientation for the species with confirmed synteny information. Gene sizes and distances are not to scale. "R" means that internal repeats are present. "R?" means that no clear repeats were identified. In D. pseudoobscura, the relative orientation of the three clustered Sgs3-like sequences GA25425, GA23426, GA23878 suggested that GA23426 could be orthologous to Sgs3 (it is inside an intron of GA11155, homologue of Mob2, which is close to Sgs3 in D. melanogaster), GA23425 to Sgs7 and GA23878 to Sgs8. The last two had more similar sequences compared to GA23426, including the repeat region. Furthermore, the latter was neighbor to GA20420, a homologue of chrb-PC, a gene adjacent to Sgs8 in D. melanogaster
Table 2 Genomic coordinates of the glue genes in 20 Drosophila species
Identification of a Sgs5 paralog
We found that Sgs5 has a tandem paralog in D. melanogaster, located ca. 300 bp upstream of Sgs5 (CG7587, hereafter named Sgs5bis), sharing 46,3% identity and 66,9% similarity at the protein level. Similar expression profiles from Gbrowse (flybase.org) and FlyAtlas (flymine.org) show that Sgs5bis is co-expressed with Sgs5 during the late third larval instar in dissected salivary glands, and both genes harbor two introns in all species. To our knowledge, this paralog has not been mentioned earlier. The Sgs5/5bis pair is widely distributed in our species sample, and is therefore probably ancestral to most of the species studied. Occasional losses of either Sgs5 or Sgs5bis occurred at least four times (Fig. 1): 1) loss of Sgs5bis in D. mauritiana, where a relictual sequence may still be recognized, 2) loss of Sgs5bis in D. elegans, 3) loss of Sgs5bis in D. rhopaloa, 4) loss of Sgs5 in D. erecta. These patterns of loss suggest that Sgs5 and Sgs5bis can replace each other functionally. There is no Sgs5 nor Sgs5bis in D. willistoni. In D. ananassae, the orthologous sequence of Sgs5bis (formerly Dana\GF19880 in FlyBase release R1.3, with a different intron/exon structure) has been withdrawn from the genome annotation for reasons unknown to us though it is in conserved synteny relative to D. melanogaster. In D. virilis and D. pseudoobscura, a single Sgs5/5bis gene was identified. A phylogeny of all Sgs5 and Sgs5bis amino acid sequences (Fig. 2) revealed a clear separation in the gene sequences of the two groups, Sgs5 and Sgs5bis. The D. virilis gene (annotated as uncharacterized protein Dvir\GJ24445) and the D. pseudoobscura Sgs5/5bis gene (annotated as uncharacterized protein Dpse\GA20459) were clustered with the Sgs5bis genes and they shared with most other Sgs5bis sequences a motif Gln-Ala-Thr in the signal peptide. This suggests that D. virilis and D. pseudoobscura possess an ortholog of Sgs5bis. The D. virilis and D. pseudoobscura lineages diverged first in our sample (Fig. 1), but it cannot be determined whether they have lost Sgs5 or if the Sgs5-Sgs5bis gene duplication arose after their separation. In this case, the ancestral gene before the duplication was probably Sgs5bis.
Maximum likelihood (ML) tree of aligned Sgs5 and Sgs5bis amino acid sequences (repeated parts removed when present). Numbers along branches are the posterior probabilities. The tree was rooted between the Sgs5 cluster and the Sgs5bis cluster
Gains and losses of Sgs3, Sgs7, and Sgs8 genes
The genes Sgs3, Sgs7 and Sgs8 form a tight cluster, 4.5 kb long, on the 3 L arm in D. melanogaster [31] and share sequence similarities [16] in their N-terminal and C-terminal parts, however Sgs3 contains internal repeats whereas Sgs7 and Sgs8 do not (Sgs7 and Sgs8 are small proteins, about 75 amino acids in length). When the internal repeats of Sgs3 are excluded, the amino acid identity amongst the three genes in D. melanogaster is 51.3% between Sgs3 and Sgs7, 48.7% between Sgs3 and Sgs8, and 46.7% between Sgs7 and Sgs8. Additionally Sgs3, Sgs7 and Sgs8 share a phase 1 intron position, interrupting the signal peptide sequence [16]. In the clade D. yakuba / santomea / erecta, Sgs7 and Sgs8 are inverted with respect to the D. melanogaster arrangement (Fig. 1). Sgs7 is duplicated in D. yakuba (Dyak\GE20214 and Dyak\GE21218) and D. santomea (Fig. 1) with the two copies being inverted relative to each other and having only one, nonsynonymous, nucleotide difference. Sgs8 lies between the two Sgs7 copies, and has the same orientation as Sgs3. In species outside the D. melanogaster subgroup, all the Sgs3, Sgs7 and Sgs8 sequences also have the same intron, with slightly different positions depending on codon indels before the intron. Notably, D. suzukii is the only species in our study that has lost Sgs3. D. suzukii retained Sgs8 and has undergone an amplification of Sgs7, containing three identical copies.
In D. pseudoobscura, D. ficusphila, D. rhopaloa (see Fig. 1), Sgs7 and Sgs8 could not be identified. However, a BLAST search using the Sgs7 or Sgs8 sequences of D. melanogaster as queries, returned several Sgs3-like genes (i.e. long proteins with internal repeats showing N-terminal and C-terminal parts similar to Sgs3). In those species with no Sgs7, no Sgs8 and several Sgs3-like genes occupying the physical location of Sgs7 and Sgs8, it is tempting to infer that the ancestral Sgs7 and Sgs8 have gained internal repeats. According to such a hypothesis, at least in some cases, the non-repeated parts of those Sgs3-like protein sequences are expected to cluster with Sgs7/8.
To disentangle the relationships among Sgs3–7-8 paralogs, we constructed a phylogeny using an alignment of the non-repeated parts of the protein sequences (Fig. 3). The tree is discordant with the assumed species phylogeny, but shows a clear separation between Sgs3/Sgs3-like and Sgs7/Sgs8 genes. The exceptions are D. bipectinata and D. willistoni, whose Sgs7/Sgs8 sequences are clustered with the Sgs3 sequences, with low support due to short sequence lengths. This suggests that those Sgs7/Sgs8 sequences are old Sgs3-like sequences which have lost their internal repeats. However, with such low support throughout the tree, we cannot confirm this hypothesis or infer whether there were two ancestral Sgs3 and that subsequent losses occurred.
ML tree of aligned Sgs3 (repeats removed), Sgs7 and Sgs8 amino acid sequences. Numbers along branches are the posterior probabilities. The tree was rooted between the main Sgs7-Sgs8 cluster and the cluster containing all the Sgs3 sequences
Sgs1 is related to Sgs3/7/8
We find that Sgs1 is only present in the melanogaster subgroup and so-called Oriental subgroups (with a loss in D. erecta), which suggests that it originated in the ancestor of this clade. The Sgs1 sequence identified by BLAST search in the genome database (see Materials and Methods) was found to have many stop codons in the second half of the repeat region and had not been annotated as a coding sequence. However, based upon the surrounding repeat sequences, we found that inserting a C at position 1829 (from start) would restore the reading frame, translating into a putative 2245 amino acid protein. Inspection of another D. suzukii genome sequence [36] (contig CAKG01017146) showed that there is indeed a C at position 1829 and that Sgs1 is 2245 amino acid long, pointing to an error in the original annotation. Since position 1829 lies in the middle of a long repeat-containing region which prevents PCR amplification, we did not try to check experimentally for the missing C in the first D. suzukii genome sequence.
In all the Sgs1 genes identified, except in D. elegans, an intron was found at the same position and phase as in Sgs3, Sgs7 and Sgs8. There is also a loose similarity in the N-terminal and C-terminal parts of Sgs1 and Sgs3 (in D. melanogaster about 14% identity between Sgs3 and Sgs1 excluding the repeats). This suggests that Sgs1 belongs to the same family as Sgs3/Sgs7/Sgs8 genes.
Origins of Sg4 and Eig71Ee genes remain elusive
Sgs4 is intronless and is not present outside the D. melanogaster subgroup (Fig. 1, Table 2). We find no similarity between Sgs4 and any other sequence in any genome. Previously some sequence similarity between Eig71Ee and Sgs4 had been reported [27], but only in the low complexity repeat regions of the genes. Eig71Ee is found in all the D. melanogaster subgroup species and in some of the so-called Oriental species, where it has been annotated as mucin2, or extensin in D. takahashii, or even, erroneously, as Sgs3 in D. suzukii. We also detected the N-terminal parts of the gene in the D. ananassae group thus making the phylogenetic distribution of the gene unclear (Table 2). More interestingly, we noticed that Eig71Ee harbors an intron at the same position as the ones found in Sgs3, Sgs7, Sgs8 and Sgs1. This result argues for a certain relatedness among those genes. However, using Eig71Ee as a TBLASTN query did not retrieve any hits from any Sgs genes and the Eig71Ee amino acid sequence does not align with the Sgs sequences.
Rate of gene gains and losses in the glue gene families
Our analysis reveals that the seven annotated genes that code for glue proteins can be grouped into three gene families. Sgs1, Sgs3, Sgs7, Sgs8, and Eig71Ee comprise one of the three families since all of them share a phase 1 intron at the same position, interrupting the signal peptide sequence. Sgs4 then forms its own family and the Sgs5 and 5bis comprise the third family. We used CAFE [37] to reconstruct ancestral copy numbers throughout the Drosophila phylogeny and to test whether these three gene families evolve at an accelerated rate along any Drosophila lineage. For the CAFE analysis Eig71Ee was not included due to uncertainties about its presence in some species. We find that the Sgs4 and Sgs5-5bis families do not evolve faster compared to other gene families present in the Drosophila genomes (p = 0.58 and p = 0.107, respectively; Additional file 1: Table S1), however the Sgs1–3–7-8 family was found to evolve rapidly (p = 0.005; Additional file 1: Table S1). Overall, this family seems to be prone to duplication and loss (Additional file 2: Figure S1) and we find that this signal for rapid evolution is driven mostly by small changes on many lineages (i.e. a gain or loss of 1 gene) rather than large changes on one or a few lineages.
Characterization of the glue proteins and their repeats
Sgs1, Sgs3 and to a lesser extent, Sgs4 and Eig71Ee, are characterized by long repeats often rich in threonine and prone to O-glycosylations, in addition to their signal peptide and conserved C-terminus (Table 3 summarizes the characteristics of the repeats). We checked that across all populations in PopFly, the D. melanogaster Sgs5 protein is devoid of internal repeats while most other species, including close relatives of D. melanogaster, contain repeats mostly consisting in Pro-(Glu/Asp) pairs. Indeed the Sgs5 protein length is highly variable across species. For example, D. kikkawai harbors a long additional stretch (127 amino acids) containing 60% of acidic residues. Interestingly, the paralog Sgs5bis never has repeats. Sgs7 and Sgs8 are much smaller proteins, without any repeats and are rich in cysteine (12–14%). The conserved C-terminal sequences of Sgs proteins are important to characterize because the repeats are quite variable in motif, length and number, even between closely related species, meaning that, most often, glue proteins may be retrieved only based on their conserved C-terminal part. The C-terminal segments are about 120 amino acids long in Sgs1, 50 amino acids in Sgs3, 120 amino acids in Sgs4, 115 amino acids in Sgs5/5bis and 135 amino acids in Eig71Ee. The longest Sgs protein is Sgs1 in D. suzukii (2245 aa), which harbors ca. 63 repeats of a 29 amino acid, threonine-rich motif so that threonine makes up 40% of the residues. In D. melanogaster, Sgs1 is also very long (1286 aa) due to 86 repeats of a motif of 10 amino acids, also threonine-rich (46%). The shortest Sgs1 protein is the one of D. sechellia (492 aa). In all the species where it exists, Sgs1 is also rich in proline (12–18%). Sgs3 has a similar amino acid composition as Sgs1.
Table 3 Characteristics of glue proteins in the species studied (except Sgs7 and Sgs8)
Repeats can also be quite different between paralogs. For example, in D. eugracilis, while the two Sgs3-like genes are physically neighbors, Sgs3a has several repeats of CAP(T)n, whereas Sgs3b has ca. 65 KPT repeats. In D. elegans, the three Sgs3-like proteins also have quite different repeats (Table 3). Sgs4 is richer in proline than in threonine (18% vs. 16% in D. melanogaster) and contains 10% cysteine residues.
Interspecific variation in number and sequence of repeats
Between closely related species the number of repeats varied enormously and the repeated sequence diverged sometimes rapidly (Table 3). In the following we examine some specific examples to highlight these patterns. D. simulans, D. sechellia, and D. mauritiana form a clade, which split less than 300,000 years ago [38]. Their Sgs1 genes harbor the same repeated sequence but the number of repeats ranges from 40 in D. simulans to 13 and 22 in D. mauritiana and D. sechellia, respectively. Likewise, Sgs3 is very similar in the three species, except in the number of repeats. There are no repeats in D. simulans, but threonine-rich stretches; in the published sequence of D. mauritiana, there are three tandem occurrences of CAPPTRPPCTSP(T)n; in D. sechellia, several CKP(T)6 repeats. Sgs4 shows shared repeats C(D/N)TEPPT among these species, with many more repeats in D. mauritiana. In contrast, in the sibling species D. yakuba and D. santomea, which diverged 0.5 million years ago [39, 40], Sgs3, Sgs4 and Sgs5 harbor the same repeat sequences and the same number of repeats (Table 3). Sgs4 genes show 91% identity at the protein level with the same 23 repeats; Sgs5 97% identity and no repeats.
Another pair of species worth of interest is D. suzukii/D. biarmipes, considered to have diverged ca. 7.3 mya [41]. As mentioned above, only Sgs1 and Sgs5 can be compared because D. suzukii has lost Sgs3, and Sgs4 is limited to the melanogaster subgroup. Despite a longer divergence time than for the previous comparisons, the Sgs1 29 amino acid repeats are similar in the two species but D. suzukii has many more repeat units. In the non repeat parts, identity is 69.3%; Sgs5 is well conserved even in the repeat region, with an overall identity of 76.4% in amino acids, and 84.8% in the non-repeat parts. A last pair of related species (despite their belonging to different subgroups) is D. elegans/D. rhopaloa. We estimate their divergence time to be roughly 12 million years based on molecular data (see Methods) and find that their Sgs proteins are very similar overall. This similarity extends to the repeat regions, with the exception being the repeats in Sgs3, which exists as four gene copies in D. rhopaloa. Their Sgs5 proteins have a high overall identity (75%), including repeats (Glu-Pro)n. In the non-repeat regions, identity rose to 82%. Indeed we often found more divergence among paralogs within a genome than across orthologous proteins.
Structure prediction programs (IUPred [42], PrDOS [43], disEMBL [44], PONDR [45]) indicate that the repeat regions of Sgs1, Sgs3, Sgs4, Sgs5 and Eig71Ee are intrinsically disordered (Fig. 4). Only IUPred and PrDOS indicate Sgs4 repeats to be ordered, in disagreement with the other predictors.
Example of predictions for disordered regions by PONDR. The X axis represents the protein length; the Y axis shows the score of the predictor VL-XT, which compares two predictors based on neural networks. The higher the value (closer to 1), the most disordered it is. The thick bars show the best predicted disordered regions. The VL-TX algorithm is more accurate for stretches longer than 30 amino acids. Regions shaded in light blue are the signal peptide regions; regions shaded in light orange are the conserved C-terminal regions. a: The glue proteins with internal repeats of D. simulans, except Sgs5; b: example of an Sgs5 protein with large internal repeats (D. kikkawai) compared to the one of D. simulans
Intraspecific variation in number of repeats
Owing to the difficulty of short-read sequencing methods to deal with the repeated sequences found in glue genes, we could not get a species-wide insight of repeat number variation (RNV) in D. melanogaster. Therefore, we resequenced Sgs3 and Sgs4 in strains from various geographic locations using classical Sanger sequencing (Table 4). We find striking inter- and intrapopulation variation in the number of repeats: for Sgs3 (Additional file 3: Figure S2 and Additional file 4: Figure S3, Table 4), there was at least 9 repeat difference between the shortest and the longest allele (22 to 31); for Sgs4, we find a range of 18 to more than 26 repeats (Additional file 5: Figure S4 and Additional file 6: Figure S5, Table 4). Regarding the data from the Drosophila Genome Nexus study (Cairo population), we observed that the repeat region of Sgs4 was erroneously reconstituted, often underestimating the repeat number, compared to our Sanger sequencing. We also sequenced the Sgs3 and Sgs4 genes in wild-caught D. mauritiana individuals. For Sgs3 we found variation in the number of stretched threonines (10 or 12) and in the number of repeats (Additional file 7: Figure S6A and Table 4). For Sgs4, we found that the actual sequences were much longer than the sequence available online, and variable in length, even at the intra-population level, ranging from 25 to 35 repeats of the 7 amino acid motif (Additional file 7: Figure S6B and Table 4).
Table 4 List of strains used for PCR amplification. Number of repeats and repeat motifs in Sgs3 and Sgs4 in populations of D. melanogaster and D. mauritiana. Sequences of Sgs4 for Oregon R and Samarkand strains are from [83]. * indicate lines also used in the Drosophila Nexus project. @ indicate suspected artifactual repeat losses during cloning. PTC indicates the presence of a premature termination codon
Nonsense mutations in the Sgs genes
Despite the rather low quality of sequences in the Drosophila Genome Nexus data set, we searched for putative premature termination codons (PTC) in Sgs genes of D. melanogaster, which could lead to non-functional proteins. The search was limited to non-repeat regions. We find PTC in Sgs4 of several lines that truncated the protein at the beginning of its conserved C-terminal part. We confirmed experimentally the presence of this PTC in 10 lines of the Cairo population EG (K165stop) (Additional file 6: Figure S5 and Table 4). We also found putative PTC for Sgs5 in a few lines (W161stop, that is sub-terminal, and maybe not detrimental), and experimental verification confirmed it in one Ethiopian line (EF66N); in Sgs5bis, we found a putative PTC (C33stop) in six African lines from Rwanda (RG population) and Uganda (UG population). We also find a putative PTC for Sgs1 in a few lines from USA and Cairo (P49stop), which was confirmed by resequencing the Egyptian line EG36N. This nonsense mutation required two substitutions from CCA to TAA in all cases. Interestingly, EG36N also has a truncated Sgs4, warranting more careful investigation of its glue gene.
In Sgs3, no PTC was found, but putative PTC were found for Eig71Ee in two lines, EA90N (S345stop) and RAL894 (W380stop), both in the C-terminal region. One putative PTC was found in Sgs7 (Q47stop, line USI33), but was not checked experimentally. No PTC was found in Sgs8 sequences. Stretches of Ns found in non-repeat regions could possibly, at least in some cases, turn out to be true deletions, which deserves further investigation. There is a possibility that some PTCs could experience stop codon readthrough [46] leading to translation of the correct protein. For instance this is possible in Sgs4 because the nonsense mutation was not accompanied by other mutations, which would be expected in case of relaxed selection (unless the nonsense mutation is very recent). Further studies of the protein content of the salivary glands in those strains will be needed to check whether Sgs4 is produced and if it is full-size.
Evolutionary rate of Sgs protein sequences
Given that glue proteins harbor RNV and our hypothesis that they could be putative targets for fast selection, we wanted to test whether glue gene coding sequences evolve quickly. To this end, we computed substitution rates of the Sgs genes between D. melanogaster and D. simulans (Table 5). We did not include Sgs3, because the internal repeats were very different and not alignable between the two species. This, at any rate, shows that this particular gene has evolved rapidly. Although it had the biggest size and the highest number of repeats, we were able to make an estimate for Sgs1 because the repeats were rather similar in D. melanogaster and D. simulans. We removed the unalignable parts before computation, therefore underestimating the real evolutionary rate. Rate calculations were similarly performed for Eig71Ee, Sgs4 and Sgs5. The computed values for the Sgs genes were compared to the genome-wide distributions of dN/dS between these species (Fig. 5) using data from the flyDIVaS database [47]. All dN values were within the highest quartile, and Sgs1, Sgs4 and Sgs8 were within the highest three centiles. Furthermore, high dN/dS values were found for Sgs1 (dN/dS = 1.393) and Sgs8 (dN/dS = 1.259), indicating accelerated protein evolution. The dN value of Sgs8 (0.1789) contrasts with the one of its close relative Sgs7 (0.0475).
Table 5 Non-synonymous (dN) and synonymous (dS) substitution rates, and the dN/dS ratio for glue genes between D. melanogaster and D. simulans in pairwise alignments. Sgs3 was not included, and unalignable regions were removed
Distribution of dN/dS for the pair D. melanogaster/D. simulans from the flyDIVaS database with the position of glue genes. Vertical axis: number of genes. Genes are binned into rate value categories with increment of 0.005
We wondered if Sgs8 had also evolved faster than Sgs7 in other pairs of related species. Table 6 shows the results for other species pairs known to be close relatives: D. melanogaster/D. sechellia, D. simulans/D. sechellia; D. yakuba/D. erecta; D. biarmipes/D. suzukii. Comparisons between D. yakuba/D. erecta and D. biarmipes/D. suzukii showed no evolutionary rate difference between Sgs7 and Sgs8. However we found that between D. simulans and D. sechellia Sgs7 has a dN ten times higher than that of Sgs8. This pattern is opposite that the D. simulans vs. D. melanogaster comparison. In fact, D. sechellia Sgs7 is more divergent than D. simulans from D. melanogaster Sgs7, whereas Sgs8 has not diverged further. Obviously, the small number of substitutions points to a high variance, and the difference may be not significant.
Table 6 Non-synonymous (dN) and synonymous (dS) substitution rates and the ratio dN/dS for Sgs7 and Sgs8 between related species pairs in pairwise alignments
To test for adaptive evolution after the "out of Africa" event of D. melanogaster [48], we measured the nucleotide diversity π and divergence Dxy between one population from Zambia, (ZI) thought to be within the original geographical area of D. melanogaster, another African population (EF, Ethiopia) and two derived populations, from France (FR) and USA (Raleigh, RAL). This study was limited to the coding sequences of Sgs5 and Sgs5bis because these genes conveniently lack internal repeats and the gene size is not too short, (as opposed to Sgs7 and Sgs8). Due to the numerous residual unidentified nucleotides in the Drosophila Genome Nexus data, the number of sites taken into account was actually much smaller than the sequence size, e.g. for Sgs5bis, 278 sites left over 489 in RAL. We compared the overall π and Dxy between these populations [49]. Roughly, for both genes π is higher in ZI than in EF, FR and RAL (Tables 7 and 8). This matches the pattern observed for the whole genome and is as expected for the region of origin of this species. We found that divergences Dxy are less than expected from the whole genome, except for the ZI/EF comparison of Sgs5 (Tables 7 and 8). Both genes gave similar results. Therefore, we find that the glue genes Sgs5 and Sgs5bis do not show particular divergence pattern across populations, which could have been related to a change in population environment.
Table 7 Nucleotide diversity π of Sgs5 and Sgs5bis in four populations, computed from Jukes and Cantor [84] using DnaSP
Table 8 Nucleotide divergence between populations Dxy computed from Jukes and Cantor [84]in DnaSP
We also searched for episodic diversifying selection (EDS) among species for the three genes entirely devoid of repeats, Sgs5bis, Sgs7 and Sgs8 using the branch-site REL test (BS-REL) from the HyPhy package. No accelerated evolution was detected for Sgs5bis, whereas one branch (D. santomea-D. yakuba clade) underwent EDS for Sgs7 (corrected p-value 0.012) and one branch (D. erecta-D. yakuba-D. santomea) underwent EDS for Sgs8 (corrected p-value 0.015) (Additional file 8: Figure S7). These results must be considered with caution given the small size of the data set, but anyway do not favor a specific selection regime, regarding single nucleotide (or amino acid) polymorphism.
We have investigated the presence and characteristics of Sgs genes and proteins in several Drosophila species belonging to the two main subgenera Sophophora and Drosophila, with particular emphasis on species closer to D. melanogaster. We have identified the various Sgs genes through sequence similarity with D. melanogaster. While this study is extensive, it is of course possible that we may have missed glue genes completely different from the ones of D. melanogaster. In order to get the full collection of glue genes we require transcriptional evidence from late larval salivary gland RNA for each species studied. Interestingly, according to our census, the seven genes characterized for years in D. melanogaster are far from being always present in the other genomes, although the seven members are generally preserved in the D. melanogaster subgroup. Our results are in disagreement with the succinct interspecific study of Farkaš [50]. We also propose here an eighth glue gene, Sgs5bis. Based on its close sequence homology and its co-expression with Sgs5 we propose that these two genes are tandem paralogs. We notice that Sgs5bis never contains internal repeats whereas Sgs5 often harbors more or less developed repeat motifs, although not in D. melanogaster. Given our data, and notwithstanding the unbalanced taxonomic sampling which may mislead us, we suggest that the ancestor of the species studied here had only Sgs3 and Sgs5bis (Fig. 1). It is likely that Sgs7, Sgs8, and perhaps also Sgs1 and Eig71Ee, originated from duplications of Sgs3. The important differences in repeat motifs between duplicate Sgs3 (e.g. in D. eugracilis) are striking and suggest a high rate of evolution, or independent acquisition of repeats from a repeatless or repeat-poor parental gene. A part of the sequence we named Sgs3-like in D. willistoni is reported in FlyBase as GK28127, with transcription on the opposite strand, and without a homolog in D. melanogaster. Thus, it is possible that some duplicates of Sgs3 may have been actually recruited for other functions other than glue production. In this respect, it is also possible that Eig71Ee, which has been studied mostly for its immune functions, could be an ancient glue protein, which gained new functions.
The repeat-containing glue proteins are typical of secreted mucins. Mucins are highly glycosylated proteins found in animal mucus and they protect epithelia from physical damage and pathogens [51]. In D. melanogaster, more than 30 mucin-like proteins have been identified [52] but the precise function of most of them remain unknown. It would be interesting to compare the glue genes with the other mucin-like genes in terms of protein domains and sequence evolution. In D. melanogaster, repeats similar to those of Sgs3 (KPTT) are found in the mucin gene Muc12Ea. The high level of glycosylation is thought to favor solubility at high concentration while accumulating in salivary glands ([50]). The richness in cysteines suggests that, upon release in the environment through expectoration, disulfide bridges between glue proteins may be formed by cysteine oxidation by air, making a complex fibrous matrix. Intramolecular disulfide bonds can also be predicted ([50]). Examination of the amino acid composition of the glue proteins suggests that the numerous prolines may induce a zigzag-like shape while serine and threonine, which are very abundant, besides being prone to O-glycosylation, make them very hydrophilic and favor interaction with the solvent and then solubility while preventing folding. The presence of regularly scattered arginines or lysines (or sometimes aspartic and glutamic acids) would add charge repulsion, helping the thread structure to be maintained flat and extended. This is similar to linkers found between mobile domains in some proteins [53]. The shorter Sgs7/Sgs8 would, considering their richness in cysteine, bind the threads together through disulfide bonding.
In the frame of an intrinsically disordered structure (Fig. 4), it is not surprising to observe a high level of repeat number variation (RNV) even at the intra-population level. It has been reported ([54, 55]) that in proteins with internal domain or motif repeats, if these repeats form disordered regions and do not interact with the rest of the protein chain (for a cooperative folding for example), they are more prone to indels which are better tolerated, and favored by the genetic instability of repeated sequences. It is likely that, within a certain repeat number range, variations in repeat numbers might have little effect on the chemical and mechanical properties of the glue. In fact it is likely that the differences in repeat motif sequences rather than the number of repeats would change the mechanical and physical properties of the glue. Accordingly, we measured rather fast rates of evolution, but found no clear indication of positive selection. One reason why the evolution of the repeats is fast (across related species or across paralogs) might be that the constraints to maintain disorder and the thread-like shape are rather loose ([54]).
We do not know the respective roles of the different Sgs proteins in the final glue. Farkaš [50] mentioned that Sgs1 could have chitin-binding properties, which is in line with the function of the glue. He also proposed roles of specific components before expectoration, inside salivary gland granules, related to packaging, solubility. The absence of some glue components may have consequences on its properties and may play a role in adaptation, as suggested by [50]. Gene loss, gene duplication, or repeat sequence change may modify the strength of the glue or its resistance to water or moisture, to acidity (of a fruit) and therefore might be linked to pupariation site preference. D. suzukii lacks both Sgs3 and Sgs4, and has duplications of Sgs7. D. suzukii pupae are found mostly in the soil just below the surface, and less rarely within ripe and wet fruits such as cherries or raspberries, the pupa half protruding [56, 57]. The extensive loss of Sgs genes in D. suzukii may be related to its pupariation in soil. Shivanna et al. ([58]) have related pupariation site preference to the quantity of glue and, counter-intuitively, have reported that species that prefer to pupariate on the food medium in the laboratory produce more glue than species that pupariate on the glass walls of the vials. However, the chemical glue content was not investigated. Another study [59] compared pupariation site preferences between the sibling species D. mauritiana, D. sechellia and D. simulans. While D. simulans populations from the native region share pupariation preference in fruits with D. mauritiana and D. sechellia, worldwide populations preferably pupariate off-fruit, i.e. on a drier and harder substrate. Although the QTL associated with pupariation site preference in D. simulans and D. sechellia do not map to glue genes [59], it would be interesting to see whether, secondarily, significant variations in glue composition or quantity occurred and might be contrasted across D. simulans populations. Given its worldwide expansion associated with adaptation to multiple local environments including diverse pupariation sites, D. melanogaster is an interesting model to study the intraspecific evolution of Sgs genes in relation to adaptation. Interestingly, absence of Sgs4 protein was reported in a few strains from Japan and USA [33], most likely due to deletions or mutations in the promoter region. Our resequencing of a few Nexus lines revealed nonsense mutations within the coding sequence at position 165 in Sgs4, deleting the well conserved C-terminal part. The translational consequences for this protein and for final glue properties remain unknown. In addition to such qualitative protein variations, it is possible that the relative proportions of the Sgs proteins in the glue may change in D. melanogaster according to the ecological circumstances. In this respect, collecting wandering larvae from various substrates, analyzing their glue composition and designing adhesion assays to compare adhesive properties between various glues will be valuable.
In conclusion, the pupal glue appears as a genetically and phenotypically simple model system for investigating the genetic basis of adaptation. The present work provides a first exploration of the evolution of glue genes across Drosophila species and paves the way for future studies on the functional and adaptive consequences of glue composition variation in relation to habitat and geographic and climatic origin.
Identification of Sgs genes in Drosophila species
The seven annotated glue genes of D. melanogaster (Sgs1 (CG3047); Sgs3 (CG11720); Sgs4 (CG12181); Sgs5 (CG7596); Sgs7 (CG18087); Sgs8 (CG6132)) and Eig71Ee (CG7604) were used as BLAST queries for retrieving their orthologs in 19 other Drosophila species. The genome data used for each species is indicated in Table 1. BLAST searches were performed directly through GenBank, FlyBase [60], the SpottedWingFly base for D. suzukii [61] or using local BLAST program (v2.2.25) after downloading the genomes for D. santomea [62] and D. mauritiana [63]. The BLASTP and TBLASTN programs were used [64], without filtering for low complexity, which otherwise would have missed the repeated regions. Repeats, when present, were often quite different from the repeats present in D. melanogaster Sgs sequences. Consequently, BLAST results were often limited to the C-terminal part of the targeted gene, which was the most conserved part of the proteins, and to a lesser extent to the N-terminal end. For each species, a nucleotide sequence containing large regions upstream and downstream of the BLAST hits was downloaded from InsectBase [65] or from species-specific websites when genome data was not present in InsectBase (Table 1). We used Geneious (Biomatters Ltd.) to identify by eye the coding regions, the start of which was identified by the signal peptide sequence. Putative introns were also identified manually, guided by the intron-exon structure of the D. melanogaster orthologs. In cases of uncertainties or missing sequence data, we extracted DNA from single flies of the relevant species (Table 4) and the questionable gene regions were amplified with primers chosen in the reliable sequence parts (Additional file 9: Table S2), and sequenced by the Sanger method using an ABI 3130 sequencer. For instance, we characterized the exact sequence corresponding to N stretches in the published sequence of D. mauritiana Sgs4; we found that the published premature termination codon (PTC) of D. biarmipes Sgs3 was an error and that three frameshifts found within 50 bp in D. sechellia Sgs1 were erroneous.
Evolutionary relationships between genes and estimate of evolutionary rates
Alignments of DNA or protein sequences were done using MUSCLE [66] implemented in Geneious and protein trees were computed using PhyML, as implemented in the online server Phylogeny.fr [67], drawn using iTOL [68], and rooted at midpoint. The substitution rates dN and dS values for over 10,000 coding sequences computed for D. melanogaster/D. simulans comparisons were retrieved from the flyDIVaS database [47] but Sgs genes were not included in this dataset. Thus, dN and dS were computed using yn00 in the PAML package ([69]), removing the unalignable parts. We tested for episodic diversifying selection across species using the branch-site random effect likelihood (BS-REL) algorithm implemented in the HyPhy package [70, 71] at the Datamonkey server (classic.datamonkey.org) [72]. We used only genes devoid of repeats to ensure reliable aligments, and we supplied species trees for the analysis.
Test for accelerated gene turnover
To infer ancestral gene counts in the three newly classified Sgs gene families and to determine whether the three newly classified Sgs gene families are evolving rapidly we first need to determine the average rate of gene gain and loss (λ) throughout Drosophila. Previous studies have estimated λ from 12 Drosophila genomes and found rates of 0.0012 gain/losses per million years [4] and 0.006 gains/losses per million years after correcting for assembly and annotation errors [37]. However, since those studies numerous additional Drosophila genomes have been published. In order to update the gene gain/loss rate (λ) for this genus, we obtained 25 available Drosophila peptide gene annotations from NCBI and FlyBase. The latest versions at the time of study for the genomes of the original 12 sequenced species (ananassae v1.05, erecta v1.05, grimshawi v1.3, melanogaster v6.10, mojavensis v1.04, persimilis v1.3, pseudoobscura v3.04, sechellia v1.3, simulans v2.02, virilis v1.06, willistoni v1.05i, and yakuba v1.05) were downloaded from FlyBase [73] and 13 other species (arizonae, biarmipes, bipectinata, busckii, elegans, eugracilis, ficusphila, kikkawai, miranda, navojoa, rhopaloa, suzukii, and takahashii) were downloaded from NCBI [74].
To ensure that each gene from the 25 Drosophila species was counted only once in our gene family analysis, we used only the longest isoform of each protein in each species. We then performed an all-vs-all BLAST search [75] on these filtered sequences. The resulting e-values from the search were used as the main clustering criterion for the MCL (Markov cluster algorithm) program to group peptides into gene families [76].This resulted in 17,330 clusters. We then removed all clusters not present in the Drosophila ancestor, resulting in 9379 gene families. An ultrametric phylogeny with branch lengths in millions of years (my) was inferred using MCL in a similar fashion, with the addition of the genome of the house fly, Musca domestica, as an outgroup and utilizing single-copy orthogroups between all 26 species [77]. Calibration points at the split of D. pseudoobscura/D. melanogaster (49–59 my), D. melanogaster/D. grimshawi (64–74 my), and Musca domestica/D. melanogaster (156 my) were from Timetree.org [78, 79].
With the gene family data and ultrametric phylogeny as input, we estimated gene gain and loss rates (λ) with CAFE v3.0 [4]. This version of CAFE is able to estimate the amount of assembly and annotation error (ε) present in the input data using a distribution across the observed gene family counts and a pseudo-likelihood search. CAFE is then able to correct for this error and obtain a more accurate estimate of λ. We find an ε of about 0.04, which implies that 4% of gene families have observed counts that are not equal to their true counts. After correcting for this error rate, we find λ = 0.0034. This value for ε is on par with those previously reported for Drosophila (Additional file 10: Table S3; [37]). However, this λ estimate is much higher than the previous reported from 12 Drosophila species (Additional file 10: Table S3; [4, 37]), indicating a much higher rate of error distributed in such a way that CAFE was unable to correct for it, or a much higher rate of gene family evolution across Drosophila than previously estimated. The 25 species Drosophila phylogeny was then manually pruned and modified to represent the 20 Drosophila species in which Sgs gene families have been annotated. Some Sgs gene families are not present in the ancestor of all 20 species, so additional pruning was done to the phylogeny for each family as necessary (see Additional file 1: Table S1). The phylogeny, Sgs gene copy numbers, and the updated rate of gene gain/loss (λ = 0.0034) were then used by CAFE to infer p-values in each lineage of each family (Additional file 11: Table S4). Low p-values (< 0.01) may indicate a greater extent of gene family change along a lineage than is expected with the given λ value, and therefore may represent rapid evolution.
Search for polymorphism and repeat number variation in D. melanogaster and D. mauritiana
Polymorphism in D. melanogaster was investigated in the coding regions, especially the repeat number variation (RNV). We intended to use the data from the Drosophila Genome Nexus study ([49, 80], available at the Popfly web site [81]) to assess RNV. This database contains resequenced and aligned genomes of hundreds of D. melanogaster lines from about 30 populations from all over the world. Those data, like most D. melanogaster populations' and other species' genomes were obtained using NGS technologies, which yielded short reads. The data were often not accurate in repeat regions, likely because short reads may be not properly assembled when there are numerous short tandem repeats, and thus could not be used for counting RNV. Thus, experimentally, using single-fly DNAs, we amplified and sequenced the repeat-containing Sgs3 and Sgs4 from one or a few individual flies from several strains or natural populations available at the laboratory (French Guyana, Ethiopia, France, Benin, Ivory Coast, India, Comores, and the laboratory strain Canton S), and from a number of lines used in the Drosophila Genome Nexus study (Table 4). In addition, we investigated the occurrence of possible premature termination codons in gene alignments from the Drosophila Nexus database [49, 80], available at the Popfly web site [81] and checked the results by PCR in Sgs4 and Sgs5 (Table 4). We also used data from the Drosophila Nexus database to study polymorphism and divergence in Sgs5 and Sgs5bis, which are devoid of repeats, and are not too short. Four populations represented by numerous lines were retained for analysis: ZI (Siavonga, Zambia), for the ancestral geographical range, EF (Fiche, Ethiopia), which shows overall rather large differentiation (Fst) with most other populations [49], and FR (France) and RAL (Raleigh, USA) for the worldwide populations. Diversity and divergence indices were computed with DnaSP [82]. Experimental sequences were deposited to GenBank with accessions MH019984-MH020055.
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Delport W, Poon AF, Frost SD, Kosakovski Pond SL. Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics. 2010;21(10):2531–3.
Gramates LS, Marygold SJ, Santos GD, Urbano JM, Antonazzo G, Matthews BB, Rey AJ, Tabone CJ, Crosby MA, Emmert DB, et al. FlyBase at 25: looking to the future. Nucl Ac Res. 2017;45(D1):D663–71.
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The authors thank Dr. Georges Feller for comments on the disordered protein regions, and Dr. Amir Yassin for critical reading of the manuscript. The authors are grateful to three anonymous reviewers for their fruitful comments.
The research leading to this paper has received funding from the regular annual funding of CNRS to JLDL, MB and VCO and from the European Research Council under the European Community's Seventh Framework Program (FP7/2007–2013 Grant Agreement no. 337579) to VCO. GWCT is supported by NSF DBI-1564611. The funding bodies had no role in study design, analysis and interpretation, or writing the manuscript.
Available upon request to the authors
UMR 9191 Évolution, Génomes, Comportement, Écologie. CNRS, IRD, Université Paris-Sud. Université Paris-Saclay, F-91198, Gif-sur-Yvette, France
Jean-Luc Da Lage & Magalie Bonneau
Department of Biology and Department of Computer Science, Indiana University, Bloomington, IN, 47405, USA
Gregg W. C. Thomas
Institut Jacques Monod-CNRS UMR7592-Université Paris Diderot, 15 rue Hélène Brion, 75013, Paris, France
Virginie Courtier-Orgogozo
Magalie Bonneau
VCO and JLDL designed the study and analyzed data; JLDL and MB performed experimental work; GWCT performed CAFE analysis; JLDL, VCO and GWCT wrote the manuscript. All authors have read and approved the manuscript.
Correspondence to Jean-Luc Da Lage.
The authors declare that they have no competing interest.
Table S1. Number of gene copies for each family, and results of CAFE analysis for the glue gene families. (XLSX 161 kb)
Figure S1. Ancestral states for the Sgs1–3–7-8 gene family inferred by CAFE. Species tips are labeled with the observed gene count and internal nodes are labeled with inferred gene counts. Orange branches represent gene losses, blue branches represent gene gains, while black branches represent lineages in which no change in gene copy number is observed. Branches marked with asterisks have marginally significant p-values (< 0.05). (PDF 173 kb)
Figure S2. Partial alignment of Sgs3 sequences with translation in D. melanogaster individuals. EF: Ethiopia; Chavroche and Gally: France; Cotonou: Benin; Delhi: India; Cayenne: French Guyana. (PDF 2258 kb)
Figure S3. Partial alignment of Sgs3 sequences with translation in the EG population (Cairo) of D. melanogaster. (PDF 1733 kb)
Figure S4. Partial alignment of Sgs4 sequences with translation in D. melanogaster individuals. EF: Ethiopia; Chavroche and Gally: France; Cotonou: Benin; Delhi: India; Cayenne: French Guyana; Tai: Ivory Coast. (PDF 2312 kb)
Figure S5. Partial alignment of Sgs4 protein sequences in the EG population (Cairo) and ZI (Zambia) of D. melanogaster. The reference sequence is shown. Asterisks indicate premature stop codons. (PDF 966 kb)
Figure S6. Partial alignment of Sgs3 (A) and Sgs4 (B) amino acid sequences in D. mauritiana individuals. Sgs3 mau and Sgs4 mau are the sequences from the online genome. Sgs4 mau has been corrected with our resequencing. Xs are undetermined amino acids. (PPTX 452 kb)
Figure S7. Output trees of Branch-Site-REL analyses (classic.datamonkey.org). "The hue of each color indicates strength of selection, with primary red corresponding to ω>5, primary blue to w = 0 and grey to w = 1. The width of each color component represent the proportion of sites in the corresponding class. Thicker branches have been classified as undergoing episodic diversifying selection by the sequential likelihood ratio test at corrected p ≤ 0.05". MEL: melanogaster, SIM: simulans, SECH: sechellia, SAN: santomea, YAK: yakuba, ERE: erecta, TAK: takahashii, SUZ: suzukii, BIAR: biarmipes, FIC: ficusphila, KIK: kikkawai, ANA: ananassae, BIP: bipectinata. (PDF 51 kb)
Table S2. List of primers used for this study. Different combinations were used to amplify glue genes. All primers were chosen outside the repeated regions. D. sechellia, D. santomea, D. virilis and D. biarmipes were resequenced because of uncertainties or putative errors in the online sequences. D. melanogaster and D. mauritiana were resequenced for studying RNV in Sgs3 and Sgs4. (DOCX 102 kb)
Additional file 10:
Table S3. Assembly/Annotation error estimation and gene gain/loss rates in a single λ model in the 25 Drosophlia species included in this study compared to previous studies using fewer species. (DOCX 48 kb)
Table S4. Summary of gene gain and loss events inferred after correcting for annotation and assembly error across all 25 Drosophila species. The number of rapidly evolving families is shown in parentheses for each type of change. (DOCX 107 kb)
Da Lage, JL., Thomas, G.W.C., Bonneau, M. et al. Evolution of salivary glue genes in Drosophila species. BMC Evol Biol 19, 36 (2019). https://doi.org/10.1186/s12862-019-1364-9
Internal repeats
Disordered protein
Eig71Ee
Gene family
Gene copy number


The CSSS Alphonse-Desjardins visitors (including the Hôtel-Dieu de Lévis), the Centre de congrès et d'expositions de Lévis visitors (whose square footage was doubled in 2015) and the ferry passengers can easily converge towards Les Galeries Chagnon. The mall is home to major retailers such as Pharmaprix, Sports Experts/Atmosphere, Renaud-Bray and Walmart. It hosts also the supraregional service center of Desjardins Caisse de Lévis. Sales per square foot are approximately $510. Annual visits exceed 5 million people.


text:
Penalty cut for Notre Dame in videographer's death
Stacy St. Clair
The University of Notre Dame today reached agreement to pay a significantly smaller penalty for its role in the death of Declan Sullivan, a football team videographer who died last fall when a hydraulic lift he was using toppled over in high winds.
The school will now pay a $42,000 fine as part of a settlement deal with the Indiana Occupational Safety and Health Administration.
In exchange for the smaller fine, the university is required to launch a national education campaign on the safe use of scissor lifts. The school will also make a donation to Sullivan's memorial fund and designate a liaison between Notre Dame's athletic department and the risk-management division to make sure all employees are properly trained.
The university will not disclose how much it will donate to the memorial fund, university spokesman Dennis Brown said.
"What remains ever-present throughout this process is the reality of the loss experienced by Declan Sullivan's family and friends, all of whom remain in the thoughts and prayers of the Notre Dame community," university spokesman Dennis Brown said in a statement. "Notre Dame is committed to doing all we can to ensure that a tragedy like this never happens again and is working with IOSHA and others on a national education initiative on aerial-lift safety in regard to setup, training and weather-related risks."
Sullivan's family, which has been steadfast in support of Notre Dame since the accident, released a statement saying it was satisfied with the settlement.
"We also look forward to seeing the launch of the education campaign and expect it will provide a safer work environment for institutions around the country," Sullivan's uncle Mike Miley said in a statement. "There can be no better way to remember Declan than to help others avoid future tragedies."
Notre Dame also will provide IOSHA with a list of other areas of the university where scissor lifts are in use and complete a refresher training for all operators of those lifts within 90 days, according to the agreement.
Though the university already indicated plans for a national program, enhanced safety measures and the Sullivan memorial months ago, the resolution requires that those steps be taken and sets a timetable for their execution. Without the agreement, the state would have had no legal authority to compel the school to follow through, IOSHA officials said.
"Notre Dame has said multiple times publicly that it wants to ensure nothing like Declan's death occurs again on its watch, and that it wants to honor Declan's memory," said IDOL Commissioner Lori Torres. "We believe this unique agreement allows Notre Dame to live up to those statements, and it allows our agency to carry out its primary mission, which is to advance the safety of employees throughout the state."
The agency initially fined the school $77,500 in March for ignoring industry standards that could have prevented Sullivan's death. The university filed a notice of contest with IOSHA in April, shortly before it released the results of its own internal investigation.
Notre Dame specifically objected to IOSHA's finding that it "knowingly" put Sullivan in harm's way because there are no national standards for wind-speed protocols when using hydraulic lifts. IOSHA agreed to eliminate that language as part of the settlement, even though the winds that day far exceeded international and industry standards for using the lifts.
The university's internal report blamed the accident on a "sudden and extraordinary" wind and long-standing policies.
Sullivan, a 20-year-old film and marketing student from Long Grove, was working Oct. 27 as a paid employee of the school's athletic department when he went up in an aerial scissor lift to record the football team's practice.
The National Weather Service had issued a wind advisory for the day, and gusts reached 53 mph about the time of Sullivan's fall. The lift crashed through a fence and landed on a street. In keeping with international standards, the lift industry recommends grounding the equipment when winds exceed 28 mph.
According to the university's internal report, the football program's policy was to keep the videographers off the hydraulic lifts if the winds reached more than 35 mph. The weather data accessed by the staff never crossed that threshold, school officials said. Sullivan had learned of the wind advisory from weather.com shortly before practice and tweeted his concerns about the conditions before and during the training session.
The football staff accessed the same website several times during the day but missed the wind warnings on the page, according to the university's internal report.
No one was disciplined for their role in the accident, school officials said. The report also did not blame any individuals for breaking safety protocols.
In light of the internal report, the university proposed new safety protocols, including the installation of on-field wind gauges and the adoption of the international wind-speed standards when operating lifts. It also has banned the use of lifts to record practices and replaced them with a remote-controlled camera system.
"This agreement goes far beyond the requirement to pay fines," said Torres. "It requires one of the nation's most well-known universities to create and implement a mechanism for spreading education about scissor lifts that are used in a range of educational and athletic settings, it requires changes at Notre Dame to ensure employee safety going forward, and it formally recognizes Declan Sullivan's memory."
Guilty plea lays bare ex-state Sen. Martin Sandoval's greed in red-light camera bribery scheme. 'So why don't I get that offer?'

redpajama_set_name: RedPajamaCommonCrawl


Wulverblade is an old school side scroller beat 'em up with a twist: instead of walking the seedy back alleys of a modern city, this game is set in 120 AD, where you play one of three characters belonging to a tribe of Northern Britons fighting off a Roman invasion.
I used to adore side scroller games like Double Dragon, even though I totally sucked at video games as a kid. As 3D gaming quickly became the norm, the 2D brawler went out of style, as expected, but there are gamers and developers out there who have a soft spot for side scrollers.
Enter Fully Illustrated and Darkwind Media, taking this genre, adding in an incredible amount of violence plus loads of historical research, and you get Wulverblade.
So Wulverblade plays like an old school beat 'em up. You walk to the right, you are swarmed with enemies, and you need to fight your way out. You use a sword or any other weapon you can pick up, and timing your attacks can lead to combos. Aside from hoes, knives, and axes, you can also pick up dismembered body parts like hands and heads to use as weapons.
Don't let the cartoonish art style fool you. This game is bloody. Every kill spills an enormous amount of blood on the ground, splattering body parts everywhere. If that wasn't enough, you can go into berserker mode to attack your enemies without worrying about getting damage, or you can even call on wolves once every level to add to the carnage. It's insane and it's great.
It's not all mindless violence, though. Set during a very real period in the Roman occupation of Britain, Wulverblade is full of well-written and well-researched historical notes that should satisfy any history buff. Heck, I never even thought about this time period since I live on the other side of the world, and I found reading through the notes engaging.
Now if there's anything Wulverblade needs to fix, it would be the loading times. They're long, and if they manage to cut down the loading times, it'll greatly improve the game.
The Roman Empire is about take control of the north of Britannia and quash the last remaining tribes – and you must stop them dead in their tracks! Play as either Caradoc, Brennus or Guinevere – members of a family originating in the northern tribe of the Caledonii. This family of warriors has been the guardian of the most northerly tribes of Britannia for generations. The Romans have invaded Britain and conquered all that lies before them… until now.
Defend the homeland from the Roman army with strength, courage and a healthy number of wolves. Unleash deadly combos and devastating finishers to turn the tide of battle as you fight through eight scenic levels. Explore a rich, historically inspired story filled with all-out mayhem, manic action, and plenty of murder in this faithful homage to classic side-scrolling beat 'em ups.
Wulverblade brings back the glory days of Golden Axe, Sengoku, and Knights of the Round with over 8 varied levels filled with sharp blades, enemy hordes, and blood-soaked arenas. Slice through the invading Roman Legions in graphic detail on your own – or enlist a friend for the slaughter in two-player co-op!
I have a more in-depth review over on Play Without Apology. You can get Wulverblade for the Nintendo Switch here!
You can also get Wulverblade for Steam, PS4 and Xbox One.


For one day, the California family court is not taking anyone's kids away. It is not causing its usual destruction. The judges have been furloughed because the state is short of money, and they each make about $150k per year. They are supposed to give up a day's wages, but some judges are refusing.
I say the state should just shut down the family court permanently, and everyone would be better off.


"We've shut more sites down faster than ever," said Mat Crocker, head of waste at the agency.
"We put additional resources to it, we got more people in with the right skill sets, and we've also targeted our efforts at the right people so we can deal with it faster."
"We target our prosecutions at the most serious offenders," Mat Crocker explained. "Quite often by providing clear information and following it up with the right type of encouragement, people stop.
"Prosecution is just one of the tools in the bag, really."
The agency identified 820 active sites in March this year - the vast majority around these pivotal transport routes.


Tonight, Elisabeth Hasselbeck will debut the line of women's clothing she designed on QVC at 9 PM EST. She previewed some pieces on The View this morning. Our critique, after the jump.
Like a lot of celebrity-designed lines on QVC, Elisabeth's line is affordable, with price points ranging from about $40 - $80. "Affordable" doesn't have to mean "cheap," but in this instance, it does. And I swear on my superficially-aborted fetuses that my opinion on this is in no way swayed by Elisabeth's opinions on anything, although admittedly, it's equally perfunctory.
Anyway, this is Elisabeth's modernized version of a nautical jacket ($59), and two different ways to wear it. She also designed both pairs of pants ($44, $49).
There are lots of animal prints in this line—like this cardigan ($59)—and three-quarter length tops and capri pants, because when she was designing the line, Elisabeth thought of something that Joy often says: "The wrists and ankles are the last to go."
Elisabeth calls this shirt ($39.94), "A little T-shirt with alotta flirt." Of the design, she said, "I thought, 'Heck, let's give it a try.'"
This pinstripe shirt ($48)—perfect for a Concerned Women for America meeting—has a metallic flair.
"I took animal to the next level," Elisabeth said of this sheer blouse ($54.54). She told Joy that "it'll let you survive any hot flash." Seriously, the jokes are writing themselves here.
She called this top ($39.84) the "anchor piece of the collection." Maybe it makes you look heavy, no matter your body type.
Sherri totally wants out of there.


text:
Category Archives: General Idiocy
This is where a coaching hire winds up when you don't use a search firm.
Well, this is, um, something: Chad Plein, a sports anchor for KY3/KSPR, is reporting that Bobby Petrino will become the new head coach at FCS Missouri State. The football scoop site is saying it's gonna be Art Briles who gets announced tomorrow as the new head coach.
— Bruce Feldman (@BruceFeldmanCFB) January 15, 2020
There ain't enough popcorn in the world for this one.
Any other time a school would have hired Bobby Petrino, the reaction would be, "I can't believe a school would hire that sleazebag."
But when it's over Art Briles it's like, "Well, at least it's not Art Briles!!!" https://t.co/y4PGsLY6vo
— Jim Weber (@JimMWeber) January 15, 2020
Filed under General Idiocy
The new normal doesn't seem that normal.
To paraphrase Robin Williams, this quote is Gawd's way of telling you you have too much money:
Texas Tech's football coaching staff turnover between the 2018 and 2019 seasons will wind up costing its athletics department nearly $6.6 million…
"It's something that was budgeted for, so prepared for," Tech senior associate athletics director Robert Giovannetti said. "It's part of the business, right? You understand it's a part of the business and you chalk that up to cost of doing business and don't let it affect the way you run your operation."
Um… dude, your football team finished 5-7 and 4-8 those two seasons. Maybe you should let it affect the way you run your operation.
I'm pretty much convinced that a school could pick at random a GTP commenter as its next AD and wind up with a better run athletic department. The sad thing is these guys are all convinced they're doing a bang-up job.
Filed under General Idiocy, It's Just Bidness
If you can't throw a pass, throw a punch.
Punches fly in bowl games after dark. Ref looking for that workers comp. pic.twitter.com/wKF37CMGED
— Warren Sharp (@SharpFootball) December 27, 2019
What a way to go.
Eastern Michigan quarterback Mike Glass III was ejected with 10 seconds left in Thursday night's Quick Lane Bowl in Detroit after throwing punches at two Pittsburgh players.
Glass smacked Panthers linebacker Cam Bright in the face mask, then hit defensive back Paris Ford across the helmet. He also appeared to make contact with an official, who was trying to keep players separated.
A brief scuffle ensued, and Glass was ejected from the game, a 34-30 Eastern Michigan loss.
And to think some folks believe minor bowl games are meaningless.
At least that happened in the heat of battle. Mississippi State didn't even wait for the bowl game to get into it.
If at first you don't succeed, you're probably qualified to be an athletic director.
If you wonder why I feel like most college athletic directors are generally clueless when it comes to hiring/firing decisions, here are just a few recent snapshots to ponder:
One nugget I picked up today in the postmortem of the Arkansas coaching search: Among the candidates the Razorbacks interviewed, I'm told, was Deion Sanders.
— Dan Wolken (@DanWolken) December 9, 2019
Colorado State, with help from Urban Meyer, met with five candidates – Butch Jones, Dave Aranda, Tony Alford, Steve Addazio and Kevin Wilson. There's a strong indication CSU favors a hire with HC experience.
— Steven Godfrey (@38Godfrey) December 9, 2019
We have confirmed that #Mizzou will pay new coach Eliah Drinnkwitz $4 million per year, which represents a more than 400 percent raise for Drinkwitz. Story: https://t.co/DAjfOwbUey
— Mitchell Forde (@Mitchell4D) December 9, 2019
Hard to argue with this conclusion:
The coaching market is so messed up.
Nothing against Drinkwitz, but if you offer a guy with 1 year's experience $3M and he turns you down .. there are other choices. https://t.co/TVfFdUgkFA
— Stewart Mandel (@slmandel) December 9, 2019
Does this really strike you as a pattern of guys who have the first clue as to what they're doing? Colorado State's probably the closest to being rational, but, then again, Corch as a consultant? Only thing he's got insight on is that he had a couple of those guys on his staffs. I'm sure he's gonna step right up and say he made a mistake hiring them.
Mandel's also right about this.
Early signing period is causing schools with openings to make rush hires that, if they get wrong, will set the program back way further than 1 recruiting class.
Ordinarily I'd say clueless and pressured is no way to go through life, son, but this is college football we're talking about. If things don't work out, they'll just pay the buyout and make the same mistakes again in hopes that they get lucky next time around.
Filed under College Football, General Idiocy
It's gonna be some offseason in Oxford.
I see the football gods were in Starkville, Mississippi last night.
This Egg Bowl ending was INSANE 🤯
Ole Miss scored with 4 seconds left and had a chance to tie the game with the PAT.
After a flag for the excessive celebration moved the PAT back, the kicker missed wide right and Mississippi State held on to win the Egg Bowl 21-20. pic.twitter.com/z4U7c8JukQ
— ESPN (@espn) November 29, 2019
Jon Solomon is calling that the "Piss and Miss", which ain't bad, but, really, the jokes are gonna write themselves for a while.
I just hope somebody asks that kid if it was worth it and, after pondering the question for a moment, he answers, "totally". Might as well own it at this point.
Filed under General Idiocy, SEC Football
#BoycottGameDay?
Man, first I thought people were getting worked over what's been a mediocre, tired show, something I haven't bothered to watch for years, and decided to stick it to Mickey. Good show!
Then I found out it's just some whiny Ohio State fans.
At his first-ever headgear pick on College GameDay, Coach Lee Corso pulled on Brutus the Buckeye's mascot head and correctly picked the Ohio State Buckeyes to beat the Penn State Nittany Lions. That was 1996, and since then, Ohio State University has hosted ESPN's pregame show 18 times. On November 23, 2019, OSU hosts for the 19th time, which is the most all-time and five more than Alabama.
With that resume, you'd think Buckeye fans would be excited for Rece Davis, OSU alum Kirk Herbstreit and the gang to set up shop in Columbus, right? Apparently, a lot of fans hate the idea of ESPN and the network's "SEC bias" coming to their town, and some are planning a full-on boycott of the upcoming show.
Get a life, snowflakes.
Filed under ESPN Is The Devil, General Idiocy
"He added that alcohol is believed to be a factor in the incident."
Shot. (See what I did there?)
Two lifelong friends house-sitting east of Bend got in an alcohol-fueled argument, then a fight over a college football game Saturday afternoon, prompting one man to get a shotgun and shoot the other, Deschutes County sheriff's deputies said. The Bend man was jailed on attempted murder and other charges…
Detectives learned Honey and the other man were at the home, watching the LSU-Alabama football game. An argument escalated into a physical fight, which in turn led Honey to get a shotgun with a birdshot shell and shoot the other man once in the head and neck area as the man walked away in the driveway, Bailey said.
Bailey added that the man was not barricaded in the home and reportedly went back inside to finish watching the game before surrendering to deputies.
What are the odds PAWWWLLL!!! books this dude for his show this week?
(h/t)
Filed under Crime and Punishment, General Idiocy, SEC Football


text:
Q: Как в С++ вызвать шаблонную функцию напрямую? [продолжение] Добрый день.
Я уже задавал похожий вопрос вот ссылка на мой старый вопрос, и снова он возник.
Итак, есть код:
#define OPERATIONWRAPPER(name)                         \
void name(ob_savewrapper* a){                             \
var* tmpptr=ptr->name##_v(a->ptr);                     \
if(tmpptr==ptr){ a->clear();} else {clear();transfer(a);} \
}

    //some realization
    class savewrapper {
        var* ptr;
    public:
        savewrapper() :ptr(NULL) {}
        savewrapper(var* p) :ptr(p) {}

        void setptr(var* s) {
            if ((ptr != NULL) && (s != NULL)) {
                clear();//for eliminate double delete
                ERR(ob_err78)
            }//if
            ptr = s;
        }

        void clear() { delete ptr; ptr = NULL; }
        ~savewrapper() { delete ptr; }

        void transfer(savewrapper* d) {
            setptr(d->ptr);
            d->setptr(NULL);
        }

        OPERATIONWRAPPER(add);
        OPERATIONWRAPPER(sub);
        OPERATIONWRAPPER(mul);
        OPERATIONWRAPPER(div);
        OPERATIONWRAPPER(pww);

    };

Здесь есть иерархия классов, в которой класс var является базовым.
Класс savewrapper это обертка над классами этой иерархии.
В классах иерархии есть функции add_v, sub_v, mul_v, div_v, pww_v.
В классе savewrapper должны быть функции add, sub, mul, div, pww, в которых вызываются соответствующие функции из классов иерархии var.
Сейчас это сделано через макрос, как видно в примере. Но я хочу быть передовым программистом, и поэтому я решил переделать макрос в шаблон. Пишу шаблон:
template<var* (var::*method)(var* ptr1)> inline void operation_wrapper(savewrapper* a) {
            var* tmpptr = (ptr->*method)(a->ptr);

            if (tmpptr == ptr) {
                a->clear();
            }
            else {
                clear();
                transfer(a);
            }
        }

Инстанцирую шаблон для примера для метода add:
void add(savewrapper* a) { operation_wrapper<&(var::add_v)>(a); }

Все даже работает, но есть лишний вызов по сравнению с решением на основе макроса. А именно, хотя шаблон объявлен как inline, но инлайн не гарантируется компилятором. А если шаблон будет не инлайн, то будет лишний вызов функции, по сравнению с решением на основе макроса.
Вопрос:

*

*Как сделать так, чтобы не было лишнего вызова функции? Имя add для метода класса savewrapper должно сохраниться, так как во многих местах оно используется и подставлять по всему коду вместо add явный вызов шаблона
operation_wrapper<&(var::add_v)>(a);
это очень плохой вариант.
Спасибо.
UPD1:
Вобщем, как не крути, но получается что для шаблонов функций нужно иметь еще одну возможность. А именно, при инстанцировании шаблона функции нужно иметь возможность дать получившейся функции новое имя. Иначе шаблоны проигрывают по гибкости макросам.

redpajama_set_name: RedPajamaStackExchange


text:
Farewell August '14
This is the part where I usually engage in some discussion about tea, not any old tea but pu'er, the vaunted king of teas or perhaps I should say queen of teas, as pu'er is the mother source of all tea throughout the world.
My tea exploits are comprised of two parts: one is primarily for health professionals; the other is for tea enthusiasts. In both instances, the objective is to not only provide quality and value but also uniqueness, stuff that you're not likely to find on-line.
To this end, I'd like to introduce you to the veal of tea, Yabao, stuff you can have without a guilty conscience. Yabao is comprised of the shoots of the tea tree branch, as opposed to the leaf. It yields certain distinguishing features, a resinous perfume being primary among them. Quick infusions produce a crystal clear liquor ranging from a champagne to goldenrod caste. Enough cannot be said about the uniqueness of its flavour, not in that "do I like this" uniqueness but in a way more usually associated with the flavoured Indian teas. For me it really evokes a sense of sandalwood and cedar.
YaPaoDay
Just recently, I've started taking the spent leaves, which yield about 10 infusions, and cold brewing it along with other spent pu'ers, in addition to dried longgan and hibiscus to much fanfair by patients. More than one has speculated that such pleasingly sweet a blend would be well-received in the South, though it has been my experience that Cantonese aren't the most adventuresome when it comes to these kind of things. 😉
By the way, cursory search for longgan did not turn up any appealing prices, so just reach out if you're really interested. As far as the Yabao is concerned a 357/g cake goes for $39 and I'll be running a discount special of 15% until the full moon of October.
More pu'er tea pics? Visit Universotea.com


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Posted inW42ST Daily
NY Comedy + Candidates West Side Stories
by Phil O'Brien August 17, 2022
Good morning and happy Wednesday. Yet another wonderful summer day. High of 80 degrees and a chance of a light shower this afternoon. Go have fun in the sun!
It's time to vote in the Democratic primary for New York District 12. We asked the three main candidates to tell us their West Side Stories. Check out what Carolyn Maloney, Jerry Nadler and Suraj Patel had to say.
But seriously, folks — Mayor Eric Adams announced the return of the New York Comedy Festival this fall, ahead of Carolines on Broadway's 40th anniversary year. Read more…
What we've been reading
It's 43 years since a special unit was created on Rikers for transgender people — now it is failing. (THE CITY)
More electric buses are heading to the streets of NYC. (TimeOut)
It's National Thrift Shop Day — here's how to dispose of your old clothes in a sustainable way. (New York Times)
Soul Cycle changes gear and closes locations. (NBC)
Check out this cool list of vinyl stores in New York. (Secret NYC)
We checked out the new Hard Rock Hotel in Times Square for the first time yesterday. Delighted that a local girl made good took center stage in the lobby. "Born January 25, 1981, in Hell's Kitchen, New York, Alicia Keys became an international success story from the day her first album dropped in 2001. Songs in A Minor went platinum five times over and earned her five Grammys. Alicia Keys wore this yellow Max Azria Atelier gown during her appearance at the 2009 Academy Awards Vanity Fair after-party," said the plaque. The bar up the stairs looked amazing too!
Thank you for your readership. If you can support our ongoing work to keep Hell's Kitchen connected, updated and upbeat — please hit the button below.


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They're also playing host to Arkansas, which has lost eight consecutive Southeastern Conference games, before likely heading into the SEC Tournament with a first-round bye.
The No. 6 Rebels face a much tougher opponent this week, Texas A&M, but two or three victories and a decent performance in the SEC Tournament could be enough to catapult them into the conversation for being worthy of a national seed.
Ole Miss hoping to "make some more magic" vs. Texas A&M
Or they could face consequences because of how good their conference is. Florida and Texas A&M are the top two teams, respectively, while the Bulldogs are No. 4.
"While in a traditional year Ole Miss may have the resume to be a slam dunk type of national seed — a top-5 RPI assuming they finish strong this week, they'll be near the top of the SEC standings — we don't really know how the committee will gauge going four-, five-deep in a conference in your top eight nationally," Etheridge said.
"Three conference teams have made the top eight several times, but we've never had four. In this case, we could be talking about five candidates (including LSU) in the top eight in the SEC."
Southern Miss has never hosted a regional as a No. 1 seed but did so as a No. 2 seed in 2003. The Golden Eagles need to keep winning to hold off No. 17 Florida Atlantic and No. 21 Rice but have Conference USA's top batting average (.305) and fifth-best ERA (4.20).
"We control our own destiny," USM first baseman Tim Lynch said. "That's what everybody wants. If we go out and play our game, we'll get the outcome we want. We just need to keep our blinders on, keep playing good baseball and stay focused on our goals."
Surging in polls, USM to host UL-Monroe
If the No. 19 Golden Eagles host a regional, they may be paired with a nationally-seeded Mississippi State. The winner of that super regional would advance to the College World Series.
Etheridge calls the Golden Eagles one of the feel good stories of the season. It's a fitting description for the success all three Mississippi teams are having in baseball, three seasons that are proving all of the preseason polls and predictions incorrect.
"You wouldn't have predicted (all three hosting before the season began)," Etheridge said. "You probably wouldn't have predicted two of the three."
The Clarion-Ledger's Michael Bonner and the Hattiesburg American's Jason Munz contributed to this story.
Contact Daniel Paulling at dpaulling@jackson.gannett.com. Follow @DanielPaulling on Twitter and Daniel Paulling on Facebook.
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