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Figure S1

Diagram of the experimental design to test if the crowding cues experienced by hermaphrodite mothers change the sex ratios of the F1 generation. Dauer larvae were isolated onto five 6 cm plates under each condition (control and crowded) and left to mature into a hermaphrodite. F1 eggs were then collected and placed individually onto non-treated 3.5 cm plates. Egg collection occurred >3 d and hermaphrodite mothers were moved to new plates (of their original condition) each day. Eggs were allowed to develop until adulthood and sexed.
Diagram of the experimental design to test if the crowding cues experienced by hermaphrodite mothers change the sex ratios of the F1 generation. Dauer larvae were isolated onto five 6 cm plates under each condition (control and crowded) and left to mature into a hermaphrodite. F1 eggs were then collected and placed individually onto non-treated 3.5 cm plates. Egg collection occurred >3 d and hermaphrodite mothers were moved to new plates (of their original condition) each day. Eggs were allowed to develop until adulthood and sexed.

Figure S2

Visualization of the contamination present in the four genomic libraries using BlobTools. The libraries were generally free from contaminants except for a few (<2%) proteobacteria most likely corresponding to the nematode food source (E. coli strain OP50-1).
Visualization of the contamination present in the four genomic libraries using BlobTools. The libraries were generally free from contaminants except for a few (<2%) proteobacteria most likely corresponding to the nematode food source (E. coli strain OP50-1).

Figure 1

Pictures of a (A) male, (B) female, and (C) hermaphrodite of Auanema melissensis on day 1 of adulthood (100× magnification). A dauer larva (D), fated to develop as a hermaphrodite, is depicted at a magnification of 112×.
Pictures of a (A) male, (B) female, and (C) hermaphrodite of Auanema melissensis on day 1 of adulthood (100× magnification). A dauer larva (D), fated to develop as a hermaphrodite, is depicted at a magnification of 112×.

Figure 2

Differences between females and hermaphrodites during the first three days of adulthood. (A) Representative example of the size and pigmentation differences between A. melissensis females and hermaphrodites over the first 3 d of adulthood (100× magnification). The scale bar is the same for all images. (B) Hermaphrodites’ body length is longer than that of females during the first three days of adulthood (repeated measures ANOVA F(1, 8) = 26.580, P < 0.05). As the worms aged, they also became longer (repeated measures ANOVA F(2, 16) = 19.151, P < 0.05).
Differences between females and hermaphrodites during the first three days of adulthood. (A) Representative example of the size and pigmentation differences between A. melissensis females and hermaphrodites over the first 3 d of adulthood (100× magnification). The scale bar is the same for all images. (B) Hermaphrodites’ body length is longer than that of females during the first three days of adulthood (repeated measures ANOVA F(1, 8) = 26.580, P < 0.05). As the worms aged, they also became longer (repeated measures ANOVA F(2, 16) = 19.151, P < 0.05).

Figure 3

Male tail characters of A. melissensis. Micrographs (A) and (B) show the spicules. Male tail in ventral (C) and lateral (D) orientation. “ph” = Phasmid, “CO” = cloaca, “gb” = gubernaculum, “vh” = ventral hook. Genital papillae/rays are designated by arrowheads and named r1-r8.
Male tail characters of A. melissensis. Micrographs (A) and (B) show the spicules. Male tail in ventral (C) and lateral (D) orientation. “ph” = Phasmid, “CO” = cloaca, “gb” = gubernaculum, “vh” = ventral hook. Genital papillae/rays are designated by arrowheads and named r1-r8.

Figure S3

Whole body length of males (n = 10), females (n = 10) and hermaphrodites (n = 10).
Whole body length of males (n = 10), females (n = 10) and hermaphrodites (n = 10).

Figure 4

Hermaphrodite production is promoted when A. melissensis hermaphrodite mothers are cultured in the presence of a crowding cue. The boxplots were drawn from the proportion of hermaphrodites (number of hermaphrodites/number of hermaphrodites and females) under each condition for each replicate.
Hermaphrodite production is promoted when A. melissensis hermaphrodite mothers are cultured in the presence of a crowding cue. The boxplots were drawn from the proportion of hermaphrodites (number of hermaphrodites/number of hermaphrodites and females) under each condition for each replicate.

Figure 5

Phylogenetic position of A. melissensis. Single-copy BUSCO orthologs (2071) were used to build a concatenated alignment, which was subsequently used to construct the phylogenetic tree. The supermatrix alignment was 1,018,758 amino acids in length.
Phylogenetic position of A. melissensis. Single-copy BUSCO orthologs (2071) were used to build a concatenated alignment, which was subsequently used to construct the phylogenetic tree. The supermatrix alignment was 1,018,758 amino acids in length.

Whole body measurements of ten males, females and hermaphrodites.

ID Sexual morph Length (μm)
MM1 Male 408.007
MM2 Male 417.763
MM3 Male 459.784
MM4 Male 416.338
MM5 Male 448.795
MM6 Male 440.899
MM7 Male 461.569
MM8 Male 446.739
MM9 Male 431.713
MM10 Male 440.548
FM1 Female 823.88
FM2 Female 762.546
FM3 Female 761.116
FM4 Female 853.252
FM5 Female 945.703
FM6 Female 918.242
FM7 Female 894.754
FM8 Female 912.291
FM9 Female 741.757
FM11 Female 926.158
HM1 Hermaphrodite 1,006.355
HM2 Hermaphrodite 960.354
HM3 Hermaphrodite 1,001.369
HM4 Hermaphrodite 1,024.498
HM5 Hermaphrodite 856.356
HM6 Hermaphrodite 1,010.322
HM7 Hermaphrodite 839.704
HM8 Hermaphrodite 986.148
HM10 Hermaphrodite 982.561
HM11 Hermaphrodite 963.425

Number of F1 males, females and hermaphrodites produced by selfing mothers in the absence (control) and presence (crowding) of a crowding cue.

Replicate Male (%) Female (%) Hermaphrodite (%) Total
Control 1 9 (11.7) 68 (88.3) 0 (0) 77
Control 2 3 (7.7) 35 (89.7) 1 (2.6) 39
Control 3 1 (7.7) 9 (69.2) 3 (23.1) 13
Control 4 4 (23.5) 13 (76.5) 0 (0.0) 17
Control 5 0 (0.0) 45 (97.8) 1 (2.2) 46
Total Control 17 (8.9) 170 (88.5) 5 (2.6) 192
Crowding 1 0 (0.0) 15 (88.2) 2 (11.8) 17
Crowding 2 0 (0.0) 15 (68.2) 7 (31.8) 22
Crowding 3 1 (2.2) 44 (95.7) 1 (2.2) 46
Crowding 4 0 (0.0) 27 (56.3) 21 (43.8) 48
Crowding 5 0 (0.0) 8 (19.5) 33 (80.5) 41
Total Crowding 1 (0.6) 109 (62.6) 64 (36.8) 174

BUSCO scores of the genomes used to construct the phylogeny.

Complete Complete and single copy Complete and duplicated Fragmented Missing
A. melissensis (this study, GCA_943334845.1) 2,784 (88.9%) 2,771 (88.5%) 13 (0.4%) 45 (1.4%) 302 (9.7%)
A. freiburgensis (Talal Al Yazeedi, pers. comm.) 2,789 (89.1%) 2,756 (88.0%) 33 (1.1%) 48 (1.5%) 294 (9.4%)
A. rhodensis (GCA_947366455) 2,812 (89.8%) 2,796 (89.3%) 16 (0.5%) 39 (1.2%) 280 (9.0%)
O. tipulae (GCA_013425905.1) 2,843 (90.8%) 2,786 (89.0%) 57 (1.8%) 47 (1.5%) 241 (7.7%)
C. elegans (GCF_000002985.6) 3,113 (99.4%) 3,096 (98.9%) 17 (0.5%) 3 (0.1%) 15 (0.5%)
P. pacificus (GCA_000180635.4) 2,587 (82.6%) 2,537 (81.0%) 50 (1.6%) 38 (1.2%) 506 (16.2%)

Preprocessing of the genomic libraries.

Library (insert size) Number of raw pairs Number of pairs after trimming (quality and size)* Number of pairs after contamination removal
Pair-end DNA library (200 bp) 68,587,064 68,109,105 (99.30%) 67,271,297
Mate-pair DNA library (3 kb) 16,195,093 9,920,448 (61.26%) 9,766,526
Mate-pair DNA library (5 kb) 11,655,487 7,484,279 (64.21%) 7,372,147
Mate-pair DNA library (8 kb) 10,769,133 7,125,674 (66.17%) 7,017,505

Genomic and transcriptomic raw data used in this study.

Library (insert size) Number of raw pairs GC content (%) Run accession number (experiment acc)
Pair-end DNA library (200 bp) 68,587,064 37 ERR9709439 (ERX9258761)
Mate-pair DNA library (3 kb) 16,195,093 37 ERR9709942 (ERX9259264)
Mate-pair DNA library (5 kb) 11,655,487 37 ERR9710904 (ERX9260226)
Mate-pair DNA library (8 kb) 10,769,133 37 ERR9709954 (ERX9259276)
RNA-seq library of mixed stages 22,509,195 44 ERR9712246 (ERX9261548)

Basic statistics on the genome of A. melissensis compared to that of C. elegans. The nematoda.odb10 database was used for the BUSCO analysis.

A. melissensis PRJEB51845/ GCA_ 943334845.1 C. elegans PRJNA13758/ GCF_ 000002985.6
Number of scaffolds 7,511 6 + MT
Span (Mb) 59.7 100.3
GC content (%) 37.1 35.4
N50 (bp) 404,820 (n = 39) 17,493,829
Longest scaffold/ chromosome 2,171,611 20,924,180
N counts 5,769,006 0.00
Gaps 3,945 NA
Repeats 4,816,819 bp (8.07%) (21.95%)
BUSCO (v5.2.2) score (on genome) C: 88.9% [S: 88.5%, D: 0.4%], F: 1.4%, M: 9.7%, n: 3,131 C: 99.4% [S: 98.9%, D: 0.5%], F: 0.1%, M: 0.5%
No. of protein-coding genes 11,040 20,184
BUSCO (v5.2.2) score (on the proteome, using nematoda_ odb10, n = 3,131) C: 89.7% [S: 77.2%, D: 12.5%], F: 1.1%, M: 9.2% C: 100.0% [S: 74.8%, D: 25.2%], F: 0.0%, M: 0.0%

Crosses were performed between A. melissensis and A. rhodensis or A. freiburgensis. The number of crosses performed is denoted by “n.”

Males
A. melissensis A. rhodensis A. freiburgensis
Females A. melissensis No offspring (n = 5) No offspring (n = 11)
A. rhodensis No offspring (n = 5)
A. freiburgensis No offspring (n = 8)

Putative X scaffolds. Scaffolds containing at least 3 Nigon X BUSCO genes were considered putative X scaffolds.

Scaffolds Number of Nigon X BUSCO genes Number of BUSCO genes of other Nigons
scaffold120 (CALQYR010007222.1) 10 0
scaffold167 (CALQYR010007273.1) 18 0
scaffold125 (CALQYR010007227.1) 5 0
scaffold146 (CALQYR010007250.1) 5 0
scaffold91 (CALQYR010007503.1) 12 0
scaffold79 (CALQYR010007489.1) 7 0
scaffold72 (CALQYR010007482.1) 6 0
scaffold42 (CALQYR010007449.1) 3 0
scaffold41 (CALQYR010007448.1) 3 0
scaffold190 (CALQYR010007299.1) 3 0
scaffold168 (CALQYR010007274.1) 3 0

Classification of the repeats by Repeat Masker.

Category Number of elements* Length occupied (bp) Percentage of sequence
SINEs (all) 30 7,757 0.01
SINEs (ALUs) 0 0 0.00
SINEs (MIRs) 0 0 0.00
LINEs (all) 78 22,828 0.04
LINEs (LINE1) 0 0 0.00
LINEs (LINE2) 10 4,088 0.01
LINEs (L3/CR1) 14 5,507 0.01
LTR elements (all) 1,799 727,003 1.22
LTR elements (ERVL) 0 0 0.00
LTR elements (ERVL-MaLRs) 0 0 0.00
LTR elements (ERV_classI) 0 0 0.00
LTR elements (ERV_classII) 0 0 0.00
DNA elements 430 161,038 0.27
DNA elements (hAT-Charlie) 0 0 0.00
DNA elements (TcMar-Tigger) 1 807 0.00
Unclassified 6,850 2,109,448 3.53
Total interspersed repeats NA 3,028,074 5.07
Small RNA 792 186,973 0.31
Satellites 167 46,755 0.08
Simple repeats 24,687 1,082,694 1.81
Low complexity 8,083 464,828 0.78
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