Caenorhabditis monodelphis sp. n.: defining the stem morphology and genomics of the genus Caenorhabditis
Author
Dieter Slos
Author
Walter Sudhaus
Author
Lewis Stevens
Author
Wim Bert
Author
Mark Blaxter
text
BMC Zoology
2017
2017-02-23
2
4
1
15
journal article
10.1186/s40850-017-0013-2
38f37f62-465c-4b02-ac7c-974bb0b90869
1156816
0E6F137B-9975-4A8E-91F2-D588A572076E
Caenorhabditis monodelphis
1 sp. n. Slos & Sudhaus
=
Caenorhabditis
sp. SB341 [
7
]
=
Caenorhabditis
sp. SB341 and
Caenorhabditis
sp. n.
SB341 [
36
]
=
Caenorhabditis
sp. n.
1 (SB341) and (lapse)
Caenorhabditis
sp. n.
4 (SB341) [
10
]
=
Caenorhabditis
sp. 1 SB3 41 [
6
,
8
,
37
]
=
Caenorhabditis
sp. 4 SB3 41 [
38
]
(
Figs. 1
,
2
,
3
and
4
;
Table 1
)
Adult
Small species (female 0.7 2 - 1.0 4 mm, male 0.6 5
–
0.7 7 mm); cuticle thin, ca. 1 μm wide and finely annulated, 0.8 μm wide at midbody. Lateral field inconspicuous, about 9% of body width, consisting one ridge that can be traced anteriorly to the level of the median bulb and posteriorly at level of rectum in females and about 1½ spicules length anterior of the cloacal aperture in males. Six lips slightly protruding, each with one apical papilliform labial sensillum and a second circle of four sublateral cephalic sensilla in both sexes; amphids opening on the lateral lips, hardly discernible. Buccal tube long and slender, more than twice the width in lip region, pharyngeal sleeve envelopes nearly half of the stoma, the anterior as well as the posterior end of the tube appear slightly thickened, cheilostom inconspicuous, arcade cells forming the gymnostom sometimes visible; glottoid apparatus completely absent. Pharynx with a prominent median bulb, diameter more than 90% of diameter of terminal bulb; terminal bulb pyriform, with double chambered haustrulum, the anterior chamber smallish; cardia conspicuous, opens funnel-like in intestine. Nerve ring encircles isthmus in its anterior part in living specimens, more to the middle of the isthmus in heat relaxed or preserved specimens; deirids usually conspicuous in the lateral field at level of beginning of terminal bulb, sometimes not visible in heat relaxed animals; pore of excretory-secretory system hard to discern posterior of deirid level. Two gland cells ventral and slightly posterior of terminal bulb conspicuous in live specimens. Lateral canals visible in live specimens extending anteriorly to two stoma length from the anterior end and ending at rectum level in the female. Postdeirids usually very conspicuous dorsally of the lateral field at about 7 5% of body length in both sexes and about half the length between vulva and beginning of rectum (or at level of posterior end of uterus remnant) in females, sometimes not visible in heat relaxed specimens.
Fig. 1
Line drawings of
Caenorhabditis monodelphis
sp. n.
a
Female, schematic overview.
b -i
Male,
b
: Male, lateral;
c
Anterior end in ventral view;
d
Tail in lateral view;
e
Tail in ventral view;
f
Pharyngeal region;
g
Gubernaculum in ventral view;
h
Gubernaculum in lateral view;
i
Spiculum in lateral view
Fig. 2
Caenorhabditis monodelphis sp. n., mature spermatozoa in female post-uterine sac, TEM.
Arrows
= sperm cells
Female
Maximum body diameter clearly anterior of the vulva, vulva position 65% body length, a transverse slit, bordered in both ends by cuticular longitudinal flaps, vulva lips moderately protruding, four diagonal vulval muscles conspicuous; one pseudocoelomocyte exists anterior of gonad flexure ventrally. Genital tracts asymmetrical; posterior branch rudimentary, sac like, on the left hand side of intestine, without flexure, almost as long as body diameter at the level of the vulva, containing spermatozoa (
Fig. 2
); anterior branch right of intestine, reflexed dorsally close to the pharynx, flexure more than half the length of the gonad (measured from vulva to flexure); at the flexure oocytes in several rows, downstream in one row, oocytes predominantly growing in the last position, where granules are stored inside; sphincter between oviduct and uterus, only a few sperm cells in oviduct, most of them in uterus and blind sac; oviparous, one egg at a time in uterus (rarely two), segmentation starts in the uterus. Rectum a little S-shaped, rectal gland cells very small, posterior anal lip slightly protuberant. Tail short, panagrolaimid, dorsally convex, with offset tip tapering, smooth to somewhat telescope-like by cuticle forming a sleeve-like structure; tail tip with tiny hooks, mostly one dorsal, but also subventral (compare with
Poikilolaimus
); opening of phasmids located at 60
–
65% of tail length, shortly anterior of tip, phasmid glands not reaching anus level.
Male
Testis right of intestine, ventrally reflexed in a certain distance posterior of pharynx; flexure relatively short. One pseudocoelomocyte between pharynx and flexure ventrally. Bursa well developed, peloderan, anteriorly open, with smooth margin and sometimes terminally indented, posterior part of velum transversely striated.
Nine pairs of genital papillae (GP) present, two of them anterior of the cloaca, genital papilla 1 (GP1) and GP2 spaced, GP3 to GP6 and GP7 to GP9 clustered, GP5 and GP7 point to the dorsal side of the velum, GP6 slightly bottle shaped, GP8 and GP9 fused at base, GP2 and GP8 not reaching the margin of velum. Phasmids forming small tubercles to the ventral side posterior of the last GP; formula of GPs: v1,v2/(v3,v4,ad,v5) (pd,v6,v7)ph. Precloacal sensillum small, precloacal lip simple (according to type A of W Sudhaus and K Kiontke [
39
]), postcloacal sensilla long filamentous. Spicules short and stout, tawny, separate, slightly curved, with prominent head; shaft with a transverse seam, with a prominent longitudinal ridge, a dorsal lamella, and an oval
“
window
”
, the tip notched. Gubernaculum dorsally projecting, flexible, in the distal part following the contour of the spicules, spoon shaped in ventral view.
Dauer larva
Unsheathed, mouth closed; stoma long, slender. Pharyngeal sleeve covering about half of the stoma; pharynx with welldeveloped median and terminal bulbs; corpus length ca. 52% of pharynx length. Nerve ring somewhat in the middle between the middle and terminal bulb. Genital primordium at about 60% of body length, elongated oval in shape. Tail conical. Amphids, lateral lines, position excretory pore, deirids and phasmids not observed.
Aberration
In one female a second set of
“
sensilla
”
were observed a short distance posterior to postdeirids, possibly a duplication of the postdeirids.
Type carrier and locality
Holotype and paratypes of
Caenorhabditis monodelphis
sp. n.
were isolated from the tunnels of
Cis
castaneus
(Herbst, 1793) (
Ciidae
,
Coleoptera
) in the bracket fungus
Ganoderma
applanatum
(
Polyporales
) on a stump of the common beech (
Fagus
sylvatica
) a few centimetres above the ground in Berlin-Grunewald in April 2 0 0 1. The same sample included individuals of
Diploscapter
sp.,
Plectus
sp.,
Oscheius
dolichura
and one individual dorylaimid and mononchid.
Type material
Holotype male (collection number WT 3684) and five female and four male paratypes (WT 3685, WT 3686) are deposited in the National Plant Protection Organization Wageningen, The Netherlands. In addition, four female and four male paratypes, are deposited in the collection of Museum Voor Dierkunde at Ghent University, Ghent, Belgium, five female and three male paratypes in Museum für Naturkunde an der Humboldt-Universität zu Berlin, Berlin, Germany. Additional paratypes are available in the UGent Nematode Collection (slides UGnem158, 159 & 160) of the Nematology Research Unit, Department of Biology, Ghent University, Ghent, Belgium.
Fig. 3
Light microscopic images of
Caenorhabditis monodelphis
sp. n.
a
General overview of a dauer larva;
b
: Ventral view of female reproductive system;
c
Detailed ventral view of vulva;
d
Anterior region of male;
e
Male tail in ventral view, showing seven ventral (v1-7) and two dorsal (ad, pd) genital papillae. The phasmids are not visible in this plane;
f
Male tail in lateral view;
g
Detail of the spicule;
h
Detail of the gubernaculum
Diagnosis and relationship
Caenorhabditis monodelphis
sp. n.
can be recognised as a
Caenorhabditis
based on the thickened GP6 and the clearly visible postdeirids.
Caenorhabditis monodelphis
sp. n.
is distinguished from all other described
Caenorhabditis
species by the presence of a monodelphic genital tract in the female with a blind sac posterior the vulva, a panagrolaimid female tail shape, adults with only one ridge on the lateral field, a very long and slender stoma without visible glottoid apparatus and male with short, stout spicule with bifurcate tip.
Fig. 4
Scanning electron micrographs of
Caenorhabditis monodelphis
sp. n.
a
,
d
Anterior end of female;
b
,
c
Anterior end of male;
e
Female vulva;
f
,
g
Lateral field of female and male, respectively;
h
Female anus and tail;
i
,
j
Male bursa with genital papillae indicated;
k
Detail of cloacal region with postcloacal sensillae;
l
Detail of male spiculum
Ecology and biology
Caenorhabditis monodelphis
sp. n.
is a gonochoristic species with both males and females. Females are oviparous and carry only one egg (rarely two eggs). Development from egg to adult took about 5
–
6 days in juice prepared from brown algae at room temperature. Development from dauer larva to adults was completed in less than 3 days at 20 ̊C on NA seeded with OP50. The lifespan of adults is at minimum 1 4 days for males and 1 7 days for females. One pair of adults produced 1 6 7 offspring in 8 days and the daily production of fertile eggs was 6
–
3 1 (mean 1 8;
n
= 1 4). After the reproductive phase, females lived 9
–
1 4 days (
n
= 3) with males present.
Table 1
Measurements (in μm) of heat relaxed specimens of
Caenorhabditis monodelphis
sp. n.
| Character |
Female |
Male |
Dauer |
| N |
1 1 |
1 0 |
1 0 |
| L |
870 ± 105 |
694 ± 36 |
456 ± 24 |
| A |
17.1 ± 0.8 |
22 ± 1.6 |
23 ± 1.2 |
| B |
4.9 ± 0.5 |
4.1 ± 0.3 |
3.6 ± 0.1 |
| C |
20.5 ± 2.6 |
22 ± 2.3 |
9.8 ± 0.7 |
|
c
’
|
1.99 ± 0.17 |
1.8 ± 0.2 |
3.9 ± 0.30 |
| V |
65 ± 1.8 |
- |
- |
| Body width |
51 ± 6.9 |
32 ± 3 |
20 ± 0.6 |
| Stoma length |
27 ± 2.3 |
27 ± 2 |
21 ± 1.1 |
| Stoma diameter |
1.9 ± 0.6 |
1.2 ± 0.2 |
0.6 ± 0.1 |
| Cheilostom |
2.5 ± 0.2 |
2.4 ± 0.2 |
- |
| Gymnostom |
10 ± 0.7 |
9.7 ± 0.9 |
- |
| Stegostom |
15 ± 1.5 |
15 ± 1.5 |
- |
| Pharyngeal sleeve |
12.42 ± 1.6 |
13 ± 1.2 |
- |
| Pharynx length |
150 ± 6.7 |
141 ± 9.2 |
107 ± 3.3 |
| Procorpus length |
55 ± 3.1 |
52 ± 3.6 |
- |
| Metacorpus length |
26 ± 2.1 |
22.8 ± 1.1 |
- |
| Isthmus length |
39 ± 3.2 |
40 ± 4.9 |
- |
| Nerve ring to terminal bulb |
11 ± 4.9 |
19 ± 3.3 |
- |
| Terminal bulb length |
30 ± 1.8 |
27 ± 1.7 |
- |
| Diameter of median bulb |
22 ± 2.5 |
17 ± 1.3 |
9 ± 0.5 |
| Diameter of terminal bulb |
25 ± 2 |
19 ± 1 |
11 ± 0.4 |
| Anterior end to deirid |
150 ± 8 |
150 ± 8.3 |
- |
| Postdeirid to anus |
170 ± 29.8 |
141 ± 14 |
- |
| Length intestine |
651 ± 100 |
494 ± 32 |
- |
| Rectum length |
25 ± 2.6 |
24 ± 1.9 |
- |
| Anal body width |
22 ± 2.1 |
17 ± 1.1 |
12 ± 0.6 |
| Tail length |
43 ± 4.3 |
32 ± 3.2 |
46 ± 2.4 |
| Anus to phasmid distance Gonad length a |
26 ± 2.2 303 ± 68 |
- 342 ± 44 |
- - |
| Gonad flexure length |
226 ± 67 |
46 ± 6.8 |
- |
| Postuterine sac |
45 ± 6.8 |
- |
- |
| Sperm diameter Egg length b Egg diameter b |
- 53 ± 3.1 29 ± 2.9 |
9.8 ± 1.3 - - |
- - - |
| Spicule length |
- |
25 ± 1 |
- |
| Gubernaculum length |
- |
15 ± 0.9 |
- |
Caenorhabditis monodelphis
sp. n.
has until now only been found in
Ganoderma
and
Fomes
in Germany and Belgium in relation with the ciid beetle
Cis
castaneus
. The
Ganoderma
carrying
C. monodelphis
sp. n.
from Oslo was not investigated for the presence of
C. castaneus
. In fungal fruiting bodies lacking the beetle
C. monodelphis
sp. n.
was not found. Dauers of
C. monodelphis
sp. n.
were found under the elytra of the beetle, but were not found internally when the beetle was further dissected. These findings indicate a phoretic association with the beetle. As only dauer larvae were isolated from beetles, while adults and larvae were present in the fruiting bodies, we infer that
C. monodelphis
sp. n.
exit from dauer within the mushroom, develop to adulthood and start to reproduce. The food source of the species in natural conditions is not known, but they survive and reproduce easily on
E. coli
OP50 in culture.
Genome sequence of an inbred strain of
Caenorhabditis
monodelphis
sp. n.
We sequenced the genome of an inbred strain (JU1677) of
C. monodelphis
sp. n.
using Illumina sequencing technology to ~110x coverage. The genome was assembled into 6,864 scaffolds, spanning 115.1 Mb with a scaffold N50 of 49.4 kb (
Table 2
). CEGMA (Core Eukaryotic Gene Mapping Approach) [
40
] scores suggested the assembly is of high completeness. We predicted 17,180 protein coding gene models using RNA-Seq evidence. These statistics, and the overall gene content and structure of the assembly were largely in keeping with those determined for other
Caenorhabditis
species. The genome was larger than that of
C. elegans
and
C. briggsae
, which are hermaphroditic species, but smaller than that of
C. remanei
, a gonochoristic species.
We carried out preliminary comparisons of the structure and content of the
C. monodelphis
sp. n.
genome with those of other sequenced
Caenorhabditis
species. The number of genes identified was lower than estimates for most other
Caenorhabditis
species. To compare the gene structures of
C. monodelphis
sp. n.
to that of
C. elegans
, we identified 6,174 orthologous gene pairs and calculated gene structure statistics (
Table 3
,
Fig. 5
.). To minimize bias from erroneous gene predictions (such as merged or split genes), orthologous gene pairs which differed in CDS length by 20% were considered outliers.
C. monodelphis
sp. n.
genes were typically longer than their orthologues in
C. elegans.
We also found a clear trend toward more coding exons per gene in
C. monodelphis
sp. n.
than in
C. elegans
(
Fig. 5a
). A few examples of
C. monodelphis
sp. n.
gene models compared to those of orthologues in
C. elegans
are shown (
Fig. 5b
). Although introns are, on average, shorter in
C. monodelphis
sp. n.
than in
C. elegans,
C. monodelphis
genes typically have a longer total span of introns than
C. elegans
transcripts (
Table 3
,
Fig. 5
.).
a from anus to flexure in the female; from cloaca to flexure in the male b
n
= 7
Table 2
Genome assembly statistics for
C. monodelphis
sp. n.
and other
Caenorhabditis
species
| Species |
C. monodelphis
|
C. brenneri |
C. briggsae |
C. elegans |
C. japonica |
C. remanei |
C. sinica |
C. tropicalis |
| Version |
1.0 |
WS254 |
WS254 |
WS254 |
WS254 |
WS254 |
WS254 |
WS254 |
| Mating type |
gonochoristic |
gonochoristic |
hermaphroditic |
hermaphroditic |
gonochoristic |
gonochoristic |
gonochoristic |
hermaphroditic |
| Strain |
JU1667 |
PB2801 |
AF16 |
N2 |
DF5081 |
PB4641 |
JU800 |
JU1373 |
| Span (Mb) Scaffolds (n)a |
115.12 6,864 |
190.37 3,305 |
108.38 367 |
100.29 7 |
166.25 18,808 |
118.55 1,591 |
130.76 11,966 |
79.32 660 |
| N50 (kb) |
49.4 |
381.96 |
17,485.44 |
17,493.82 |
94.15 |
1,522.09 |
25,564 |
20,921.87 |
| Genes (n) |
17,180 |
30,660 |
21,814 |
20,362 |
29,964 |
26,226 |
34,696 |
22,326 |
| GC (%) |
43.9 |
38.6 |
37.4 |
35.4 |
39.2 |
37.9 |
39.5 |
37.7 |
| CEGMA complete/ partial (%) |
89.11/ 97.98 |
98.39/ 99.60 |
97.98/ 99.19 |
96.77/ 99.19 |
78.63/ 97.18 |
94.35/ 98.79 |
95.56/ 99.60 |
97.18/ 98.79 |
a Scaffolds shorter than 500 bp were not considered
C. monodelphis
sp. n.
is sister to other known
Caenorhabditis
We clustered a total of 634,56 4 protein sequences from
C. monodelphis
sp. n.
, twenty-two other
Caenorhabditis
species, and two rhabditomorph outgroup species (
Oscheius
tipulae
; data courtesy of M. A. Félix, and
Heterorhabditis
bacteriophora
) to define putative orthologues. We identified 34,425 putatively orthologous groups containing at least two members, 303 of which were either single copy or absent across all 25 species. These single copy orthologues were aligned, and the alignments concatenated and used to perform maximum-likelihood and Bayesian inference analysis using RAxML and PhyloBayes, respectively. Both analysis methods resulted in an identical topology, with the placement of
C. monodelphis
sp. n.
arising basally to all other
Caenorhabditis
species (
Fig. 6
). All branches had maximal support except for three nodes within the
Elegans
super-group. Our analysis included data from several new and currently undescribed putative species of
Caenorhabditis
, including
C.
sp. 21 which is the sister taxon to the
Drosophilae
plus
Elegans
super-groups and
C.
sp. 31 which forms the first branch in the
Elegans
super-group.
C.
sp. 38 is placed within the
Drosophilae
super-group, while
C.
sp.
26,
C.
sp. 32 (sister to
C. afra
) and
C.
sp. 40 (sister to
C. sinica
) are all members of the
Elegans
super-group. From these analyses we conclude that
C. monodelphis
sp. n.
is sister to all other known
Caenorhabditis
.
Stemspecies pattern reconstruction
Our phylogenetic analyses were based on species with whole genome data available, and thus did not include the full known diversity of the genus. The stemspecies pattern was reconstructed based on ingroup and outgroup comparison. Previous molecular phylogenetic analyses of
Caenorhabditis
species using a small number of marker genes [
1
0] placed
C. monodelphis
sp. n.
and
C. sonorae
[
4
1
] as sister species, again arising at the base of the genus.
The following morphological synapomorphies can be hypothesised to support a
C. monodelphis
sp. n.
– C. sonorae
clade: mouth opening triangular (
Fig. 4b
), spicule having a complicated tip (notched or dentated) and a longish thin walled
“
window
”
in the blade (
Figs. 1i
,
4l
), postcloacal sensilla being filiform (
Fig. 4k
), and the female tail shortened to less than three times anal body width. Other similarities between both these species are plesiomorphic.
Caenorhabditis
and its sister group constitute the monophylum Anarhabditis within the
Rhabditina
. For convenience, we will call the sister clade of
Caenorhabditis
Protoscapter
(
Fig. 7
): it comprises
“
Protorhabditis
”
,
Prodontorhabditis
,
Diploscapter
and
Sclerorhabditis
[
4
2
]. To reconstruct the characters of the stemspecies of
Caenorhabditis
it is necessary to consider the morphologies of all these taxa, and not only the taxa for which we have molecular data.
“
Protorhabditis
”
is paraphyletic. The
Oxyuroides
group is sister taxon of
Prodontorhabditis
[
4
3
,
4
4
], and the
Xylocola
group may be sister taxon of
Diploscapter
/
Sclerorhabditis
. However, the two species
Protorhabditis
elaphri
(Hirschmann in Osche, 1952) and
P. tristis
[
4
5
] appear to represent basal branches in Protoscapter (compare [
43
]). These last two species, despite the paucity of information available for them, are crucial for comparisons that will illuminate the stemspecies patterns of Anarhabditis, Protoscapter and
Caenorhabditis
.
Table 3
Gene structure comparison of orthologous gene pairs from
C. monodelphis
sp. N.
and C. elegans
|
C. monodelphis
sp. n.
|
C. elegans |
| Gene length (bp) |
3359 |
2854 |
| Coding exon length (bp) |
109 |
144 |
| Coding exon count (n) CDS span (bp)a |
10 1167 |
6 1182 |
| Intron length (bp) |
69 |
76 |
| Total intron span per gene (bp) |
1918 |
1187 |
All values are medians
a orthologous gene pairs which differed in CDS length by 20% were not included
Fig. 5
Comparison of exon counts in single-copy orthologues between
C. monodelphis
sp. n. and
C. elegans.
a
Exon counts in 6,174 single-copy orthologous gene pairs.
C. monodelphis
sp. n. genes which had transcripts with CDS lengths 20% longer (
orange
) or shorter (
black
) than
C. elegans
were defined as outliers. Linear regression lines are shown. Inset: Frequency histogram of log2 ratio of
C. monodelphis
sp. n. exon counts to
C. elegans
exon counts.
b
Comparison of gene structures of five orthologous gene pairs. Three gene pairs were selected at random and a further two were selected because they showed a large divergence in exon count
By ingroup comparison we reconstruct the following characters of the stemspecies of Anarhabditis without differentiating them into apo- or plesiomorphies (on apomorphies see the legend of
Fig. 7
):
–
adults of small size (less than 1 mm); − lips not offset from anterior end; − four cephalic sensilla present in male and female; − stoma with pharyngeal sleeve (stegostom length nearly that of gymnostom); − median bulb of pharynx strongly developed, corpus intima with transverse ridging, terminal bulb with double haustrulum; − gonochoristic; − female tail elongate conoid; − gonads amphidelphic, the anterior branch right and the posterior left of intestine; − vulva at midbody, a transverse slit; − oviparous, usually only one egg at a time in the uteri; − male gonad on the right side, reflexed to the ventral; − bursa peloderan and anteriorly open, oval-shaped in ventral view, with smooth margin, terminally not notched; − 9 pairs of even genital papillae, two precloacal largely spaced, GP3
–
6 evenly spaced, the last three GPs forming a tight cluster; GP1, GP5 and GP7 terminate on the dorsal surface of the bursa velum; − phasmids open behind GP9, inconspicuous; − bursa formula thus v1,v2/v3,v4,ad,v5 (pd,v6,v7)ph; − male tail tip present; − 1 + 2 circumcloacal sensilla inconspicuous, precloacal lip simple; − spicules separate, stout, head not rounded, behind the shaft a slight ventral projection, dorsal part of blade weakly cuticularised (velum), its tip possibly not even (argued below); − gubernaculum simple spatulate; − dauerlarvae with double cuticle (ensheathed), not waving.