Caenorhabditis monodelphis sp. n.: defining CrossMark 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 2 4 1 15 journal article 10.1186/S40850-017-0013-2 f6d4770f-2380-4376-a80d-92745d739c09 322316 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 SB341 [ 6 , 8 , 37 ] = Caenorhabditis sp. 4 SB341 [ 38 ] ( Figs. 1 , 2 , 3 and 4 ; Table 1 ) Adult Small species (female 0.72 - 1.04 mm, male 0.65 - 0.77 mm); cuticle thin, ca. 1 pm wide and finely annulated, 0.8 pm 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 1% 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, m ore 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 term inal 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 75% 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 ateral view Fig. 2 Cae o abolts mo ode ph s sp. n., matu e spe matozoa fema e post- ıte e sac, TEM A ovvs=spe m ce s 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: vl,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 2001. The same sample included individuals of Diploscapter sp., Plectus sp., Oscheius dolichura and one individual dorylaimid and mononchid. Type material Holotype male (collection num ber W T 3684) and five female and four male paratypes (WT 3685, W T 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 o verview of a dauer larva; b: Ventral view of fem ale reproductive system; c Detailed ventral view of vulva; d Anterior region of male; e Male tailin ventral view, showing seven ventral (v1-7) and tw o dorsal(ad, pd) genital papillae. The phasmids are not visible in this pl ane; f Male tai lin lateral view; g Detai l o f the spicu le; h Detail of the gubernacu lum s____________________________________________________________________________________________________________________________________________________________________________________________________________________ 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 fem ale; b,c Anterior end of male; e Female vulva; f.g Lateral field of female and male, respectively; h Female anus and tail; ij Male bursa with genital papillae indicated; k Detail of cloaca region with postcloacal sensillae; I 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 m inim um 14 days for males and 17 days for females. One pair of adults produced 167 offspring in 8 days and the daily production of fertile eggs was 6-31 (mean 18; n = 14). After the reproductive phase, females lived 9-1 4 days (n = 3) with males present. Table 1 Measurements (in |jm) of heat relaxed specimens of Caenorhabditis monodelphis sp. n.

Character	Female	Male	Dauer
N	11	10	10
870 ± 105	694 ± 36	456 ± 2 4
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 diam eter	1.9 ± 0.6	1.2 ± 0.2	0.6 ±0.1
Cheilostom	2.5 ±0.2	2.4 ± 0,2	-
Gym nostom	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	-
M etacorpus 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	-
Term inal bulb length	30 ± 1.8	27 ± 1.7	-
Diam eter o f median bulb	22 ± 2.5	17 ± 1.3	9 ± 0 5
Diam eter o f terminal bulb	25 ± 2	19 ± 1	11 ± 0,4
Anterior end to deirid	150 ± 8	150 ± 8.3	-
Postdeirid to anus	1 70 ± 29.8	141 ± 1 4	-
Length intestine	651 ± 100	494 ± 32	-
Rectum length	25 ± 2.6	24± 1.9	-
Anal body width	22 ±2.1	1 7 ± 1.1	12 ± 0.6
Tail length	43 ± 4.3	32 ± 3.2	46 ± 2.4
Anus to phasm id distance	26 ± 2.2	-	-
Gonad length3	303 ± 68	342 ± 4 4	-
Gonad flexure length	226 ± 67	46 ± 6.8	-
Postuteiine sac	45 ± 6.8	-	-
Sperm diam eter	-	9.8 ± 1.3	-
Egg lengthb	53 ± 3.1	-	-
Egg I liam eterb	29 ±2.9	-	-
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 £ 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 ~110 x 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. W e 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. W e also found a clear trend toward more coding exons per gene in C . monodelphis sp. n. than in C . elegans ( Fig. 5 a). A few examples of C . monodelphis sp. n. gene models compared to those of orthologues in C . elegans are shown ( Fig. 5 b). 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 .). afrom anus to flexure in the female; from cloaca to flexure in the male bn = 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	W S254	W S254	W S254	W S254	W S254	W S254	W S 254
Mating type	gonochoristic	gonochoristic	herm aphroditic	herm aphroditic	gonochoristic	gonochoristic	gonochoristic	herm aphroditic
Strain	JU1667	PB2801	AF 16	N2	DF5081	PB4641	JU800	JU1373
Span (Mb)	115.12	190.37	108.38	100.29	166.25	118.55	130.76	79.32
Scaffolds (n)a	6,864	3,305	367	7	18,808	1,591	11,966	660
N 50 ( 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
G C (%)	43.9	38.6	37.4	35.4	39.2	37.9	39.5	37.7
CEGM A com plete/ 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

“Scaffolds shorter than 500 bp were not considered C . monodelphis sp. n. is sister to other known Caenorhabditis We clustered a total of 634,564 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. afin) 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 num ber of marker genes [ 10 ] placed C . monodelphis sp. n. and C . sonorae [ 41 ] 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: m outh opening triangular ( Fig. 4 b), spicule having a complicated tip (notched or dentated) and a longish thin walled “window” in the blade (Figs, li, 41), postcloacal sensilla being filiform ( Fig. 4 k), 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 [ 42 ]. 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 [ 43 , 44 ], and the Xylocola group may be sister taxon of Diploscapter ! Sclerorhabditis . However, the two species Protorhabditis elaphri (Hirschmann in Osche, 1952) and P. tristis [ 45 ] 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)	10	6
CDS span (bp)a	1167	1182
Utron length (bp)	69	76
lotal intron span per gene (bp)	1918	1187

All values are medians aorthologous 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 w hich had transcripts with CDS lengths 20% longer (orange) or shorter (black) than C. elegans w ere defined as outliers. Linear regression lines are shown. Inset: Frequency histogram o f log2 ratio of C. monodelphis sp. n. exon counts to C. elegans exon counts, b Comparison of gene structures o f five orthologous gene pairs. Three gene pairs w ere selected at random and a further tw o w ere selected because they show ed 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, GP 3 -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 vl, v 2/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. Fig. 6 Phylogenetic relationships of Caenorhabditis species and two outgroup species. M aximum -likelihood based tree from RAxML. Bootstrap support values (1000 replicates) were 100 all branches, unless noted otherw ise as numbers on branches. Bayesian Posterior Probabilities were 1 for all branches and are not shown. Coloured boxes indicate supergroups, as defined in [3], Strain names from which protein sequence were derived are noted Discussion Taxonomy of Caenorhabditis monodelphis sp. n. Caenorhabditis monodelphis sp. n. is a new species of Caenorhabditis supported by its phylogenetic position as inferred from 303 molecular markers, morphology, habitat and specific association with Cis castaneus ( Coleoptera ). Morphologically, it could be confused with “ Protorhabditis " species because of the absence of a clear glottoid apparatus. A glottoid apparatus has been lost 5-6 times independently within “ Rhabditidae ” [ 46 ] and, as illustrated here , also in C. monodelphis sp. n. This species resembles species from “ Protorhabditis " With a very long stoma without glottoid apparatus , but differs from the Oxyuroides- group within ‘ Protorhabditis " in having an open bursa and GP1 not anterior of the bursa. It is differentiated from the Xylocola- group within “ Protorhabditis " in having nine genital papillae. Previously , Caenorhabditis has been characterised as having the following apomorphic characteristics: the presence of a dorsal velum on the spicule, a lateral field with three ridges, an unsheathed dauer juvenile and a slightly thickened GP6 [ 42 ], W ith the discovery and description of C . monodelphis sp. n. the num ber of lateral ridges is no longer an apomorphic character of Caenorhabditis , since C . monodelphis sp. n. only has one lateral ridge. Association with fungivorous beetles Species of Caenorhabditis are known to occur in soil , compost, cadavers of insects, some plant material and the intestine of birds [ 10 ], and can most easily be isolated from rotting fruits, flowers and stems [ 3 ], Caenorhabditis elegans has also been found infesting cultures of the m ushroom Agaricus bisporus [ 47 ]. Wild mushrooms are an under-explored habitat for this genus, but our limited geographical sampling indicates that they could be an important habitat. Caenorhabditis monodelphis sp. n. was present in galleries made by Cis castaneus inside Ganoderma applanatum in Belgium, Norway, Germany and in an old fruiting body of Fomes fomentarius in Belgium . Although the true distribution of C . monodelphis sp. n. is not yet known, it is expected that this species will be found throughout Europe where Ganoderma (or in lesser extent Fomes ) co-occurs with the mycophagous beetle Cis castaneus . That Caenorhabditis species have phoretic relationships with insects and other invertebrates is well known [ 10 ]. For C . monodelphis sp. n. , all records are from m ushroom fruiting bodies that are also inhabited by different insect groups, and dauer larvae were found under the elytra of Cis castaneus . Based on this evidence, we propose that C. monodelphis sp. n. propagates in galleries generated by Cisidae and the dauer larvae are transported by these beetles to uninfested mushrooms. Records of C . monodelphis sp. n. in both Ganoderma and Fomes , respectively the preferred [ 9 ] and the known [ 48 ] host indicate a beede-specific rather than a mushroom-specific relationship. The only other known Caenorhabditis species which appears to be phoretically associated with fimgivorous organisms, most likely insects, is C. auriculariae Tsuda & Futai, [ 49 ] of the Elegans super-group. This species was found only once in the fruit bodies of Auricularia polytricha (Agaricomycetes) in Japan, but the vector needed to infest the mushroom is unknown [ 49 ], C. elegans was also found to infest cultures of the champignon mushroom Agaricus bisporus [ 47 ], but most likely originated from mushroom compost where it can be frequently found. Several samples of different mushrooms on wood in Europe, USA and Japan did not yield other Caenorhabditis spp. However, given that many more insect species are known to feed and reproduce on mushrooms [ 50 ] and Rhabditida are known to use insects as a phoretic transport carrier [ 51 ], it is possible that mushroom species are habitats for many other rhabditid species, including new species of Caenorhabditis . Genome sequence and gene structures of C. monodelphis sp. n. Using next generation sequencing technologies and advanced bioinformatics toolkits, we have generated a good first-draft genome sequence for an inbred line derived from the type strain of C . monodelphis sp. n .. Although assembly metrics and CEGMA scores indicate the assembly is relatively contiguous and complete, it is likely that a proportion of C . monodelphis sp. n. genes are assembled only partially. This may have affected gene prediction, with the number of predicted gene models (17,180) being lower than estimates from most other Caenorhabditis species with available sequence data [ 25 ]. Comparisons of orthologous gene pairs revealed a significant divergence in gene structure between C . monodelphis sp. n. and C . elegans. C. monodelphis sp. n. genes are typically longer, contain more coding exons and a longer span of introns than C . elegans genes ( Table 3 ). This increase in gene length may, in part, account for the difference in genome span between C . monodelphis sp. n. and C. elegans . The clear trend towards more coding-exons in C. monodelphis sp. n. relative to C . elegans ( Fig. 5 ) could be explained by extensive intron loss or gain in either species. Previous studies using a small number of genes have shown that intron losses have been far m ore common in Caenorhabditis evolution than intron gains [ 7 , 52 , 53 ], Thus, it is possible that the gene structures seen in C. monodelphis sp. n. reflect an intron-rich ancestral state, and intron loss has predominated during the evolution of C . elegans. In Pristionchus pacifions , which is distantly related to Caenorhabditis , genes typically have roughly twice as many introns as their orthologues in C . elegans [ 54 ], Further analysis using genomes from more closely related outgroup species and other Caenorhabditis species is necessary before we can infer the dynamics of intron evolution in the genus.