Alloionema similis n. sp., a genetically divergent sibling species of A. appendiculatum Schneider, 1859 (Rhabditida: Alloionematidae) from invasive slugs in California, USA

A new species of Alloionema Schneider, 1859, A. similis n. sp., and the known species A. appendiculatum Schneider, 1859 were isolated from cadavers of invasive slugs in California. Both species are described based on morphology, morphometrics and molecular data. Alloionema similis n. sp. is morphologically very similar to A. appendiculatum but can be distinguished by a more posterior position of the excretory pore in the Kleinform females and longer tail in the Kleinform males. Substantial differences between the two species are, however, found in both 18S and 28S rDNA sequences. Sequence analysis revealed unambiguous autapomorphies in nucleotide sequence and secondary structure of rRNA genes, separating A. appendiculatum and A. similis n. sp. Molecular phylogenies were inferred from concatenated secondary-structure based multiple sequence alignments of nearly complete 18S and the D1-D3 domains of the 28S rRNA genes. Phylogenetic analyses placed these two species as sister taxa in a monophyletic clade, separately from Neoalloionema tricaudatum Ivanova, Pham Van Luc & Spiridonov, 2016 and N. indicum Nermuť, Půža & Mráček, 2016.


Introduction
Alloionema Schneider, 1859 (Rhabditida: Alloionematidae) was erected by Schneider (1859) for Alloionema appendiculatum Schneider, 1859, a nematode associated with the black slug Arion ater (Linneaus), in Germany. In a later paper, Schneider (1866) described the same species under the name Leptodera appendiculata Schneider, 1866 with a more detailed morphological description and some illustrations, apparently he erroneously marked it as a new species. Claus (1868) published an extensive account of its morphology and reproduction as well as the alternation of two different saprophytic generations previously reported by Schneider (1859). These two forms are distinguished mainly by their size and tail shape and Mengert (1953) referred to them as ''Großform'' and ''Kleinform''. Chitwood & McIntosh (1934) described from the gastropod host Succinea avara Say a variety, A. appendiculatum var. dubia Chitwood & McIntosh, 1934, intermediate in size between the two forms. Nermut' et al. (2015) made a re-description of A. appendiculatum based on material isolated from the invasive slug Arion vulgaris Moquin-Tandon (= A. lusitanicus Mabille) collected in the Czech Republic.
The first report of the genus in the United States was A. appendiculatum var. dubia recovered in 1934 from Succinea avara in Piscataway, Maryland (Chitwood & McIntosh, 1934). In 2007, surveys of slug nematode parasites in the USA  yielded a low nematode recovery (5.4%) with the majority (10 of 14) of species of Rhabditidae Ö rley, 1880 unidentified. Although found most often (34% of all isolates), A. appendiculatum was reported only from the states of Oregon (four sites) and Washington (two sites) but from neither of two sites sampled in California.
The first population of Alloionema spp. from California was recovered in 2006 from Arion rufus (Linnaeus) collected in Eureka. Specimens belonging to Großform were prepared for morphological and molecular studies but the culture was lost, making it impossible to study Kleinform specimens. A subsequent statewide gastropod survey in 2013 resulted in the recovery of multiple Alloionema isolates from Deroceras reticulatum (Müller) (four isolates), Lehmannia valentiana (Férussac) (three isolates) and Arion hortensis species complex (the latter complex comprises A. hortensis Férussac, A. distinctus Mabille and A. owenii Davies) (four isolates) collected in San Mateo. The most recent population was isolated from Arion rufus collected in McKinleyville, California during a 2015 survey.
With the exception of one isolate (ITD225), which was lost before it could be subjected to sequencing, all populations from 2013 sampling were found to be genetically identical to each other on the basis of their rRNA genes (nearly full length 18S rRNA gene and partial 5' section of 28S rRNA gene encompassing D1, D2 and D3 domains), but different from previously described populations of Alloionema appendiculatum from Europe (Laznik et al., 2009(Laznik et al., , 2010Nermut' et al., 2015;Ross et al., 2010;Spiridonov et al., personal communication)  The objectives of this paper were: (i) to describe the two genotypes of Alloionema from California, giving additional information on morphology, morphometrics and genetic variability of the genus; (ii) to compare the present material with previously described populations of A. appendiculatum; and (iii) to designate a new species for the genetically divergent population of Alloionema collected in San Mateo in 2013.

Materials and methods
Collection and maintenance of gastropods Statewide invasive slug and snail surveys were conducted during 2006, 2007, 2013, 2014 and 2015 in California. Gastropods were collected primarily from nurseries and garden centers by examining the area under potted plants and taxa were identified using Mc Donnell et al. (2009). Gastropod specimens collected during these surveys yielded a total of 13 strains of Alloionema (Table 1). The first population was collected in 2006 from Arion rufus in Eureka while the most recent sample was also recovered from A. rufus collected in McKinleyville in 2015. In addition to A. rufus, specimens of Alloionema were recovered from A. hortensis agg., D. reticulatum and L. valentiana in California. Slugs and snails were reared on organic carrots in plastic containers (26.5 9 15.5 9 6.5 cm) lined with damp paper towel, and following death of the animals, the cadavers were placed on 1% plain agar. Nematodes that emerged were isolated, subcultured, and subsequently maintained on fresh plain agar and nutrient agar (Tandingan De Ley et al., 2014). Our attempts to obtain a Großform by inoculating slugs with Kleinform specimens (isolates 175 and 295) failed; nematodes continued to propagate, the host died, but no Großform could be found in our cultures after the death of the host.
Light and scanning electron microscopy Nematodes were picked from dead slugs and culture plates, relaxed by gentle heat and fixed in cold 4% formaldehyde solution. For light microscopy (LM), specimens were transferred to pure glycerine by a slow evaporation method and mounted on permanent slides in glycerine with paraffin wax as support for the coverslip. Specimens used in this study are deposited in the general invertebrate collection (slides # SMNH-153525-SMNH-153536) of the Department of Zoology, Swedish Museum of Natural History, Stockholm, Sweden. For scanning electron microscopy (SEM), specimens from the isolate ITD176 were post-fixed in 1% osmium tetroxide (OsO 4 ) and transferred to pure acetone through an acetone/distilled water series. Specimens were critical point dried in liquid CO 2 , mounted on stubs, gold-plated under vacuum to a thickness of 200 Å in an Agar High Resolution Sputter Coater Model 20, and examined in a Hitachi S-4300 SEM at an accelerating voltage of 5 kV. All measurements in the descriptions and tables are in micrometres unless otherwise indicated.

Molecular procedures
DNA extraction and amplification were performed as described in Tandingan De Ley et al. (2007) for the 5 0 section of the 28S (covering either D2-D3 or D1-D2-D3 expansion segments) and the 18S rRNA genes (Tandingan De Ley et al., 2002). Genomic template DNA (2-3 ll) was used in a 25 ll PCR reaction using Illustra PuReTaq Ready-To-Go TM PCR beads (GE Healthcare, 800 Centennial Ave., P.O. Box 1327, Piscataway, NJ, USA) under the same PCR conditions, and using the same amplification and sequencing primers previously described (Blaxter et al., 1998;Tandingan De Ley et al., 2002). Contiguous sequences were assembled and compared with published sequences in the GenBank database using CodonCode Aligner (CodonCode Corp., 58 Beech Street, Dedham, MA, USA).

Sequence alignment
The secondary structure alignment was created based on existing secondary structure models of nearly complete 18S and partial 28S rRNA genes as described in Holovachov et al. (2015). New rRNA sequences (Table 2) were added to existing secondary structure-based alignments and aligned to maximize apparent positional homology of nucleotides. Secondary structure annotation was manually added to nonannotated sequences using 4SALE (Seibel et al., 2006); complementarity of base pairings in stem regions was manually verified for all sites.

Sequence comparison
Secondary structure-based alignments of all recent and published 18S and 28S rDNA sequences of Alloionema and Neoalloionema Ivanova, Pham Van Luc & Spiridonov, 2016 (Table 3) were visually compared in SeaView (Gouy et al., 2010). For comparative analysis, consensus sequences were created for A. appendiculatum and A. similis n. sp. Common sites were excluded from all sequences of A. similis n. sp., Neoalloionema indicum and N. tricaudatum, while variable sites were retained.
Visualization of rRNA secondary structure Secondary structures of selected domains of both 18S rRNA and 28S rRNA were visualized with the aid of   VARNA (Darty et al., 2009), saved as vector graphics and converted into raster graphic format for publication.

Phylogenetic analysis
The concatenated alignment was analyzed with Bayesian phylogenetic inference using the mcmcphase program in the PHASE 2.0 package (Gowri-Shankar & Jow, 2006). The entire concatenated alignment was partitioned into 18S rDNA and 28S rDNA partitions. Furthermore, each partition was divided into secondary partitions of ''stems'' (paired sites) and ''loops'' (non-paired sites) to account for the potential phylogenetic importance of compensatory substitutions. The REV nucleotide substitution model (Tavaré, 1986) was used for non-paired sites, whereas RNA7A (Higgs, 2000) nucleotide substitution model was used for paired sites. Model parameters were estimated independently for all sub-partitions (non-paired and paired sites of 18S rRNA gene and non-paired and paired sites of partial 28S rRNA gene). Chains were allowed to burn in for 500,000 generations, followed by 5 million generations (total 5.5 million generations) during which tree topologies, branch length and model parameters were sampled every 200 generations. The tree was rooted using Rhabditophanes sp. KR3021. General [Based on six specimens; see measurements in Table 4.] Body 2.0-2.4 mm long in females and 1.3-1.5 mm long in males. When killed by heat, females slightly arcuate ventrad and males more strongly arcuate ventrad, especially in posterior end. Cuticle finely annulated, annules less prominent in LM, in anterior body region 2.2-2.4 wide and 1.6-1.7 wide in females and males, respectively. Lateral field not seen in LM or SEM. Lip region rounded, continuous with body contour. Anterior end gradually tapering. Six rounded lips grouped in 3 pairs, 1 dorsal and 2 ventrolateral, carrying 6 inner labial, 6 outer labial and 4 cephalic papilliform sensilla and 2 small oval amphids. Stoma less than one lip region diameter long. Cheilostom broad, with thick rounded rhabdia; gymnostom short; stegostom funnel-shaped, with strongly sclerotised lining. Pharynx muscular; corpus cylindrical, 1.5-2.1 times as long as isthmus, widening posteriorly to a non-valvular metacorpus; isthmus narrower, demarcated by a break in muscular tissue; basal bulb oval, with weakly developed valves. Nervering surrounding isthmus. Excretory pore somewhat more posterior, opening in posterior part of isthmus, at isthmus-bulb junction or at terminal bulb. Deirids not observed.

Family
Female. Reproductive system didelphic, amphidelphic, ovaries reflexed. Oviducts filled with sperm. Gonads filled with oöcytes and hatched juveniles. Vulva a transverse slit, vulval lips not protruding; vagina c.1/8 of vulval body diameter (VBD). Tail conoid, tapering rapidly posteriorly to a minutely rounded terminus. Rectum short, less than one time anal body diameter (ABD) long. Phasmids in the shape of large transverse slits located at posterior third of tail length.
Male. Similar to female in most respects, except for the sexual characters. with an 18-20 long mucro ending in a pointed terminus.

Remarks
The present material agrees well with the description of the Großform of A. appendiculatum by Mengert (1953) (see Table 4). Our study of the Californian specimens also largely corroborates the results obtained by Nermut' et al. (2015) Table 5.] Body 0.7-0.9 mm long in females and 0.6-0.8 mm long in males. When killed by heat, females almost straight and males slightly arcuate ventrad, more strongly arcuate in the posterior end. Cuticle finely annulated, annules less prominent in LM, c.0.5 wide. Lateral field not seen in LM. Anterior end gradually tapering. Lip region rounded, continuous with body contour. Six rounded lips grouped in 3 pairs, 1 dorsal and 2 ventrolateral, carrying 6 inner labial, 6 outer labial and 4 cephalic papilliform sensilla and 2 small oval amphids. Stoma somewhat longer than lip region diameter. Cheilostom broad, with thick rounded rhabdia; gymnostom short; stegostom funnelshaped, with strongly sclerotised lining and small denticles in its dorsal sector. Pharynx muscular; corpus cylindrical, 1.9-2.8 times as long as isthmus, widening posteriorly to a non-valvular metacorpus; isthmus narrower, demarcated by a break in muscular tissue; basal bulb oval, with strongly developed valves. Nerve-ring surrounding isthmus. Excretory pore opening at middle or posterior part of isthmus or anterior part of basal bulb. Deirids not observed.
Female. Reproductive system didelphic, amphidelphic, ovaries reflexed, posterior ovary flexure reaching almost to vulva. Oviducts filled with sperm. Gonads filled with oöcytes and hatched juveniles. Vulva a transverse slit, vulval lips not protruding, with epiptygmata; vagina c.1/3 of VBD. Tail conoid, elongate, tapering to a finely pointed terminus. Rectum short, about one time ABD long. Phasmids at one-third to half of tail length.
Male. Similar to female in most respects, except for the sexual characters. Reproductive system Table 5 continued Species Alloionema similis n. sp.

Remarks
The new species is morphologically very similar to Alloionema appendiculatum, hence it is given the name Alloionema similis n. sp. Morphologically, it agrees well with the description by Mengert (1953) (see Table 5), except for the size of the Kleinform which are generally bigger in this study (body length 1,189-1,309 vs 922-1,073 lm for females; 910-1,029 vs 561-926 lm for males). Similar size differences were obtained by Nermut' et al. (2015) and a reason for these variations could be the food source or culture media used. Another difference is the number of male genital papillae, which were recorded as being five by Mengert (1953), but were revealed by SEM to be six (Nermut' et al., 2015;this paper). The new species is morphologically very similar to the Czech specimens of A. appendiculatum described by Nermut' et al. (2015), but there are some differences: (i) more posterior position of the excretory pore in the Kleinform females (169-188 vs 117-164 lm from anterior end); (ii) smaller anal body diameter in Kleinform females (24-26 vs 27-39 lm); and (iii) longer tail in Kleinform males (98-112 vs 56-79 lm; c = 9.2-9.9 vs 11.4-17.8; c' = 3.0-3.4 vs 3.9-6.9). There are some problems with the latter comparison since the tail length 56-79 lm and anal body diameter 27.4-35.2 lm will give a c' of about 2 and not 3.9-6.9, thus a mistake is possibly made in Table 1 of Nermut' et al. (2015). There are, however, substantial differences in both 18S and 28S rDNA sequences between Alloionema similis n. sp. and A. appendiculatum (Figs. 6-9), which will be discussed in the next section.
Our material sheds no light on the validity or status of A. appendiculatum var. dubia (Chitwood & McIntosh, 1934), which was described only from Großform. These adults were smaller in size and had a noticeably longer basal bulb than any of the material listed in our Table 1. All prior descriptions of A. appendiculatum (sensu stricto), as well as our own measurements, indicate that the basal bulb length is comparable in Kleinform and Großform. In view of the smaller body length of A. appendiculatum var. dubia, particularly in Fig. 8 Structural differences in 18S rRNA (A, B) and partial 28S rRNA (C-F) helices between Alloionema appendiculatum and A. similis n. sp.; helices numbered according to Wuyts et al. (2001Wuyts et al. ( , 2002 and Chilton et al. (2003). A, helix 18; B, helix 23e/1-23e/2; C, helix b13_1; D, helix c2_b; E, helix c2_c; F, helix d5. Compensatory substitutions marked with arrows males, we are confident that Alloionema similis n. sp. does not represent the same organism as the nematode described by Chitwood & McIntosh (1934). It is also worth noting that these authors illustrated the lip region and subcephalic region of A. appendiculatum var. dubia as being cylindrical rather than clearly tapering, which is a condition not seen in any of our material nor shown in any of the illustrations by Nermut' et al. (2015). For the time being we therefore consider it best to neither elevate A. appendiculatum var. dubia to a separate species, nor to treat as a match with A. appendiculatum (sensu stricto). The resolution of its status must await new collections from Succinea snails obtained near the location reported by Chitwood & McIntosh (a swamp near Piscataway in Maryland).

Interspecific variability of ribosomal RNA gene sequences
The four-taxa secondary-structure based multiple sequence alignments of 18S and 28S rRNA genes of all current members of the family Alloionematidae contained 1,544 and 893 positions respectively. There were 27 (12 within 18S and 15 within 28S rDNA) unambiguous autapomorphies for Alloionema similis n. sp.; 26 (20 within 18S and 6 within 28S rDNA) for A. appendiculatum; 31 (21 within 18S and 10 within 28S rDNA) for Neoalloionema indicum; and 24 (14 within 18S and 10 within 28S rDNA) for N. tricaudatum (Figs. 6-7). Not all apomorphies can be accounted for within the 28S rRNA gene due to the fact that large part of the gene was not sequenced for N. tricaudatum. Molecular differences between Alloionema appendiculatum and A. similis n. sp. are not limited to random mutations, but include a number of compensatory substitutions in the hairpins 18, 23e/1-23e/2, b13_1, c2_b, c2_c and d5 of the secondary structure of both 18S and 28S rRNA genes (Fig. 8).

Phylogenetic analysis
The combination of characters: stoma short with sclerotised anterior part and non-sclerotised funnelshaped posterior part, median bulb without valves, basal bulb with valves, female reproductive system didelphic and amphidelphic with reflexed ovaries, and male tail without bursa, makes the systematic position of Alloionema somewhat uncertain. For Alloionema and Rhabditophanes Fuchs, 1930, Chitwood & Fig. 9 Majority-rule consensus tree of the Bayesian phylogenetic analysis of concatenated alignment of 18S rRNA and D1-D2-D3 domains of 28S rRNA, rooted using Rhabditophanes sp. KR3021, branch lengths represent the mean posterior estimates of the expected number of substitutions per site McIntosh (1934) proposed a new subfamily in the family Diplogastridae Micoletzky, 1922, the Alloionematinae Chitwood & McIntosh, 1934, differing from Diplogastrinae by the presence of a basal bulb with valves. Goodey (1963) placed Alloionematinae in the family Rhabditidae Ö rley, 1880 while Andrássy (1976) raised it to superfamily and family level (Alloionematoidea Chitwood &McIntosh, 1934 andAlloionematidae Chitwood &McIntosh, 1934) in Rhabditina. Based on molecular characters, De Ley & Blaxter (2004) placed Alloionematidae in the superfamily Strongyloidoidea Chitwood & McIntosh, 1934 in the infraorder Panagrolaimomorpha De Ley & Blaxter, 2004. In a study on the molecular phylogeny of slug-parasitic nematodes, based on 18S rRNA gene sequences, A. appendiculatum clustered in a clade with species of Strongyloides Grassi, 1879 and Rhabditophanes Fuchs, 1930 (see Ross et al., 2010). In the study by Nermut' et al. (2015), the molecular evidence from several ribosomal genes also generated a strongly supported clade including A. appendiculatum and species of Strongyloides, Parastrongyloides Morgan, 1928 and Rhabditophanes. In our analysis (Fig. 9) both A. appendiculatum and A. similis were placed as sister taxa in a strongly supported clade. Neoalloionema tricaudatum Ivanova, Pham Van Luc &Spiridonov, 2016 andN. indicum Nermut', Půža &Mráček, 2016. formed a distinct strongly supported clade, in agreement with a recent study published by Nermut' et al. (2016).

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval All applicable institutional, national and international guidelines for the care and use of animals were followed.
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