The genus Acrostichus was raised by Rahm (1928) when he reported and described Acrostichus toledoi as its type species. Goodey (1951) synonymised it with the genus Diplogaster Schultze in Carus 1857. Later, Paramonov et al. (1952a) raised the genus Diplogasteritus (type species Diplogaster nudicapitatus Steiner 1914) based on the presence of longitudinal cuticular ridges, narrow and long stoma, distinctly separated cheilostom from rest of stoma, presence of a dorsal tooth distinctly larger than subventral teeth. Diplogasteritus was demarcated by another genus Diplogastrellus in the number of genital branches which were paired (opposed) in the former and single (anterior) in the latter. Paramonov et al. 1952a inсludеd ninе spесiеs in Diplogаstеritus viz., D. nudiсаpitatus (Steiner 1914), D. filiсаudаtus (Bütschli 1874), D. rhodani (Steiner 1914), D. consobrinus (De Man 1920), D. demаni (Schneider 1923), D. minutus (Kreis 1929), D. аustriасus (Fuchs 1938), D. supеrbus (Paesler 1946) and D. oсcidentalis (Steiner 1932). However, in his monograph, Paramonоv (1964) inсludеd fifteen valid spесiеs in Diplogаstеritus. Weingärtner (1950) considered eight species viz., Diplоgаstеr (Diplogastеr) dепdrophilus Weingärtner 1950, D. (D.) сonsobrinus De Man 1920, D. (D.) austriасus Fuchs 1938, D. (D.) nudiсаpitаtus Steiner 1914, D. (D.) supеrbus Paesler 1946, D. (D.) stoесkhеrti Völk 1950, D. (D.) linеаtus Fuchs 1915 аnd D. (D.) subtеrrаnеus Hnatewytsch 1929 under ‘nudicapitatus’ species group under the genus and subgenus Diplogaster. Massey (1962) synonymised Diplogasteritus with Acrostichus considering the former as a junior synonym and recognized a total of nineteen species under it with A. toledoi as type species. Goodey (1963) considered Diplogasteritus as a valid genus and endorsed Weingärtner (1950) concept of ‘nudicapitatus’ group along with listing eight species viz., D. апgustilаimus (Schuurmans Stekhoven and Teunissen 1938), D. brеviсаudаtus (Schuurmans Stekhoven and Teunissen 1938), D. еurуcеphаIus (Völk 1950), D. lаbiаtus (Соbb in Merrill and Ford 1916), D. lirаtus (Schneider 1866), D. minutus (Kreis 1930), D. occidеntalis (Steiner 1932) and D. zurstrаssеni (Saсhs 1950) thus making the number of species in the genus sixteen, however, he regarded Acrostichus as a genus inquirendum. Lazarevskaja (1965) splitted Acrostichus into Acrostichus and Filipjevella by placing 5 species under the latter and F. minimus Lazarevskaja 1965 as its type species, which was later proposed as a junior synonym of Diplogasteritus as well as Acrostichus. Andrássy (1984) recognized the two genera, and differentiated Acrostichus by having unpaired, anterior female gonad and Diplogasteritus by paired female gonad. While considering Filipjevella a junior synonym of Diplogasteritus, he diagnosed the latter genus with the presence of a large rounded, offset uterine pouch opposite vulva. Andrássy (1984) inсludеd 23 spесiеs in Diplogаstеritus. Gagarin (2002) while endorsing Andrássy's view, published a review of the genus Diplogasteritus and after several synonymizations considered eight valid species in the genus. Earlier, Fürst von Lieven and Sudhaus (2000) accepted Diplogastrina as a suborder of Rhabditida and gave an outline phylogeny of this taxon based on comparative functional morphology of the buccal cavity. Subsequently, in their catalogue of the Diplogastridae, Sudhaus and Fürst von Lieven (2003) regarded Diplogasteritus, Filipjevella and Aduncospiculum Giblin and Kaya 1984 as junior synonyms of Acrostichus and considered 28 valid species under the latter. They also accepted only 28 genera out of the 83 published genera under Diplogastridae. Three more species A. rhyncophori, A. megaloptae and A. puri have been added later to the genus by Kanzaki et al. (2009); Kanzaki et al. (2010b), Kanzaki et al. (2010a) respectively, thus making a total of 31 valid species under Acrostichus. Seinhorst et al. (1959b) raised a new family Titilleinae for the new genus Titilleus based mainly on structure of gubernaculum and considered Acrostichus, Diplogasteritus, Peterngus Ahmad et al. 2004 and Aduncospiculum as other subordinate genera. He also described three new species viz., D. major, D. microsus and D. teloporosus under the genus Diplogasteritus.

The soil and water samples were processed using Cobb (1918) sieving and decantation and modified Baermann’s funnel techniques. The nematodes were extracted and fixed in hot formalin-glycerol fixative, dehydrated by the slow evaporation method (Seinhorst et al. 1959a) and mounted in anhydrous glycerine. Permanent slides were prepared using the paraffin wax-ring method (De Maeseneer and D’ Herde 1963). The measurements were taken with an ocular micrometer. LM photographs were taken with a Jenoptik ProgRes digital camera mounted on an Olympus BX-51 DIC microscope. For Scanning Electron Microscopy (SEM), the specimens were fixed in 2% glutaraldehyde, post-fixed in 2% osmium tetroxide, dehydrated in alcohol series and critical point dried using CO₂. The mounted nematodes were coated with 10 nm gold before viewing at 10 kV with an XL30 FEG scanning electron microscope.

Thirty morphological characters were selected to compare 20 species of Acrostichus which showed relatively detailed description (Table 2). The species with insufficient morphological details could not be included in comparison. All the selected characters were important with good taxonomic value. The characters were ranked on the basis of commonality principle. Character state ‘0’’ represented the most commonly occurring trait whereas a gradual increase in value represented more deviation. Data matrix (Table 3) was prepared and single parsimonious tree was retrieved (Fig. 1) for analyses using PAUP (version 4.0 b10) (Swofford 2001) under a parsimony criterion with a heuristic search with TBR (Tree Bisection Reconnection) branch-swapping options.

Figure 1.  

Phylogram showing relationship between species of Acrostichus Rahm 1928 based on morphological data with Diplogastrellus cerea as an outgroup.

A. medius n. sp. is unique in having a blend of features of Diplogastrellus and Acrostichus. The species with nearly 1:1 sex ratio in natural population shows hermaphroditism with smaller sperms (sh) stored in spermatheca and the larger ones (sm) filling up the uterine pouch. It may be possible that like Caenorhabditis elegans, the males’ sperms besides being larger, have an edge over the hermaphrodites’ sperms in fertilizing the ova. The robust spicules with furcate distal ends, are unique for the genus as well as for Diplogastridae. The well developed and cuticularised ovijector presumably is to complement such spicules during the process of copulation. The gubernaculum, however, shows similarity to those found in several species of Acrostichus including A. superbus.

The genus Acrostichus is typified by the species A. toledoi Rahm 1928. However, the original description of the species lacks some vital information and the illustrations include only the pharyngeal region as well as the male tail region. Massey (1962) considered Acrostichus toledoi to be a true representative of the genus with stoma much longer than wide, longitudinal striations prominent, female with a reniform spermatheca and male with massive gubernaculum, almost to the size of spicules and tails of both sexes long and filiform. Andrássy (1984), Andrássy (2005) emphasized on the monovarial condition of the species and, therefore, considered Acrostichus to be representative of all monodelphic-prodelphic species. However, the reniform spermatheca of A. toledoi seems synonymous to the dorsal uterine pouch which also makes the presence of a single anterior genital branch without a posterior extension, doubtful. Thus A. toledoi seems to possess an anterior well developed genital branch and a reduced posterior genital branch occasionally represented by a post-uterine sac. In this perspective, the presence of a relatively well-built upright dorsal tooth, uterine pouch, robust spicules and equally large gubernaculum in A. toledoi confirms the status of Acrostichus as a senior synonym to Diplogasteritus thus supporting the views of Kiontke and Sudhaus (1996). Considering the criterion of single anterior gonad, the species does not seem to enjoy enough affinities with other mono-prodelphic species that belong to genus Diplogastrellus. It is also a fact that despite its placement along with other species of Acrostichus, the species A. toledoi shows some unusual features viz., relatively greater body length (1–1.9 mm vs <1mm), relatively elongate (vs ovoid) metacorpus, greater ‘V’ value (64 vs 35–50) and larger spicules (80 µm vs <40 µm in most species of the Acrostichus). Nevertheless, in the shape of spicules and gubernaculum, the species resembles A. superbus or looks like a close relative of A. nudicapitatus, A. taedus and A. gubernatus. The relative lengths of both genital branches in supposedly amphidelphic genus Acrostichus, are likely to be variable hence an unreliable character. The present species A. medius n. sp. serves a good example demonstrating a gradual reduction in posterior genital branch (Figs 6b, c, d, e, 7a) in several specimens up to the extent of a reminiscent post-uterine sac. The species also shows variation in the shape of metacorpus that ranged from ovoid to elongate-rectangular type making it a less consistent character for differentiation. It is clearly evident that the representative species of Acrostichus clade possess both narrow- and wide tubular but thick-walled stoma with strongly cuticularised dorsal tooth mostly upright and straight or arcuate. Concurrently, the metacorpus in species having narrow tubular stoma is elongate and rectangular type while the species with broader stoma possess a swollen and ovoid metacorpus. Undoubtedly, the uterine pouch is a diagnostic character of Acrostichus as also of its junior synonym Diplogasteritus. Another reliable feature of the former is the presence of very closely placed precloacals, GP1 and GP2. Thus the amended diagnosis of the genus is as follows:

Amended diagnosis of genus

Genus Acrostichus can be characterized by the presence of transverse cuticular striations, usually prominent longitudinal ridges; narrowed to truncate lip region; stoma longer than wide consisting of a cuticularized cheilostom with six adradial plates, metastegostom anisotropic, armed with thorn or dagger-like, cuticularized, dorsal tooth and usually smaller subventral teeth; pharynx typically diplogasteroid with metacorpus usually swollen and ovoid, rarely elongate; female gonad primarily amphidelphic with an elongate to bilobed uterine pouch serving as spermatheca, posterior genital branch occasionally reduced; males without bursa, with large cephalated, well-built spicules and usually massive gubernaculum, of variable shape, genital papillae GP1 and GP2 closely placed, tails of both sexes usually filiform.

Most of the species of the genus have been described on sketchy characteristics hence many of them are likely to be synonymous as suggested by Andrássy (1984). The species A. angustilaimus (Schuurmans Stekhoven and Teunissen 1938) Massey (1966) has a poor description and illustrations and has also been synonymized with D. lineatus by Gagarin (2002). Others like A. paramicrostoma (Schuurmans Stekhoven 1943) Sudhaus and Fürst von Lieven 2003, A. brevicauda (Schuurmans Stekhoven 1951) Sudhaus and Fürst von Lieven 2003, A. brevicaudatus (Schuurmans Stekhoven and Teunissen 1938) Sudhaus and Fürst von Lieven 2003, A. filicaudatus (Bütschli 1874) Sudhaus and Fürst von Lieven 2003 and A. likoi (Kokordák 1969) Sudhaus and Fürst von Lieven 2003 show insufficient morphological descriptions with the latter two reported on the basis of individuals of one sex only. A. liratus (Schneider 1866) Sudhaus and Fürst von Lieven 2003 was described with poor description and no measurements. A. lazarevskajae (Lazarevskaja 1964) Sudhaus and Fürst von Lieven 2003, Lazarevskaja 1964, without a proper description, is a new name to Acrostichus minimus Lazarevskaja 1964. Further, the species with some anomalies or atypical features were not selected for comparison. A. paxi described from five females and five males from decaying wood by Paramonov et al. (1952b) shows a gubernaculum equal to spicules but atypical of Acrostichus as also considered species incertae sedis by Gagarin (2002). A. pterygatus (Timm 1961) Massey 1966 though included in the comparison also shows some unusual features like punctations and the dissimilar stomal armature. Diplogaster minor Cobb 1893 as synonymised with A. minutus (Kreis 1930) Massey 1966 does not seem to fit in because of presence of a single gonad in female (apud Cobb 1893) whereas D. minor apud Maupas 1900 shows too wide stoma without characteristic armature. Some of the disparities of descriptions include: descriptions of A. concolor apud Massey 1962, Massey 1966 with varying number (6 vs 8) of male genital papillae and variation in the shape and size of gubernaculum. Likewise A. taedus described by Massey (1962), Massey (1966) do not correspond in the number (7 vs 9) and configuration of genital papillae. A. minimus (Lazarevskaja 1964) Sudhaus and Fürst von Lieven (2003) as described by Gagarin (2002) shows a disparity in the size of gubernaculum as stated in the text with that shown in illustration.

Of the characters taken for cluster analysis (Fig. 1, Table 2) of twenty species of Acrostichus Rahm 1928, the stomal characteristics, the presence of uterine pouch, the shape and size of spicules and gubernaculum and the genital papillae as well as the tail shape seem to be important differentiating characters. In the constructed phylogram, Diplogastrellus cerea Kiontke and Sudhaus 1996 a species of closely related genus stands out from all species of Acrostichus Rahm 1928. A. medius n. sp. occupies an intermediate position between the outgroup species and other congeners. The next close relative to A. medius n. sp. comes to be A. consobrinus (De Man 1920) Massey 1962. However, all the selected species of Acrostichus show a more or less orderly grouping. Nevertheless, in the phylogram two large and one small subgroups could be figured out that largely reflect species with closer affinities. The subgroup representing the largest aggregate includes A. halicti (Giblin and Kaya 1984) Sudhaus and Fürst von Lieven 2003, A. puri Kanzaki et al. 2010a, A. megaloptae Kanzaki et al. 2010b, A. primitivus (Gagarin 2002) Sudhaus and Fürst von Lieven 2003 and A. pterygatus (Timm 1961) Massey 1966 the former three species shows closer affinity. Another group comprises of A. dendrophilus (Weingärtner 1950) Massey 1966, A. stoeckherti (Weingärtner 1950) Massey 1966, A. miminus and A. lazarevskajae with the former three species having more relatedness. A. taedus Massey 1962 enjoys affinity with both subgroups. Rest of the species A. concolor Massey 1962, A. rhyncophori Kanzaki et al. 2009, A. gubernatus Massey 1974, A. nudicapitatus (Steiner 1914) Massey 1962, A. occidentalis (Steiner 1932) Massey 1962 and A. lineatus (Fuchs 1915) Massey 1962 show a staircase arrangement with more or less an orderly grouping except A. аustriасus (Fuchs 1938) Massey 1962 and A. superbus (Paesler 1946) Massey 1966 which show closer affinities. The clustering groups indicate similarity largely in having slightly to moderately arcuate, simple spicules, trough-shaped, robust gubernaculum of 75-100% of spicule length (Fig. 9). The species A. rhyncophori Kanzaki et al. 2009 is unique in the lot with gubernaculum having bifid processes distally. Titilleus shahinae Seinhorst et al. 1959b shows striking similarity with the latter and has been raised mainly on the basis of bifid processes of gubernaculum termed as titillae; hence the status of the genus Titilleus is doubtful and T. shahinae Seinhorst et al. 1959b seems to be a junior synonym of A. rhyncophori Kanzaki et al. 2009.

Figure 9.  

Comparison of stoma, spicule and gubernaculum of selected species of Acrostichus Rahm 1928 and Diplogastrellus cerea Kiontke and Sudhaus 1996 (modified from the original drawings). (A) A. аustriасus (Fuchs 1938) Massey 1962; (B) A. concolor Massey 1962; (C) A. pterygatus (Timm 1961) Massey 1966; (D) A. taedus Massey 1962; (E) A. rhyncophori Kanzaki et al. 2009; (F) A. gubernatus Massey 1974; (G) A. nudicapitatus (Steiner 1914) Massey 1962; (H) A. occidentalis (Steiner 1932) Massey 1962; (I) A. halicti Giblin and Kaya 1984; (J) A. megaloptae Kanzaki et al. 2010b; (K) A. puri Kanzaki et al. 2010a; (L) A. primitivus (Gagarin 2002) Sudhaus and Fürst von Lieven 2003; (M) A. superbus (Paesler 1946) Massey 1966; (N) A. dendrophilus (Weingärtner 1955) Massey 1966; (O) A. lazarevskajae (Lazarevskaja, 1964) Sudhaus and Fürst von Lieven 2003; (P) A. stoeckherti Völk 1950; (Q) A. lineatus (Fuchs 1915) Massey 1962; (R) A. consobrinus (De Man 1920) Massey 1962; (S) A. medius n. sp; (T) Diplogastrellus cerea Kiontke and Sudhaus 1996 (figures are schematic and not to scale).

The present species A. medius n. sp. seems to serve as a transitional species in the evolutionary process showing affinities to both the supposedly related yet distinct genera Acrostichus and Diplogastrellus. Besides showing distinctive features of the former viz., thick-walled stoma, a large uterine pouch, primarily amphidelphic female gonad and the closely placed GP1 and GP2, the species shows some distinctive features of Diplogastrellus too. There is a tendency of reduction of female posterior genital branch with few individuals possessing only the post-uterine sac. The narrow tubular stoma with a less prominent, triangular dorsal tooth and an elongate to rectangular metacorpus in few specimens also hint towards its affinity with Diplogastrellus. Thus the close lineage of the two taxa can further be a matter of investigation as A. medius n. sp. seems to be a link between the Diplogastrellus and Acrostichus. It is also a fact that most species of Acrostichus possess prominent cuticular ridges which are often weak or faint in species of Diplogastrellus. A. rhynchophori and A. medius n. sp. form an exception where in addition to faint longitudinal ridges, two prominent ridges are found in the lateral fields. The lip region does not appear to offer any differentiating feature between the two genera as is seen in A. rhyncophori and D. metamasius Kanzaki et al. 2008 which show striking similarities in the lip regions (apud Kanzaki et al. 2009 : Fig. 14 A–C and apud Kanzaki et al. 2008 : Fig. 7 A–C). Likewise, the dorsal tooth in D. indicus Khera 1970 and D. sikorai Khan et al. 2008 appears slightly different from those (Ahmad et al. 2005) typical of Diplogastrellus thus making the feature of stoma and dorsal tooth as weak differentiating characters. Thus the only reliable differentiating features of Acrostichus are the prominent gubernaculum, uterine pouch and the relative close position of the genital papillae GP1 and GP2.