Description of a new species of Acrostichus Rahm 1928 (Nematoda: Diplogastridae) from India with a note on its position and relationship with the congeners

Abstract Background The clade Diplogastridae Micoletzky 1922 largely represents the bacterivorous or predatory nematodes that very often demonstrate phoretic, necromenic or parasitic associations with insects (Sudhaus and Fürst von Lieven 2003). That is the reason, much of the diversity of the family remains undocumented because of their absence from routine soil samples. Due to their variable habitats and niches, these nematodes show ample variation in their stomal armature, feeding behavior and life cycle patterns. New information The paper describes and illustrates a new diplogastrid species of genus Acrostichus Rahm 1928 that appears to be the link between the genera Diplogastrellus Paramonov et al. 1952a and Acrostichus. Acrostichus medius n. sp. is characterised by hermaphroditic females and males having faintly striated longitudinal ridges, demarcated lateral fields, amalgamated lips, six adradial cheilostomal plates, moderately-built dorsal tooth, relatively smaller posterior genital branch; large oval uterine pouch and males with robust, heavily cuticularised spicules, each with hood-like capitulum, deeply forked distal end with fine extensions and a ventral attenuated arm; gubernaculum 2/3 of spicule length and rudiments of bursa confined to posterior four pairs of genital papillae. The biogeographical distribution of Acrostichus has been mapped and the relationship between congeners has been analysed cladistically and discussed.


Collection, processing, extraction and taxonomic study
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 CO . 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.
Male (Figs 2b, f, 5 and Table 1): Similar to female in general appearance but shorter in length and strongly curved in posterior region. Testis single, laterally reflexed, reflexed part 45-52 µm long. Vas deferens a long tube with a tapering ejaculatory duct joining with rectum to form cloaca. Spicules strongly built, heavily cuticularised, strongly arcuate in proximal half, 1.3-1.5 times anal body diam. long with elongated hood-like capitula, a distinct neck and distal part deeply bifurcated with fine extensions and an attenuated ventral arm separated from main body of spicule (Figs 2f, 5a, d, e). Gubernaculum stout, heavily built, 70-82% of spicule length, proximally tapering and curved and distal end with slight protuberances (Figs 2f, 5e). Tail in two parts, an anterior short, conoid part and a posterior long, filamentous part. Genital papillae ten pairs with three precloacal, one adcloacal and six postcloacal pairs. Precloacal pairs GP1 and GP2 closely placed, subventral; GP3 lateroventral. GP4 at level of cloaca. Postcloacal pairs GP5 closely posterior to cloaca; GP6 subventral, more or less one cloacal body diameter posterior to cloaca and nearly at level of phasmids. GP10 subdorsal pair placed slightly posterior to the group of subventrals GP7, GP8 and GP9 (Figs 2f, 5b, c). A membranous rudiment indicating bursa, occasionally confined to posterior most genital papillae including three subventral pairs and one dorsally directed pair (Figs 2f, 5c). Copulatory muscles representing 5-6 pairs of broad bands. Phasmids pore-like, about one anal body diam. posterior to anal opening.

Diagnosis
Acrostichus medius n. sp. is characterised by female hermaphrodites having a mediumsized body with cuticle bearing faintly striated longitudinal ridges; lateral fields demarcated with two conspicuous ridges; lips amalgamated, labial sensilla small, papilliform; cheilostom with six adradial plates; dorsal tooth moderately-built slightly larger than subventrals; posterior genital branch relatively smaller; dorsal uterine pouch large, multilocular, oval to rounded occasionally filled with sperms and males with robust, heavily cuticularised spicules with hood-like capitula, appearing deeply forked distally with fine extensions and a ventral attenuated arm; gubernaculum 2/3 of spicule length with curved and tapering proximal end and distal end with slight protuberances; bursa almost absent with rudiments confined to posterior four genital pairs.

Etymology
The species name 'medius' is a latin word that indicates the intermediate status of the species showing a blend of characters of Acrostichus and Diplogastrellus.

Distribution
Samples containing Acrostichus medius n. sp. were collected from soil rich in organic matter near State Bank of India at Aligarh, Uttar Pradesh, India at geographical coordinates 27°53'35"N, 78°4'27"E.

Ecology
Acrostichus is a genus with species reported from all the continents except Australia although biogeography of the genus indicates larger distribution in subtropical to temperate regions (Fig. 8). The individuals have been found to inhabit soil, fresh and polluted water and more specifically reported from aquatic mulm or slime flux or sewage; from soil to rotten decaying matter or from moist husk to rotten plants and from dung to frass of beetles. Most species are bacteriophagous but like other diplogastrids, may also feed on protozoa, fungi and nematodes (Bento et al. 2010).

Conservation
Due to inhabiting diverse environment types, the species show extensive diversity reflecting phenotypic plasticity. The variations in the shape of buccal cavity which can be shallow and broad, or narrower and deeper, and the variations in size and shape of dorsal tooth are few such examples.

Biology
A. medius n. sp. shows hermaphrodite females and males in the population. It demonstrates a gradual reduction in posterior genital branch in several specimens up to the extent of a reminiscent post-uterine sac. Such unique feature indicates towards its transitional status in the evolutionary process showing affinities to both the related yet distinct genera Acrostichus and Diplogastrellus.  Weingärtner 1950 andGagarin 2002).

Relationship with closely related species
The new species comes close to A. dendrophilus (Weingärtner 1950 The new species also resembles A. lazarevskajae ) Sudhaus and Fürst von Lieven 2003 in most allometric ratios but differs from it in having larger body size (L= 764-867 µm vs 365-430 µm in females and 611-715 µm vs 290-441 µm in males); greater 'ć' value (9.2-15.8 vs 6.6) and large-sized spicules (36-44 µm vs 23-25 µm) in males; spicules massive (vs slender) with elongated hood-like ( vs rounded capitula) and distal part deeply bifurcated (vs bearing very fine spines at tip) and presence [vs absence of an attenuated ventral arm separated from main body of spicules in A. lazarevskajae apud .

Notes
Most of the species of the genus have been described on very flimsy characteristics hence many of them are likely to be synonymous. We tried to have a comparative assessment of the morphological characteristics of most species. Of the total nominal species, few could not be included largely due to insufficient descriptions available.

Analysis Remarks
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.

Taxonomic status and affinities of new species
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 monodelphicprodelphic 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 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 ) 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. 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