Nematode Diversity of Phytotelmata of Nepenthes Spp. in Mount Hamiguitan Range Wildlife Sanctuary, Philippines

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Ghent University Faculty of Sciences Department of Biology

Academic Year 2011-2013

Nematode diversity of phytotelmata of Nepenthes spp. in Mount Hamiguitan Range Wildlife Sanctuary, Philippines

JOESEPH SELLADO QUISADO

Promoter: Prof. Dr. WIM BERT & Thesis submitted to obtain the degree of Dr. IRMA TANDINGAN DE LEY Master of Science in Nematology

Nematode diversity of phytotelmata of Nepenthes spp. in Mount Hamiguitan Range Wildlife Sanctuary, Philippines

JOESEPH SELLADO QUISADO Nematology Section, Department of Biology, Faculty of Sciences, Ghent University; K.L. Ledeganckstraat 35, 9000 Ghent, Belgium [email protected]/[email protected]

Summary – Nematodes from phytotelmata of Nepenthes hamiguitanensis and N. peltata in Mt. Hamiguitan, Philippines included three new species of the genera: Molgolaimus Ditlevsen 1921, Dominicactinolaimus Jairajpuri and Ahmad 1992, Tripylella (Bütschli, 1873) Brzeski & Winiszewska-Ślipińska, 1993; two known: Tylocephalus auriculatus (Bütschli, 1873) Anderson, 1966, Pelodera strongyloides (Schneider, 1860) Schneider 1866; and three uncertain species of the genera: Paractinolaimus Meyl 1957, Plectus Bastian 1865, and Anaplectus De Coninck & Schuurmans Stekhoven 1933. Measurements and illustrations are provided. Molgolaimus sp. nov. is characterized by the absence of pre-cloacal supplements, shape of the spicule with lamina widened distally, conical tail with swollen tip and without digitate prolongation, and sexual dimorphism in the shape of the cardia (elongated in male and more round in females). Moreover, a comprehensive key for the genus Molgolaimus is presented. Dominicactinolaimus sp. nov. is characterized by short body length, long tail (c = 6.6-7.6) and 6-7 pre-cloacal supplements. The generic position of Dominicatinolaimus is reaffirmed and the synonymy with Trachypleurosum or Trachactinolaimus is rejected. Tripyllela sp. nov. is morphologically close to T. iucunda but differs in the female reproductive system having reduced posterior branch. The morphology and morphometry of Tylocephalus auriculatus and Pelodera strongyloides specimens agree with the original descriptions of Anderson, 1966 and Schneider, 1866 respectively. Phylogenetic analysis of small subunit rDNA for T. auriculatus and D2-D3 expansion segment of LSU rDNA for P. strongyloides supported sister relationship with respective species sequences available in GenBank. Furthermore, Molgolaimus and Actinonema were observed from the samples which support the initial discovery of marine nematodes in the Nepenthes phytotelmata in 2008. However, detailed taxonomical identification of Actinonema is not provided due to loss of sample during the process. In addition, the presence of freshwater nematode throws a new light on a better understanding of the complex scheme of Nepenthes carnivory and enzyme production of pitcher plants. Keywords - new species, marine, freshwater, integrative taxonomy, dichotomous key, ecology

The Philippines is a tropical country located between 116° 40’ and 126° 34’ E longitude and 4° 40’ and 21° 10’ N. It is composed of 7107 islands and currently heads the world list of megadiversity countries, with a study concluding that its waters contain the world’s highest diversity of marine life (Heaney & Regalado Jr, 1998). About more than 510 species of land vertebrates are unique to the islands and estimates of endemism for vascular plants range from 45 % to 60%, an exceptionally higher level of endemism for a country of its size (Heaney & Mittermeier, 1997). In addition, Myers et al. (2000) includes the Philippines on the list of biodiversity hotspot for conservation priorities as it possesses exceptional concentration of endemic species facing also an exceptional loss of habitat. Nematodes are unfortunately overlooked in biodiversity programs and often not even mentioned as a terrestrial invertebrate (Hill et al., 2005). Philippine nematology in the past four decades focused on applied aspects (management) of plant parasitic nematodes and free- living nematodes are oftentimes neglected for taxonomical identification and characterization. Despite its reported biodiversity richness, nematodes remained unexplored in the Philippines. In the current study we will evaluate the diversity of nematodes inhabiting Nepenthes phytotelmata. The term “phytotelmata” comes from a Greek words phyton which means “plants” and telm which means “pond” (Maguire Jr, 1971). It is used to describe bodies of water impounded by plant parts and tree holes (Kitching, 1971). Phytotelmata comes in various forms Kitching and Pimm (1985), and Kitching (2000) grouped these into 5 major classes of habitats which include water-filled tree holes, plant axils, bromeliad tanks, bamboo internodes and pitchers. The latter includes the genus Nepenthes, a member of the family Nepenthaceae and the largest genus of pitcher plants with approximately 90 recognized species at present. Amoroso & Aspiras (2011) recorded 7 species of Nepenthes in Mt. Hamiguitan. Nepenthes are carnivorous plants, capable of modifying its leaves into a shape of cup or pitcher (Clarke & Lee, 2004). This serves as prey-trapping mechanism which features a deep cavity filled with liquid known as a pitfall trap (Król et al., 2012). These phytotelms of pitcher plants serve as a special kind of habitat for insect larvae, and various microorganisms including nematodes (Maguire Jr, 1971). Only a small number of studies have been conducted on nematode inhabitants of water held by Nepenthes spp. The most recent and detailed finding was the discovery of panagrolaimid nematode, Baujardia mirabilis (Bert et al., 2003). It was assumed that this nematode is naturally inhabiting the phytotelmata of Nepenthes mirabilis since it was found alive in several pitchers (Bert et al., 2003). The most comprehensive study done by Menzel

(1922) was on N. gymnamphora, who found several nematodes belonging to genera Diplogaster, Dorylaimus, Plectus and Rhabditis. He also described and considered that only Panagrellus nepenthicola is the real inhabitant of pitcher plants. A study of community structure of inhabitants of Nepenthes alata in West Sumatra recorded 25 nematode individuals but the species were unknown (Sota et al., 2006). A survey was initiated last 2010 headed by Dr. Irma Tandingan De Ley in Mt. Hamiguitan. Samples were taken from soil, litter, moss, epiphytes and from phytotelmata of Nepenthes. Unexpectedly, marine nematodes were found in the Nepenthes samples but this was never given enough attention as they thought those were just a contamination from other samples in their laboratory. This follow-up survey was initiated to provide a contribution towards the documentation of the nematodes present in Mount Hamiguitan Range Wildlife Sanctuary with an emphasis on pitcher plant phytotelmata. To verify whether nematodes associated with pitcher plants in Mount Hamiguitan are similar with the previous studies from other location; i.e. to validate the discovery of the marine nematodes from phytotelmata samples. To provide comprehensive morphological and phylogenetic information of the nematodes found in the phytotelmata. To hypothesize possible explanation how these nematodes survive if they inhabit the phytotelmata of Nepenthes which is known to produce enzymes and other compounds used to digest prey.

Materials and methods

CHARACTERISTIC OF THE AREA AND SAMPLING SITE

Mount Hamiguitan Range is one of the wildlife sanctuaries in the Philippines, located 6°40’01” to 6°46’60” N and 126°09’02” to 126°31’01” E in the province of Davao Oriental. The mountain is bordered by bodies of water, Pacific Ocean to the east and Davao Gulf to the west (Figure 1). The 6,834-hectare total surface area of Hamiguitan is characterized by five 5 vegetation types each of these forest type harbors endemic and rare species of flora and fauna, including different species of Nepenthes (Amoroso & Aspiras 2011).

Figure 1. Sampling site. Mount Hamiguitan, Davao Oriental, Island of Mindanao, Philippines

SAMPLE COLLECTION AND EXTRACTION Samples were obtained from three different species of Nepenthes (Nepenthes hamiguitanensis, N. peltata and N. micramphora). Collection was done by a random sampling of pitcher plants along the trail. A total of 13 sampling sites with respective coordinates, altitude (m) were chosen (Table 1). Each sampling site might represent one composite sample of all pitchers in the specific location while others represent only one pitcher. Three samples were recorded to have nematodes (Table 2). Liquid inside the pitchers of Nepenthes spp. were decanted into a 28 µm mesh sieves. The filtrates were washed using DESS (Yoder et al., 2006) then collected into a 100 ml falcon tubes and transported to Ghent University. Nematodes were isolated from filtrate using the Ludox floatation technique (Somerfield and Warwick, 1996). Specimens were kept and fixed back in DESS.

Table 1. Site description of Nepenthes species samples and pH value. Sample Species of Nepenthes Coordinates Elevation (m asl1) pH 1 N. peltata 06°43.615'N 126°09.167'E 639 4.8 2 N. hamiguitanensis 06°43.481'N 126°09.133'E 639 3.6 3 N. hamiguitanensis* 06°43.481'N 126°09.133'E 639 3.9 4 N. hamiguitanensis 06°43,453'N 126°09.527'E 821 5.6 5 N. peltata 06°43,453'N 126°09.527'E 821 6.2 6 N. peltata* 06°43.014'N 126°11.004'E 1188 4.4 7 N. peltata 06°43.014'N 126°11.004'E 1188 5.8 8 N. micramphora 06°00.344'N 126°10.975'E 1606 2.7 9 N. micramphora 06°00.344'N 126°10.975'E 1606 3.5 10 N. peltata* 06°00.344'N 126°10.975'E 1606 3.1 11 N. peltata 06°44.338'N 126°10.980'E 1607 3.4 12 N. hamiguitanensis 06°44.362'N 126°10.955'E 1619 2.6 13 N. hamiguitanensis 06°44.362'N 126°10.955'E 1619 3.2

1 m a.s.l = meter above sea level; * single pitcher 4

MORPHOLOGICAL CLASSIFICATION Since DESS makes the nematode shrink, preserved specimens were transferred to staining block with sterile water 3 times after every 2 hours to hydrate. Fully hydrated and preservative-free nematodes were then transferred to staining block filled with 5 – 10 drops (depends on size and number of nematodes) of sterile water and subsequently add 1 drop of solution II (95 parts of 96% ethanol and 5 parts glycerine), place it in the oven at 40°C and partially cover it to allow slow evaporation of ethanol. Add another 2 drops of solution II 4 times after every two hours. Next, leave the nematodes in the oven overnight with few drops of solution III (50 parts of 96% ethanol and 50 parts glycerine). The next day, nematodes were checked if it’s in pure glycerine before mounting on slides for light microscopy. This is almost similar technique of Seinhorst (1959) as modified by De Grisse (1969) except that, De Grisse solution 1 was never introduced in the process since it contains formalin that degrades the DNA; the use of this method is of advantage since nematode’s DNA will still be viable for molecular processes when necessary. Permanent mounts on Cobb’s slide were sealed using the paraffin wax ring technique. Multifocal video clips for morphological archiving (VCE in De Ley & Bert, 2002; De Ley et al., 2005), measurements and illustrations (photos and drawing) were accomplished using Olympus BX50 and BX51 DIC microscopes with Nikon imaging software NIS-Elements version 4.0 and the drawings were modified using Paint, Microsoft Windows® 7 version and Adobe Photoshop® 7.0 software.

ABBREVIATIONS a – body length divided by maximum body DEI – deirid diameter diam – diameter b – body length divided by pharynx length EP – excretory-secretory pore c – body length divided by tail length Gub – gubernaculum c’ – tail length divided by anal body GR – guiding ring diameter hd – head abd – anal body diameter isthm – isthmus amph – amphial fovea L – entire body length ant – distance from anterior end l - length anus – anus/cloacal opening l.c.s – length of cephalic setae blb – basal bulb lip – labial region cbd – corresponding body diameter NR – nerve ring crd – cardia odntph – odontophore corp – corpus odntsl – odontostyle p-corp – procorpus ph - pharynx m-corp – metacarpus p-rc – pre-rectum

rc – rectum ref – reflex spic – spicule tdg – digitate prolongation of tail st – stoma V – distance of vulva from anterior end as sup - supplement percentage of body length t – tail W – maximum body diameter

MOLECULAR ANALYSIS Individual nematodes preserved in DESS were placed in a separate staining block filled with sterile water for 2 hours to rehydrate and remove the preservatives. Then, they were transferred to a new staining block with sterile water for 5 minutes to remove remaining components of DESS prior to morphological archiving through multifocal video clips. However, two specimens from Dominicactinolaimus were processed to glycerin first before it was used for molecular analysis. Individual specimens of Dominicactinolaimus were recovered from permanent mounts by carefully removing the cover slip using a sharp razor blade. The recovered nematode was placed into a new staining block filled with distilled water overnight to remove the glycerol. Prior to DNA extraction was finally washed by letting it passes through 3 changes of distilled water every after 10 minutes. The nematode was cut and transferred into 500μl eppendorf tube filled with 20μl worm lysis buffer (50mM KCl; 10mM Tris pH=8.3; 2.5mM MgCl2; 0.45% NP 40 (Tergitol Sigma); 0.45% Tween 20) and was frozen for at least 10 min at -20°C. 1μl proteinase K (1.2mg/ml) was added into the sample and incubated in the PCR-machine (65°C for 1 hour and 95°C for 10 minutes). After centrifugation (1 min; 20800g), 1μl of the DNA template was added into PCR mixture (Taq DNA Polymerase, Qiagen, Hilden, Germany) with 0.5μl each of primers, G18S4 and 18P (Blaxter et al., 1998), 4F and 4R (Tandingan De Ley et al., 2002) for small subunit (SSU) rDNA gene, D2a and D3b (De Ley et al., 1999) for the D2D3 expansion segments of the large subunit (LSU) rDNA. The thermal cycling conditions for PCR were as follows: denaturation at 94°C for 4 min, followed by 50 cycles of 94°C for 30 sec, 54°C for 30 sec and 72°C for 2 min. DNA Sequencing was done as described in Munera Uribe et al. (2010) by using an Applied Biosystems ABI 3130XL Genetic Analyser (Foster City, California, USA). Additional sequences of each taxa for phylogenetic analyses were obtained from GenBank. The SSU and D2-D3 LSU sequences were aligned with the Clustal method, manually checked and edited using SeaView® version 4.2.2 (Gouy et al., 2010). Bayesian phylogenetic inference (BI) was done with MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) for 3 million generations. Phylograms were examined with TREEVIEW® 1.6 (Page, 1996) and converted into graphic files using Adobe Illustrator® 7.0 (Adobe Systems, Mountain View, CA, USA).

Results

Samples were obtained from the phytotelmata of three species of Nepenthes. Out of thirteen samples we collected, only three samples were found to have nematodes. The initial findings that phytotelmata of Nepenthes harbors marine nematode was confirmed because of the discovery of Molgolaimus and Actinonema known to be exclusively marine taxa. Simultaneously, seven other taxa belonging to genus Dominicactinolaimus, Paractinolaimus Tylocephalus, Plectus, Anaplectus, Tripylella, Pelodera and one taxon of the family Dorylaimidae were found (Table 2).

Table 2. Coordinates, elevation, pH value, lists of nematode taxa and its respective count from three samples of phytotelmata of Nepenthes spp. in Mount Hamiguitan, Province of Davao Oriental, Philippines.

N. hamiguitanensis N. peltata (sample 1) N. peltata (sample 6) (sample 4) 06°43.615'N 126°09.167'E 06°43,453'N 126°09.527'E 06°43.014'N 126°11.004'E 639 m asl 821 m asl 1188 m asl pH 4.8 pH 5.6 pH 4.4 Dominicactinolaimus sp. 8♀; 4♂ Molgolaimus sp. nov. 2♀; 3♂ Paractinolaimus sp. 2♀; 1♂ Actinonema sp. 1♀ Tripylella sp. 14♀; 4 J Anaplectus sp. 5♀ Pelodera sp. 3♂; 26 J Plectus sp. 3♀; 6J Dorylaimidae 2 J 9 actinolaimid juveniles Tylocephalus sp. 3♀; 1J

Classification of the Genus Molgolaimus Ditlevsen 1921

Jensen (1978) erected the family Molgolaimidae (Desmodorida) as characterized by the presence of a single testis and females with reflexed ovaries. Molgolaimidae contained two new subfamilies Aponematinae having single genus Aponema Jensen, 1978 and Molgolaiminae composed of two genera Molgolaimus Ditlevsen, 1921 and Prodesmodora Micoletzky, 1923. Lorenzen (1981, 1994) classified the genus Molgolaimus (Molgolaiminae) within the family Desmodoridae based on the synapomorphic feature of the superfamily Desmodoroidea in which testis is single and directed anteriorly and the antidromously reflexed ovaries and Aponema was placed to Microlaimidae because of its outstretched ovaries and Prodesmodora to Prodesmodorinae, making the subfamily Molgolaiminae monogeneric. This classification was followed by Platt and Warwick (1988). Molgolaimus differed from other taxa within the family by having the perfectly round amphidial fovea.

Molgolaimus Ditlevsen 1921 (adapted)

Diagnosis. Molgolaiminae. Cuticle finely striated to apparently smooth. Amphidial fovea round and posterior to cephalic depression. Inner labial and outer labial sensilla mostly small. Cephalic setae close to the cephalic depression either anterior or posterior. Buccal cavity small, weakly sclerotized, narrow and with small teeth. Pharyngeal corpus narrow and cylindrical, ending in a pronounced mainly spherical muscular bulb; pharyngeal lumen weakly sclerotized throughout the corpus but heavily sclerotized at the bulb. Cardia of variable length. Excretory-secretory pore anterior to nerve ring, seldom posterior to it (maybe obscure). Female reproductive system didelphic-amphidelphic, with ovaries reflexed; position of genital branches variable; anterior branch left and posterior branch right of the intestine or reversed, or both branches on the same side of the intestine either left or right; position of vulva variable. Spermatheca sometimes present. Male reproductive system monorchic, with a single anterior outstretched testis left or right of the intestine. Vas deferens long and thin. Spicule of variable length and shape from short and bent to long and sinusoidal or straight. Gubernaculum with or without apophysis. Pre-cloacal supplements often present. Tail of varying shape and length, from short conical to elongate slender with the posterior portion cylindrical (digitate); tail tip either pointed or swollen.

Molgolaimus sp. nov. (Figs 2-3)

MATERIAL STUDIED: Three males and two females: 1 holotype male, 1 allotype female and 3 paratype specimens. 2 males, 1female.

LOCALITY: Table 2

ETYMOLOGY: The species will be named after the mountain combined with pitcher plant species (Nepenthes hamiguitanensis) where these nematodes were collected.

MEASUREMENTS: Table 3

MORPHOLOGICAL DESCRIPTION Body cylindrical and rather short, gradually tapering towards the anterior end with an offset head marked by depression. Cuticle striated, cephalic setae located anterior to the cephalic depression. Amphidial fovea circular, its diameter 32 - 38% of corresponding body diameter at 1.8-2 head diameter from the anterior end. Cardia sexually dimorphic, elongated in male and spherical shaped in females. Ventral gland cell of the excretory –secretory system (E-S system) relatively large, displacing the intestine towards the dorsal side, E-S pore posterior to the nerve ring. Tail conical. Male gonads with one testis, directed anteriorly, sperm cells lemon shaped. Spicule shape with lamina widened distally and well developed capitulum, about one anal body diameter in length. Gubernaculum small, without apophysis, quite obscure under light microscope. Pre-cloacal supplements absent. Female reproductive system didelphic, amphidelphic and with reflexed ovaries. Anterior ovary slightly longer than the posterior one. Uterus filled with elongated shaped sperm cells and vulva situated at 50% of the body length. Diagnosis: Molgolaimus sp. nov. is characterized by absence of pre-cloacal supplements, shape of the spicule with lamina widened distally, short spicule about one anal body diameter long or 25-27 µm, conical tail with swollen tip and without digitate prolongation, sexual dimorphism in the shape of the cardia, elongated in male and more round in females, E-S pore located posterior to the nerve ring.

Figure 2. Molgolaimus sp. nov., A: holotype male pharyngeal region; B: allotype female pharyngeal region; C: holotype male total view; D: holotype male sperm (detailed view); E: holotype male tail region; F: allotype female total view; G: allotype female reproductive system (posterior branch). (Scale bars: A-G = 20 µm)

Figure 3. Molgolaimus sp. nov., A: allotype female anterior region; B: allotype female anterior region in surface view showing amphidial fovea and cuticle striation; C: holotype male anterior region; D: holotype male total view; E: allotype female reproductive system showing sperm in the uterus (anterior branch); F: holotype male pharyngeo-intestinal junction showing spherical basal bulb and a large ventral gland displacing the intestine towards dorsal side; G: holotype male pharyngeal region; H: holotype male gonad showing sperm cells; I: holotype male posterior region with spicule. (Scale bars: A-I = 20 µm).

RELATIONSHIPS: Among nine species without pre-cloacal supplement and with spicule length less than 70 µm. only Molgolaimus sp. nov. and M. turgofrons Lorenzen 1972 possess a ratio of spicule length and abd = 1. In addition, morphological characters such as spicule shape, well-developed capitulum and swollen tail tip are similar. However, M. turgofrons Lorenzen 1972 is distinguished from Molgolaimus sp. nov. by the following characteristics: structure of the gubernaculum which has curved apophysis, longer tail (ratio c = 7.8) with digitate prolongation, V = 46% compared to 50% in the new species, ES pore located anterior to the nerve ring.

Table 3. Morphometrics of Molgolaimus sp. nov. (all measurements are in µm except ratios and percentage: a, b, c, c’, spic.l/abd, and V%). Holotype ♂ Paratype ♂1 Paratype ♂2 Allotype♀ Paratype ♀1 L 779 770 782 789 793 W 27.8 28.0 23.9 40.1 41.0 hd cbd 9.1 9.3 8.5 8.7 9.3 amph diam 5.9 5.7 5.7 5.2 5.7 amph cbd 15.4 16.6 15.0 16.6 17.3 amph ant 16.7 15.9 18.4 19.2 17.5 l.c.s 4.5 3.9 4.1 - 3.2 NR 73.4 70.9 76.9 77.7 78.1 NRcbd 26.6 27.4 26.0 25.9 26.4 ph.l 118 113 122 121 121 ph cbd 25.0 25.7 26.4 28.9 26.9 anus ant 707 695 710 696 699 abd 23.3 23.9 22.6 22.5 23.0 spic .l♂ ; V♀ 27.2 26.1 25.9 - - sup absent absent absent - - spic.l/abd 1.2 1.1 1.1 - - t 72 75 72 93 94 a 28 27 33 20 19 b 6.6 6.8 6.4 6.5 6.5 c 11 10 11 8.5 8.5 c' 3.1 3.2 3.2 4.1 4.1

IDENTIFICATION KEY Molgolaimus species are differentiated in male by presence or absence of pre-cloacal supplements and if present with their number, by structure of the gubernaculum, with or without apophysis, by spicule length taking possible variability into accounts e.g. in M. gigaslongincus (spicule length 106-148 µm). Also the tail shape with or without digitate prolongation or tip swollen or pointed is of diagnostic importance. Females, when available can be differentiated based on the position of the vulva (range between 40% and 53%).

Furthermore, Molgolaimus species can be separated in both sexes by the degree of development of the ventral gland cell and position of ES pore with respect to the nerve ring. Currently, the genus Molgolaimus has thirty seven (37) species identified including the three species with very poor descriptions which were regarded as species inquirendae: M. labradorensis (Allgen 1957), M. tenuicaudatus (Allgen 1959) and M. tenuilaimus (Allgen 1932). The present study describes one new species found from phytotelmata of Nepenthes hamiguitanensis, a new record for the genus known for its marine habitat and a dichotomous key to species level. An examination of morphological characters combined with morphometrical data was done for M. citrus (Gerlach 1959), M. cuanensis (Platt 1973), M. lazonus (Vitiello 1971) and M. parallgeni (Vitiello 1973) to elucidate their differences since Jensen (1978) pointed out that the above mentioned species are closely related to one another and might represent only one species. The first two steps in the key made by Muthumbi (1996) were adapted with the recent key presented. Yet, there is a flaw on the following steps in relation to characters drawn which used body length alone to separate one species to another. Several studies revealed that nematode body size could be induced by different concentrations of food (Schiemer, 1982), so low food concentration and poor food quality could lead to small body size (Jensen, 1984). These variation in size likewise affect on the morphometric characters and ratios. Although, variabilities might be minimized when using ratios rather than a single character (Roggen et al., 1986). Fonderie et al. (2012) who showed that morphometric variability, even ratios, within the progeny of a single parthenogenetic female of Halicephalobus gingivalis can surpass total interspecific variability. Hence, the variability of each morphometric character must be evaluated thoroughly before utilizing it for species description (Dodson & Lee, 2006). In the case of the genus Molgolaimus, the above-mentioned requirement is not fulfilled, since most species description were based on very few specimens or even with a single specimen such as in the case of M. unicus (Fonseca et al., 2006) and M. typicus (Furstenberg and Vincx, 1992). Therefore, to discriminate one species from the other we used morphological features on top of morphometrical data to strengthen the support of individual species. In the revision made by Jensen (1978), he pointed out that the presence or absence of pre-cloacal supplements should be used to separate molgolaimid species. Because this morphological structure is easy to observe, it is a useful character to separate Molgolaimus into a group with and without pre-cloacal supplements.

Taking spicule length into account, Fonseca et al. (2006) divided the taxa into 4 major groups based on frequency distribution for spicule length. Group 1: spicules shorter than 35 µm, group 2: spicules ranging from 35 to 53 µm, Group 3: spicules between 53 to 80 µm, and Group 4: spicules longer than 80µm. However, we disagree with these groupings. Not all of the Molgolaimus species can be placed unequivocally in the appropriate group, M. xuxunaraensis (group 2; 35-53 µm), was identified only with two male specimens with spicule length of 31 µm and 39 µm in the said groupings 31 µm should be in the first group. Another example is M. autralis (group 2; 35-53 µm) identified based on only 1 male specimen with a spicule length of 36 µm, since this is at the border of the range another male individual could as well have a spicule length of less than 35 µm. Most importantly, M. gigaslongincus was identified using 14 male specimens, of which the spicule length ranged from 106-148 µm, i.e. an intra-specific variability of 42 µm (148-106 = 42) (Fig. 4). However, group 2 only has a range of 18 µm (53-35=18) in its spicule length, group 3 has a range of 27 µm (80-53=27) and group 1 has less than 35 µm variability range. Hence, the ranges of spicule length in the groupings made by Fonseca et al. (2006) are smaller than the intra-specific variability. In agreement with Dodson & Lee (2006) and Fonderie et al. (2012) to carefully evaluate all the morphometrical data to be used for species delineation, we plot the spicule length of all species and clearly we obtained two separate groups as shown in (Fig. 5). Also an intra-specific variability of 75 µm in a single species is unlikely since in this genus the highest recorded intra-specific variability was 42 µm in M. gigaslongincus. Therefore, we reduce 4 groups to only 2 groups, hereby increasing the range per group, the first group delimited by spicule length equal or lesser than 75 µm and the second group is delimitated by a spicule length greater than 75 µm based on the frequency distribution of Molgolaimus spicule length (Fig. 5). Another morphometrical data we used in the key was the ratio of spicule length over anal body diameter and before the ratio was used, we cautiously checked every ratio and the same with frequency distribution of spicule length, we found certain clustering (Fig. 6). For this reason, we include this ratio to separate species. Hence, we present a key to different species of genus Molgolaimus taking into account the morphological characters and morphometrical data. This leads to the establishment of 4 major groups: Group 1A, characterized by presence of pre-cloacal supplement and a spicule length = or < 75 µm; Group 1B, characterized by presence of pre-cloacal supplement and a spicule length > 75 µm; Group 2A, characterized by absence of pre-cloacal supplement and a spicule length = or < 75 µm; Group 2B, Characterized by absence of pre-cloacal supplement and a spicule length > 75 µm (Table 4).

Figure 4. Every bar represents the maximum intra-specific variability of the spicule length (µm).

Figure 5. Frequency distribution of Molgolaimus spicule length, showing two groups.

Figure 6. Frequency distribution of the lowest and highest ratio of spicule length over anal body diameter incurred in every Molgolaimus species, showing four groups.

Key to species of the genus Molgolaimus

1. Pre-cloacal supplement present ...... 2 Pre-cloacal supplement absent ...... 19

2. Spicule length is equal to or lesser than 75 µm ...... 3 Spicule length greater than 75 µm ...... 16

3. Spicule length/ abd = 1 ...... 4 Spicule length/ abd > 1 but lesser than (<) 3 ...... 6 Spicule length/ abd =3 or greater than (>) 3 ...... 12 Spicule length/abd > 5 ...... M. walbethi

4. Cephalic sense organ papillae form ...... M. citrus Cephalic sense organ setae form ...... 5

5. Gubernaculum slender and weakly sclerotized ...... M. cuanensis Gubernaculum distinctly sclerotized proximally with swollen ventral portion ...... M. parallgeni

6. Tail conical with digitate prolongation ...... 7 Tail conical without digitate prolongation...... 10

7. Gubernaculum with apophysis ...... 8 Gubernaculum without apophysis ...... 9 Gubernaculum very obscure; long tail (c =6); V = 42%; ventral gland cell large; spicule long and slender, (spic = 29 µm); amphidial fovea 70% of corresponding body diameter (cbd) at a distance of 2.6 to 3 x hd from anterior end ...... M. gazii

8. Long tail (c = 6.1-7.4); V = 50%; ventral gland cell large; spicule shape with distal part wider than the proximal part, (spic = 22-27µm) ...... M. galuccii Tail length intermediate (c= 7.9-8.3); V = 42%; ventral gland large; long sinusoidal to rod-like slender spicule, (spic = 50 µm) ...... M. allgeni Short tail (c = 9.8-11.1); V = 53-54%; ventral gland cell obscure; spicule shape proximally more sclerotized and tapering towards the distal part, (spic = 23-25 µm) ...... M. minutes

9. Gubernaculum long (about half of spicule length) and slender; ventral gland cell large displacing the intestine dorsally, with additional cells posterior to it; amphidial fovea 40-50% of corresponding body diameter (cbd) at a distance of 2 x hd from anterior end ...... M. exceptionregulum Gubernaculum rather short and simple; ventral gland cell small at the level of pharyngeo-intestinal junction; amphidial fovea 50-63% of cbd; at a distance of 1-1.5 hd from anterior end ...... M. kiwayui

10. Short spicule length in relation to body length (L/spic = 25-30); body very lean (a=36-43); gubernaculum not distinct; female unknown ...... M. drakus Long spicule length in relation to body length (L/spic = <21); gubernaculum different ...... 11

11. 1 pre-cloacal supplement; gubernaculum simple and straight, one third of spicule length; spicule ventrally curved, about 1.6-1.9 abd ...... M. mareprofundus 2 small papilla-formed pre-cloacal supplements; gubernaculum absent; spicule strongly bent, about 2.7 abd; ventral setae on the tail absent ...... M. typicus 2 papilla-formed pre-cloacal supplements with short terminal setae; gubernaculums claw-like with hamose front apophysis; spicule ventrally bent, about 1.3-1.4 abd; presence of rows of somatic setae ventrally on tail ...... M. hoakonmosbiensis

12. Tail without digitate prolongation ...... 13 Tail with digitate prolongation ...... 15

13. Short tail (c >10); Body slender (a = 44.1); L > 1000 µm; females unknown ...... M. unicus Long tail (c < 10); Body relatively plump (a = 25-35); L < 1000 µm ...... 14

14. Gubernaculum small and straight; spicule distal region curved, about 3.3 abd ...... M. macilenti Gubernaculum small and thin; spicule several curves, about 3.5-4.5 abd ...... M. liberalis

15. Shorter spicule length, > 15 in relation to body length (L/spic = or > 15); body long and slender, (a = 36 – 40); Tail long at 8.2 x abd; V = 40% ...... M. sabakii

Longer spicule length, < 15 in relation to body length (L/spic < 15); body short and stout, (a = 19 – 22); Tail short at 3.2 – 3.7 x; V = 49% ...... M. tyroi

16. Tail without digitate prolongation ...... M. pacificus Tail with digitate prolongation ...... 17

17. Two pre-cloacal supplements; testis with short germinal zone or unknown ...... 18 Six pre-cloacal supplements; testis with a longer germinal zone ...... M. gigaslongincus

18. Spicule length = 91- 115 µm about 3.2 to 3.8 X abd; testis with short germinal zone; V = 50% ...... M. gigasproximus Spicule length = 163 µm about 5 x abd; V = 45%; (poorly described) ...... M. tenuispiculum

19. Spicule length equal to or lesser than 75 µm ...... 20 Spicule length greater than 75 µm ...... 26

20. Spicule length/ abd = 1 ...... 21 Spicule length/ abd > 1 but lesser than (<) 3 ...... 22 Spicule length/ abd = 3 or greater than (>) 3 ...... M. nettoensis

21. Long tail length (c=7.8)with digitate prolongation; gubernaculum with curved apophysis; pharynx short (b=9); V = 46%; amphidial fovea 45-50% of corresponding body diameter ...... M. turgofrons Short tail (c= 9.1-9.7) with digitate prolongation; gubernaculum straight surrounding the distal portion of spicule, without curved apophysis; intermediate pharynx length (b=7.2-7.7); V = 45%; amphidial fovea with little obvious inner coil ...... M. lazonus

Short tail length (c=10-11) without digitate prolongation; gubernaculum small and simple; long pharynx (b=6.4 – 6.8); V = 50%; amphidial fovea 32-38% of corresponding body diameter ...... M. hamiguitanensis sp. nov

22. Tail with digitate prolongation ...... M. falliturvisus Tail without digitate prolongation ...... 23

23. Ventral gland cell small in size ...... 24 Ventral gland cell large in size ...... 25 Ventral gland cell absent ...... M. abyssorum

24. Shorter spicule length in relation to body length (L/spic > 15); male testis right of the intestine; female unknown ...... M. carpediem Longer spicule length in relation to body length (L/spic < 15); male testis left of the intestine ...... M. sapiens

25. Spicule ventrally curved (S-shaped) 31-39 µm; long cylindrical tail about 6 x abd; amphidial fovea 2-2.5 hd from anterior end ...... M. xuxunaraensis Spicule sinusoidal (several curves) 36 µm; short conical tail about 3.8 x abd; amphidial fovea 1-1.5 hd from anterior end ...... M. australis

26. Conico-cylindrical tail with pointed tip; cephalic setae more posterior, located just behind the head depression; body more stout (a = 24); spicule length 84 µm about 4-5 x abd; females unknown ...... M. tanai Conico-cylindrical tail with swollen tip; cephalic setae very closed to anterior end; body slender (a = 36); spicule length 154 µm about 9 x abd ...... M. longispiculum

ECOLOGY OF MOLGOLAIMUS

In 1961, Timm found one species of Molgolaimus in the bottom mud of the Bay of Bengal and in Siphonocladus a marine green macro-alga occurs in subtropical or tropical seas, in shallow intertidal and subtidal habitats (Leliaert et al., 2012). Lorenzen (1971) identified M. turgofrons from sea area of North-West of Helgoland. Two years after, Platt (1973) identified M. cuanensis from intertidal sandflat in Strangford Lough, Northern Ireland and on the same year M. parallgeni was found in the muddy sediments of Port-Miou near Cassis by Vitiello (1973). A decade after he made the revision of Microlaimidae and erected Molgolaimidae, Jensen (1988) found M. minutus from Norwegian Sea. Furstenberg & Vincx (1992) and Muthumbi & Vincx (1996) identified several species from sand in Port Elizabeth in South Africa and Indian Ocean respectively. Fonseca et al. (2006) did an intensive study on taxonomy and biogeography of Molgolaimus where they identified 17 new species from Wenddell Sea and Pacific Ocean. Moreover, he reported that the genus had high densities until 2000 meter deep. Gambi et al. (2003) illustrated that this genus is relatively abundant with about 10-35% of total nematode community occurring in all oceans from shallow water and deep sea. Moreover, its abundance is often associated with recently colonized sediments and where physical disturbance occurs constantly (Vanreusel et al. 1997, Lee et al. 2001). Schratzberger et al. (2003) demonstrated from an experimental observation that a species of Molgolaimus is an opportunistic colonizer in shallow sandy sediment. Recently, one new species was recorded from deep-sea Håkon Mosby Mud Volcano (Portnova, 2009). With all records presented, Molgolaimus is certainly a marine taxon. The presence of Molgolaimus in the phytotelmata of a pitcher plant is a new record.

PHYLOGENETIC ANALYSIS

Remarkably, phylogenetic analysis places Molgolaimus sp. nov. (the only available sequence for this genus) as sister to a crown group of four Daptonema spp. and one Metadesmolaimus sp. and nested within a maximally supported group that includes three more species of Daptonema (Metadesmolaimus is probably a misidentification according to Meldal et al. (2006) although it belongs to the same family with Daptonema). Molgolaimus isolate differs in 47-51 (3.5-3.8%) nucleotides from the five Daptonema isolate (Fig 7). The two sequences of Molgolaimus demani in GenBank should be renamed because Lorenzen

(1981, 1994) placed this species into Microlaimidae on the basis of number of testis and outstretched ovaries. However, since both Molgolaimus and Microlaimus belong to Desmodorida, our isolate should be closely related to Molgolaimus (Microlaimus) demani. The current result is difficult to understand since our Molgolaimus sequence is the only available for this genus. Meldal et al. (2006) showed that Monhysterida and Desmodorida are in a separate clade although branch separating these two orders using Bayesian Inference could be regarded as unresolved since the posterior probability value is only 74%. In addition, Desmodorida was never observed as a monophyletic group with Microlaimidae and Monopostiidae forming another clade outside the order and all taxa belonging to Monhysterida has been observed to include Desmoscolecidae and Comesomatidae. Morphologically our specimen is totally different from Daptonema by the absence of terminal setae vs. 2 terminal setae, pharynx with terminal bulb vs. without bulb, small and narrow stoma vs. spacious funnel shape buccal cavity, 2 testes vs. single testis and didelphic amphidelphic reflexed ovaries vs. single anteriorly directed ovary. It is unlikely that our PCR or sequencing reactions were contaminated since we do not have monhysterids in our sample and we placed a single nematode in the tube during the process. Furthermore, this species is vouchered so we have a morphological record. Additional Molgolaimus sequences are warranted. More taxon sampling will be needed and other alignment and phylogenetic algorithms will be explored.

Figure 7. Bayesian inference 50% majority rule consensus of phylogeny of Molgolaimus sp. nov. from MH sample and other Monhysterida and Desmodorida sequences from GenBank based on SSU rDNA data. Anaplectus grandepapillatus was designated as outgroup. Branch support values are indicated with posterior probability.

Table 4. Morphometrics of all species under genus Molgolaimus separated into 4 respective groups (all measurements are in µm except ratios and percentage: a, b, c, spic/abd, and V%). L W hd cbd ph.l abd spic.l anus ant sup a b c c' V spic.l/abd Group 1A

M. allgeni 2 ♀ 790 26 7 24 22 696 - 30 8.7 8.4 - 42% - (Allgen 1935) 7 ♂ 875-890 25-30 6-7 23-24 21-26 50 770-778 present 29-36 7.8-8.3 7.9-8.3 4-5.3 - 1.9 - 2.3 M. citrus - ♀ 493 18 6 16 15 - 411 - 27 6.3 6 5.5 46% - (Gerlach 1959) - ♂ 455 17 6 17 15 15 390 present 27 5.6 7 4.3 - 1 1500 30 9 25 23 - 1386 - 50 13.8 13.1 - 47% - M. cuanensis 4 ♀ (Platt 1973) 4 ♂ 1210 26 8 24 24 25 1125 present 46.5 11.5 14.2 - - 1 12,0- M. drakus 3 ♂ 495-575 5 14 13 19-20 425-490 present 36-43 6.1-7.0 7-7.9 5.3-6.2 - 1.4 - 1.6 (Fonseca et al. 2006) 15 565-605 18-23 4-5 17-21 13-15 - 495-535 - 26-31 6-6.7 7.9-8.8 4.6-5.5 50% - M. exceptionregulum 3 ♀ (Fonseca et al. 2006) 10 ♂ 595-600 20-21 5 20-21 18 30-31 520 present 30-32 5.9-6.1 8-8.9 4.3-4.6 - 1.8 380-440 15-20 4-5 13-17 10,0-13 - 325-375 - 21-26 5.3-5.6 6.5-7.1 4.4-5.9 50% - M. galluccii 6 ♀ (Fonseca et al. 2006) 12 ♂ 355-450 14-18 3-4 14-17 13-15 22-27 300-380 present 22-31 5.2-6.3 6.1-7.4 3.9-4.8 - 1.6 - 2 435 14 5 14 10 - 366 - 31.1 6.3 6.3 - 40% - M. gazii 2 ♀ (Muthumbi & Vincx 1996) 3 ♂ 385 18 5 15 13 29 311 present 27.5 5.6 6 5.5-7.7 - 2.2 551-581 31-43 9 24-29 20-28 - 490-520 - 14-18 9-9.5 9-9.5 2.2-3 42 - 53% - M. hoakonmosbiensis 3 ♀ (Portnova 2009) 5 ♂ 536-612 26-28 9 23-24 21-23 30,5 474-551 present 21-22 8.4-10 8.75-13.2 2.7-2.9 - 1.4 H. kiwayui 1 ♀ 312 20 5 17 10 - 267 - 16 5 6.8 - 51% - (Muthumbi & Vincx 1996) 2 ♂ 289-321 16 5-6 15-16 12,0-13 20-22 249-270 present 17-20 4.2-4.9 5.8-6.3 3.9-4.6 - 1.7 M. liberalis 14 ♀ 450-660 15-22 5-6 15-20 11-15,0 385-550 - 24-35 5.4-7.3 5.5-7.7 5.4-8.3 40% - (Fonseca et al. 2006) 15 ♂ 445-640 16-23 5-6 16-20 13-16 53-64 370-535 present 25-32 5.4-6.9 5.6-7.5 4.8-7.3 - 3.4 - 4.2 420-580 14-26 5-6 15-33 10-15,0 - 355-490 - 21-31 5.0-7.5 5.7-8.0 5.2-7.1 40-50% - M. macilenti 16 ♀ (Fonseca et al. 2006) 8 ♂ 415-550 15-22 5 13-19 11-16,0 37-52 360-475 present 24-30 5-6.4 6.3-7.7 4.5-6.2 - 3.1 - 3.5 M. mareprofondus 1 ♀ 630 22 5 20 17 - 545 - 28 6.5 7.6 4.9 50% - (Fonseca et al. 2006) 2 ♂ 610-630 20-25 5-6 20-21 17-18 29-32 525-540 present 24-31 5.8-6.7 7.2-7.3 4.6-5.1 - 1.6 - 1.9

Table 4. (Continued.) L W hd cbd ph.l abd spic.l anus ant sup a b c c' V spic.l/abd M. minutus 2 ♀ 410-440 26 5 23 14 - 343 - 16-17 5.1-5.2 5.8-6.4 - 43-46% - (Jensen 1988) 6 ♂ 420-480 17 5 16 12 23-25 350 present 24-30 4.6-5.9 6.2-6.7 - - 2 1345 - 1477 32 9 26 24-26 - 1227-1361 - 42-46 11.3-12.6 14-15.2 - 42% - M. parallgeni 12 ♀ (Vitiello 1973) 10 ♂ 1373 30 9 26 26 28 1268 present 46 13 14.6 - - 1 M. sabakii 1 ♀ 578 18 5 18 12 - 469 - 36 6.4 5.3 - 40% - (Muthumbi & Vincx 1996) 6 ♂ 641 16 5 16 13 37-44 528 present 40 7.1 5.8 8.2 - 3.1 - 3.4

M. typicus 1 ♂ 456 13 4 13 11 30 381 present 35 5.6 6.1 - - 2.7 (Furstenberg and Vincx 1992) M. tyroi 5 ♀ 280 15 4 13 8 - 250 - 1 5.6 9.3 - 49% - (Muthumbi & Vincx 1996) 4 ♂ 237 11 4 11 10 29-32 200 present 22 4.7 6.4 3.2-3.7 - 3

M. unicus 1 ♂ 1060 24 7 21 19 56 985 present 44 8.9 14.6 3.9 - 3 (Fonseca et al. 2006) M. walbethi 4 ♀ 540-610 18-23 4-5 18-19 12-15 475-530 - 27-32 5.9-6.3 7.2-9.8 4.1-6.6 50% - (Fonseca et al. 2006) 2 ♂ 540-560 17-18 5-6 17-18 12-13 65-71 465-475 present 31-33 5.6-5.8 6.7-7.2 6.2-6.6 - 5.1 - 5.8 Group 1B

M. gigaslongincus 14 ♀ 945-1350 31-46 5-7 23-32 19-31 890-1255 - 25-39 9.1-11.4 11.1-19.1 2.6-4.3 50% - (Fonseca et al. 2006) 14 ♂ 850-1240 23-35 5-7 21-30 19-26 106-148 780-1145 present 32-42 8.5-10.8 10.6-15.7 2.8-4.2 - 4.2 - 6 810-1260 31-45 6-7 22-37 21-31 735-1145 - 26-29 9.2-10.4 10.3-12.1 2.8-3.7 50% - M. gigasproximus 5 ♀ (Fonseca et al. 2006) 12 ♂ 845-1190 29-41 5-7 28-34 26-32 91-115 755-1075 present 27-32 7.7-10.4 8.8-11.4 2.9-3.7 - 3.2 - 3.8 M. pacificus 5 ♀ 865-1060 22-29 6 17-23 16-19 795-965 - 36-43 9.8-11.2 10.8-12.6 4.6-5.3 50% - (Fonseca et al. 2006) 5 ♂ 780-1055 19-26 6 17-25 17-21 88-95 705-965 present 36-44 9.6-10.3 10.4-12.2 4.1-4.7 - 4.5 - 5.3 M. tenuispiculum 3 ♀ 790 - 24.7 9 7.6 - 45% - (Ditlevsen 1921) 1 ♂ 750 163 present 23.5 9.4 9.4 - - 5

Table 4. (Continued.) L W hd cbd ph.l abd spic.l anus ant sup a b c c' V spic.l/abd Group 2A

292 16 5 14 11 - 245 - 18 5.3 6.2 - 46% - M. abyssorum 1 ♀ (Muthumbi & Vincx 1996) 5 ♂ 342 14 5 14 10 18-23 290 absent 24 5.7 6.6 4.7-5.5 - 1.8-2.3 M. australis 3 ♀ 490-525 24-26 5-6 20-22 14-16 440-460 - 20-21 5.7-6.3 8.2-9.3 3.4-4.6 50% - (Fonseca et al. 2006) 1 ♂ 525 25 5 21 16 36 465 absent 21 6.1 8.6 3.8 - 2.2

M. carpediem 3 ♂ 450-510 19-23 4-5 19-21 16 23-26 390-445 absent 20-27 5.2-5.7 7-8.3 3.7-4.1 - 1.4 - 1.6 (Fonseca et al. 2006)

M. falliturvisus 4 ♀ 520-615 20-24 5 20-21 14-16 - 450-520 - 25-27 5.6-6.3 6.8-8.3 4.8-5.7 50% - (Fonseca et al. 2006) 1 ♂ 495 22 5 20 15 26 425 absent 23 5.7 6.9 4.8 - 1.7 Molgolaimus sp. 2 ♀ 789-793 40-41 9 27-29 22-23 - 696-670 - 19-20 6.5 8.5 4.1 50% - nov. 3 ♂ 770-779 24-28 9 25-26,38 22-24 26-27 694-710 absent 27-33 6.4-6.8 10.2-10.8 3.08-3.2 - 1 M. lazonus 7 ♀ 724 29 6 26 20 - 652 - 24.9 7.7 10 3.5-3.6 45% - (Vitiello 1971) 7 ♂ 707-783 25-28 7 23-24 22 21 630-730 absent 28 7.2-7.7 9.1-9.7 3.5-3.6 - 1 500 23 5 16 13 415 - 22 6.7 6.2 6.4 50% M. nettoensis 1 ♀ - (Fonseca et al. 2006) 1 ♂ 515 19 6 20 15 50 430 absent 28 5.9 6 5.7 - 3.3 M. sapiens 2 ♀ 435-620 22-25 6-7 18-21 15-17 - 385-560 - 20-24 5.4-6.8 8.8-10.5 3.3-3.5 50% - (Fonseca et al. 2006) 1 ♂ 500 24 7 21 19 35 455 absent 21 5.9 10.2 2.6 - 1.9 M. turgofrons 3 ♀ 930 27 8 17 17 - 762 - 34 8.8 8.1 - 46% - (Lorenzen 1972) 6 ♂ 900 23 9 21 20,5 21 785 absent 40 9 7.8 - - 1 M. xuxunaraensis 5 ♀ 600-705 18-20 5 15-17 12-13,0 - 530-610 - 32-38 5.8-7.6 7.2-8.3 5.9-7.4 50% - (Fonseca et al. 2006) 2 ♂ 670-700 16-19 5-6 17-18 15 31-39 585-605 absent 37-42 6.9 7.3-7.7 5.7-6.2 - 2 - 2.6 Group 2B

M. longispiculum - ♀ 732-738 6 - 28-28 6.9-8.2 5-5.9 5-7.0 45-50% - (Timm 1961) - ♂ 737 13,8 6 154 absent 54 7.4 8.2 4.7 - 9

M. tanai 3 ♂ 684 28 7 25 22 83-94 597 absent 24 6.6 7.9 4.0-4.8 - 3.8 - 5 (Muthumbi & Vincx 1996)

Dominicactinolaimus sp. nov. (Fig 8) MATERIAL STUDIED: Eight females and two males.

LOCALITY: Table 2

MEASUREMENTS: Table 5

MORPHOLOGICAL DESCRIPTIONS Body open C shape after fixation, male tail region sharply curved (Fig 8D). Cuticle finely striated. Longitudinal ridges absent. Lip region offset. Entrance of stoma encircled by ribbed annulus (Fig 8B). Buccal cavity contains four massive onchia, few tiny denticles and massive odontostyle. With cuticularized guiding ring. Pharynx typical actinolaimid with cardia partly embedded in the intestine. Gland at pharyngeo-intestinal junction absent. Pre- rectum and rectum 3 and 1.3 anal body diameter long respectively. Tail tapered rapidly towards filiform shape. Female reproductive system didelphic, amphidelphic with reflex ovaries and with pars refringens vaginae. Vulva situated 50% of the body length. Vagina nearly half of corresponding body diameter. Spermatozoa visible in the uterus. Male testis outstretched filled with sperm. Spicule paired, dorylaimoid, about 41 µm long. Pre-cloacal supplements arranged in ventral series of 6 or 7.

RELATIONSHIPS: The very fine transverse striation of the cuticle, male tail region sharply curved after fixation, offset lip region, the presence of corrugated annulus at the entrance of stoma, double guiding ring, the female reproductive system and the outstretched testis in males are all shared characters of our specimen and that of D. dominicus. However, morphometrically, our population is different. Our specimen is much smaller, about half of the size of D. dominicus while the tail is much longer (c = 6.6 – 7.6 vs. 9.8 – 12). Furthermore, the tail has fewer pre-cloacal supplements (6-7 vs. 9). Similarities on morphological structure suggest that our specimen is closely related to D. dominicus. However, because of the differences in the number of pre-cloacal supplements, length of vagina in relation to body diameter and most ratios’ (a, b, c, and c’) we propose this as a new species.

Paractinolaimus sp. (Fig 9) MATERIAL STUDIED: Two females and one male.

LOCALITY: Table 2

MEASUREMENTS: Table 6

MORPHOLOGICAL DESCRIPTIONS Body C shape after fixation. Cuticle about 3.5 - 4.5 μm thick throughout the body and marked with very fine transverse striation. Lip region offset by a distinct depression; lip region provided with a corrugated cheilostom ring. Amphidial fovea funnel- shaped, with a slit-like opening occupying 50 - 60% of lip region diameter. Buccal cavity contains four massive sclerotized onchia. The stomatal wall covered with tiny, sparse denticles. Odontostyle massive, 1.3 times the corresponding diameter of lip region, with aperture occupying 40 - 45% of its length. Odontophore rod-like, simple almost twice as long as odontostyle (1.7-1.9). Guiding ring double. Pharynx typical dorylaimoid, Cardia consisting of a discoid, partly glandular section at pharyngeo-intestinal junction, posterior conoid part embedded into the intestine. Prerectum 2.7 - 4 times and rectum 1.4 – 2 times as long as anal body diameter. Female reproductive system didelphic, amphidelphic, each branch possessed reflexed ovary. Vagina occupying about half of corresponding body width: pars proximalis rhomboid, pars refringens weakly sclerotized, and pars distalis very reduced (almost indistinguishable). Tail convex-conoid becoming filiform, about 4.5-6 long as anal body diameter (c’=4.4–6.0). Male testis filled with small lemon shaped sperm (Fig 9K). Pre-cloaca contains series of nearly contiguous 8 ventromedian supplements. Spicules typical of the genus, about 1.5 times the anal body diameter. Tail convex conoid with blunt terminus.

RELATIONSHIPS: Most morphological characters would conform with description of the genus Paractinolaimus by having smooth cuticle without longitudinal ridges, denticulated cheilostom with walls weakly sclerotized but without sclerotized ventral plate, corrugated lip region, distinct onchia, filiform tail in female and short in male with supplements arranged in series. However, the pars refringens vaginae were not strongly sclerotized as is usually the case for the genus and most morphometrical data were different from other described species. This has the fewest number of pre-cloacal supplements among the species described with males (8 vs. 9-24). Nevertheless, L, ratios c and c’ and odntsl.l are comparable to P. vulvapapillatus and ratios a, c, c’ and spicule length to P. proximus based on male specimen. In this genus, male should be used for further morphological classification because females of Paractinolaimus and Dominicactinolaimus are indistinguishable.

Figure 8. Dominicactinolaimus sp. nov. A-C: head at different focus (A: showing odontostyle and onchia, B: showing corrugated annulus at opening of buccal cavity, C: view showing amphid and smooth cuticle); D: male and female entire body; E: pharyngeo-intestinal junction; F: showing pore in line of pharyngeal lumen; G: male spicule; H-I: female reproductive system (H: showing clear view of vagina and the two opposing uterus, I: anterior branch with clear view of par refringens vaginae). (Scale bars = 20 µm except D = 100 µm).

Table 5. Measurements (in µm) of Dominicactinolaimus sp. MH specimen and D. dominicus Hunt, 1978. Dominicactinolaimus MH specimen D. dominicus Hunt, 1978 8♀ 4♂ 2♀ 4♂ L 1374 ± 95 1288 ± 23 2580 2430 (1206 - 1497) (1261 - 1318) (2520 - 2640) (2310 - 2560) W 37.6 ± 6.2 37.2 ± 4.9

(28.5 - 47.1) (31.3 - 42.5)

a 37 ± 6.1 35 ± 5 49.6 53 (27.5 - 45.8) (30.3 - 41.1) (48 - 53) (49.5 - 56) b 3.9 ± 0.2 3,8 ± 0.3 4.2 4.2 (3.6 - 4.1) (3.5 - 4.2) (4.2 - 4.3) ( 4.0 - 4.3) c 7.2 ± 0.7 7.2 ± 0.5 7.8 10.6 (6.2 - 7.9) (6.6 - 7.6) (6.9 - 9.1) (9.8 - 12) c' 9.4 ± 0. 5 7.1 ± 0. 8 11.2 7 (8.8 - 10.1) ( 5,9 - 7,8) (9.6 - 12.7) (6.2 - 7.7) V 50 ± 1 - 52

(48 - 51) (51 -52)

lip cbd 14.8 ± 0.2 14.9 ± 0.8

(14.3 - 15) (14.3 - 16.1)

odntsl.l 21.2 ± 1.3 20.6 ± 1.2 27

(19.1 - 23) (19.6 - 22.1) (25 - 29)

odntph.l 41.0 ± 2.3 41 ± 2.1 26

(37.1 - 43.5) (38.4 - 43.3) (21 - 27)

GR ant 14.4 ± 1.1 13.4 ± 0.6

(13.3 - 16.5) (12.8 - 14.2)

ph.l 355 ± 31.2 339 ± 30.9

(306 - 403) (304 - 370)

ph.daim 22.9 ± 2.3 20.5 ± 3

(18.2 - 24.1) (17.8 - 24.4)

ph cbd 35.8 ± 2.4 29.9 ± 4.7

(33.1 - 40.3) (23.7 - 33.7)

abd 20.5 ± 1.3 25.4 ± 2.1

(18.6 - 22.6) (23.2 - 28.2)

p -rc.l 58.5 ± 5.6 67.4 ± 11.8

(50.4 -63.6) (50.5 - 75.5)

rc.l 27.3 ± 3.3 38.2 ± 2.8

(23.2 - 33.5) (34.5 - 40.4)

t.l 193 ± 17.6 179 ± 13.5

(168 - 224) (166 - 197)

spic.l - 41.2 ± 3.5 58

(36 - 43.1) ( 53 - 62)

sup - 7

(6 - 7) 9

Figure 9. Paractinolaimus sp. A-C: head at different focus (A: showing odontostyle, B: showing corrugated annulus at opening of buccal cavity, C: view showing amphid and smooth cuticle); D: pharyngeo-intestinal junction; E: pharynx; F: male and female entire body; G: male tail region with spicule; H: female tail; I: pre-cloacal supplements; J: female reproductive organ; K: sperm. (Scale bars = 20 µm except F = 100 µm). 31

Table 6. Measurements (in µm) of Paractinolaimus sp. MH specimen. Paractinolaimus sp. MH specimen 2♀ 1♂ 2♀ 1♂ L 1419 - 1647 1548 GR ant 13.8 - 15.6 14.2

W 45.6 - 46.7 40.0 ph.l 354 - 425 394

a 30 - 36 39 ph.diam 20.8 - 23 22.1

b 3.9 - 4.0 3.9 ph cbd 39.9 - 40.3 36.4

c 12.6 - 14.9 52 abd 21.6 - 21.9 32.4

c' 4.4 - 6.0 0.92 p-rc.l 70 - 89 87.3

V 52 - 54 rc.l 32.7 - 43.0 45.3

lip cbd 16.3 - 17.2 17.5 t.l 96 - 131 29.7

odntsl.l 21.4 - 22.1 20.6 spic.l - 49.3

odntph.l 37.3 - 37.8 39.4 sup - 8

PHYLOGENETIC ANALYSIS

Our phylogenetic analysis indicates a monophelytic group of all four Dominicactinolaimus sp. nov. isolates and a maximally supported clade of two isolates of Paractinolaimus sp. from phytotelmata. However, there relationship together with other actinolaimid isolates from GenBank is presented as unresolved having a posterior probability of less than 95%. There were no nucleotides differences among the four isolates of Dominicactinolaimus and the differences in branch length attributed to sequence ambiguities (“t, W, Y” instead of resolved nucleotides in few positions). The two sequenced individuals of the Paractinolaimus sp. MH specimen differed in 48 (6.4%) nucleotide positions, and are most likely two different species (Fig 10). Morphologically we could distinguish only two different populations of actinolaimids from our samples. However, phylogenetic analysis indicates that there might be more than two species from our sample as shown in the phylogenetic tree forming a single clade of all Dominicactinolaimus individuals representing one species and a highly supported sister clade of potentially two new species of our Paractinolaimus (Paractinolaimus MH1 and Paractinolaimus MH2) with 48 nucleotide differences from 751 positions.

Figure 10. Bayesian inference 50% majority rule consensus of phylogeny of Paractinolaimus sp. and Dominicactinolaimus sp. from MH sample and other Actinolaimidae sequences from GenBank based on D2-D3 expansion of LSU rDNA data. Labronema vulvapapillatum and Dorylaimus stagnalis were designated as outgroups. Branch support values are indicated with posterior probability. Drawing represents the morphological differences of the structure of male tail of Dominicactinolaimus (after Hunt, 1978) and Paractinolaimus (after Andrássy, 1964).

ECOLOGY OF ACTINOLAIMIDAE

All species belonging to Actinolaimidae are typical freshwater or semi-freshwater although they can also be found in wet moss and wet soils. Actinolaimids are most abundant and species rich in Africa but they are still considered cosmopolitan (Andrássy, 2009). Actinolaimids are regarded to have a c-p value of 5 and considered to be “persisters” which is characterized by a low reproduction rate, low colonization ability and sensitivity to disturbance; all accounting for their occurrence in habitat with durational stability (Bongers, 1990). Indeed, majority of the species were found from mountains. Paractinolaimus peruvianus at an elevation of 3800 meter above sea level (m a.s.l) (Gadea, 1965); P. acutus found associated with Paeonia japonica’s rhizosphere at an elevation of 1080 m a.s.l (Khan & Park, 1999); P. magistris on wet moss in Chile (Vinciguerra et al., 2013); P. macrolaimus which was considered the most common Palaeartic species of the genus, occurring in various limnic habitats and in wet soils and mosses and was recorded in Fertö-Hanság National Park in Hungary (Andrássy, 2002). The type species of genus Dominicactinolaimus was found in a wet area of experimental station and from soil in the virgin forest of Dominica (Hunt, 1978). Another characteristic of a nematode known to have a c-p value of 5 is that they never belong to the dominant species in the sample that is why most identification was based on few individuals. However, we found 8 females and 4 males of Dominicactinolaimus and 2 females and 1 male of Paractinolaimus excluding the 9 juveniles whose genus we could not identify since female tails are similar for both genera. In addition, all juveniles found have long filiform tail. This turns out to be the dominant species of specimen in our sample. However, individuals from this group also came from a composite sample of more than one pitcher. It is therefore only possible to assign juveniles to a genus after sequencing them individually. In 1922, Menzel recorded Dorylaimus in a sample from Nepenthes gymnamphora, although not from similar family with actinolaimids but more or less dorylaims shared the same habitat, which also occurs in water bodies, wet soils and moss. Therefore this was not the first record of species from Dorylaimoidea in phytotelmata of Nepenthes. Moreover, two juveniles assumed to be under Dorylaimidae were found in another sample.

Note on the generic status of Dominicactinolaimus Jairajpuri & Ahmad, 1992: synonymy with Trachypleurosum or Trachactinolaimus rejected.

Trachypleura was erected for genus with filiform tail for both sexes, presence of transverse rows of denticles in the cheilostom and the absence of onchia in the buccal cavity (Thorne, 1939). In 1959 Andrássy changed the genus name from Trachypleura to Trachypleurosum because he found out that it was a junior homonym. Andrássy (1963) erected the genus Trachactinolaimus for actinolaims with filiform tail for both sexes, but separated from Trachypleurosum by the presence of four large onchia in the buccal cavity. Hunt (1978), unaware of Andrássy’s 1963 paper described a new species Paractinolaimus dominicus characterized by the presence of four onchia and cheilostom walls with denticles (a typical characteristic of Paractinolaimus) coupled with filiform tail in both sexes (as observed in Trachypleurosum). He considered the paractinolaimid type stomatal structure more important than tail shape and provisionally placed the newly found species under Paractinolaimus Meyl, 1957. Vinciguerra (1988) designated three subfamilies under family Actinolaimidae Thorne, 1939: Hexactinolaiminae for species with six onchia, Actinolaiminae for species with four onchia including Trachactinolaimus and Paractinolaimus, and Trachypleurosinae for Trachypleurosum having no onchia. However, Coomans et al (1990) redescribed and resolved the status of Paractinolaimus Meyl, 1957; Trachypleurosum Andrássy, 1959 and Trachactinolaimus Andrássy, 1963. It was found out that buccal cavity of Trachypleurosum conforme, type species of the genus Trachypleurosum possesses four large onchia and that the only difference from Trachactinolaimus was the absence of denticles in the wall of cheilostom. Moreover, he studied type specimens of P. dominicus and concluded that this species falls under Trachactinolaimus since it agrees with T. radulatus in stomatal structures and tail shape, to which it was transferred by Vinciguerra (1988). Clearly, Jairajpuri & Ahmad (1992) was not aware of Coomans’ 1990 paper and still considered Trachypleurosum the type and only genus under Trachypleurosidae and described as, buccal cavity without onchia but with corrugated ring and several transverse rows of mural denticles. In line with the scheme, he erected four subfamilies under Actinolaimidae: Actinolaiminae, Neoactinolaiminae, Brittonematinae and Paractinolaiminae. The latter consists of genera with cheilostom bearing minute denticles and cuticle without longitudinal striations. Paractinolaimus was designated as type genus of the subfamily together with other genera: Trachactinolaimus Andrássy, 1963; Westindicus Thorne, 1967; Afractinolaimus

Andrássy, 1970; and newly erected genus Dominicactinolaimus. The new genus was somewhat similar to Trachactinolaimus having filiform tail in both sexes but was distinguished by the absence of glands in the pharyngeo-intestinal junction. Vinciguerra (2006) followed her earlier classification regarding Actinolaimidae as a parallel family with Dorylaimidae within Dorylaimoidea. However, she did not discriminate subfamilies within Actinolaimidae. Although Andrássy (2009) accepted the same scheme of Vinciguerra, to lower the rank of Actinolaimoidea to family status, he proposed that the family should be divided into three subfamilies where genera without longitudinal ridges and with filiform tail for both sexes should be regarded under Trachypleurosinae. Furthermore, his perception towards the presence or absence of denticles in the cheilostom of genera with filiform tail for both sexes contradicted Vinciguerra’s description (see table below). Andrássy’s key 2009 Vinciguerra’s key 2006 Trachypleurosum Cheilostom walls denticulated Cheilostom walls adenticulated Trachactinolaimus Cheilostom walls adenticulated Cheilostom walls denticulated Dominicactinolaimus syn. Trachypleurosum (2009) syn. Trachactinolaimus (1998)

Undoubtedly, the genus Dominicactinolaimus possesses a cheilostom with denticles as observed in the synonymy of the two authors. It was clearly described by Hunt (1978) that stomatal structure was similar to those of Paractinolaimus but differed from the shape of tail for male. In addition, the absence of glands at pharyngeo-intestinal junction differentiated it from Trachactinolaimus and Trachypleurosum. These glands were very prominent in latter genera as illustrated by Andrássy (1963) for T. radulatus and Trachypleurosum venezolanum (Coomans et al., 1990; Fig. 11B and 11A, respectively). Therefore, Dominicactinolaimus (Fig. 11C-E) differ from genera under Trachypleurosinae because of the absence of glands. One might question whether these character differences are of generic importance. Unless the conflicting description of Andrássy and Vinciguerra about possession of cheilostomal denticles won’t be addressed and the morphological importance of gland in the pharyngeo- intestinal junction will not be assessed, we propose to regard Dominicactinolaimus as a separate genus. Furthermore, availability of DNA sequences for both Trachactinolaimus and Trachypleurosum will help resolve the taxonomy of this group.

Figure 21. Differences in Pharyngeo-intestinal junction. A: Trachypleurosum venezolanum (after Coomans, Vinciguerra & Loof, 1990); B: Trachactinolaimus radulatus (after Andrássy, 1963); C: Dominicactinolaimus dominicus (after Hunt, 1978) Jairajpuri & Ahmad, 1992; D-E: Dominicactinolaimus sp. Mt. Hamiguitan specimen (D: drawing, E: photo).

Pelodera strongyloides (Schneider, 1860) Schneider, 1866 (Fig 12) MATERIAL STUDIED: Two males

LOCALITY: Mt. Hamiguitan, Davao Oriental, Philippines

MEASUREMENTS: Table 7

MORPHOLOGICAL DESCRIPTION Body short and plump. Cuticle finely annulated with very fine longitudinal striations. Lips differentiated from body contour. Amphid very small, pore-like. Metastegostom with parallel walls and with three setose denticles. Pharyngeal corpus swollen. Excretory and secretory pore located just behind the isthmus.

Male reproductive system monorchic with reflexed testis. Spicule fused distally, 67% of its length. Bursa peloderan, well developed with 10 pairs of rays, 2 pairs situated anterior to cloaca and 8 pairs posterior. Female not found.

Figure 12. Pelodera strongyloides MH specimen. A: entire body; B: pharyngeal region; C: swollen isthmus and E_S pore situated posterior to isthmus; D: cuticle annulations; E: Bulbus; F: Testis terminus showing reflex; I&J: different focus on bursal rays; G&H: spicule. Scale bars = 20 µm except A = 100 µm.

Table 7. Measurements (in µm) of Pelodera strongyloides MH specimen. Pelodera sp. 2♂ L 535 ± 5.8 anus ant 500 ± 4.3 (531 - 540) (497 - 503)

W 51.4 ± 0.35 abd 27.8 ± 0.25 (51 - 52) (27.6 - 28.0)

EP 88 ± 1.8 t.l 35.4 ± 1.5 (87 - 90) (34.4 - 36.5)

NR 80 ± 1.4 tdg.l - (79 - 81) -

st.l 20.1 ± 0.40 a 10 ± 0.18 (19.8 - 20.3) (10.3 - 10.5)

l.c.s - b 3.7 ± 0.02 -

ph.l 145 ± 2.5 c 15 ± 0.48 ( 143 - 146) (14.8 - 15.5)

p-corp.l 54 ± 1.1 c' 8.3 ± 0.18 (53 - 54) (8.1 - 8.4)

m-corp.l 33.0 ± 0.64 V - (32.6 - 33.5) -

isthm.l 43.2 ± 1.2 spic.l 58 ± 0.57 (42.4 - 44.1) (57 - 58)

blb.l 31.9 ± 0.55 gub.l 42.2 ± 0.95 (31.5 - 32.3) ( 41.5 - 42.8)

corp.l 86 ± 2.4 testis ref 54 ± 0.11 (84 - 88) testis.l 230 ± 2.8

Corp.l/isthm.l 2.0 ± 0.00 (228 - 232)

PHYLOGENETIC ANALYSIS

Phylogenetic analysis places two individuals of Pelodera strongyloides MH specimen (adult male and juvenile) in a highly supported (100%) monophyletic group together with sister Pelodera strongyloides (posterior probability = 98%) which was sister to P. punctata. The Pelodera spJ (MH specimen juvenile) and Pelodera spM (male MH specimen) differ in only one nucleotide (0.2%) while P.strongyloides (GenBank) differ two (0.4%) nucleotides from juvenile isolate and one nucleotide (0.2%) from male isolate. The P. punctata from GenBank differ from juvenile isolate 17 nucleotides (3.4%). However, the clade of Pelodera does not form a supported basal group, resulting in unresolved early branching (Fig. 13).

Figure 33. Bayesian inference 50% majority rule consensus of phylogeny of Pelodera strongyloides from MH sample and other Rhabditidae sequences from GenBank based on D2-D3 expansion of LSU rDNA data. Nemertinoides elongatus was designated as outgroup. Branch support values are indicated with posterior probability. Drawing represents the differences of spicule shape and arrangement of bursal rays of Pelodera strongyloides group (after Sudhaus & Schulte, 1986), Cruznema (after Weighärtner, 1953), Poikilolaimus (after Sudhaus, 1980), Teratorhabditis (after Sudhaus, 1974 & 1985).

RELATIONSHIPS AND DIAGNOSIS: Pelodera was divided into three monophyletic species groups (Sudhaus, 2011) on morphological basis. According to the pictorial key of Scholze & Sudhaus (2011) and Sudhaus (2011), and based on the numbers of the bursal rays and its arrangement, the two pairs of pre-rectal rays coupled with the percentage of the fused region of spicule, anteriorly open bursa, and the shape and length of gubernaculum which is as long as the fused part of the spicule, our specimen belongs to the Pelodera Schneider, 1866 Strongyloides - group. Currently, there are 11 species identified within this group. Based on the identification key of Andrassy (1983)our specimen are close to Pelodera punctata Cobb, 1914 and P. strongyloides Schneider, 1866 since they share the same number of pre-anal bursal rays, the length of fused spicule which is 2/3 of its length. Moreover, our specimen agree with the original description of Sudhaus et al. (1987) for P. strongyloides based on bursal papillae not radially arranged, distance of bursal rays 1 and 2 is more than 5 µm, and a spicule length of 57-58 µm which is within the range (55-82 µm). Highly supported with our molecular analysis, which strongly place the two MH specimens as sister taxa of P. strongyloides with only 1-2 nucleotide differences. Hence, we could classify our specimen as P. strongyloides.

ECOLOGY OF PELODERA

Based on the recent phylogenetic systematization of Sudhaus 2011, Pelodera belongs to Rhabditidae. This family has a c-p value of 1 “enrichment opportunist” (Bongers, 1990). Hence, most species are found associated with bacteria-rich ephemeral terrestrial habitats. Our specimen matches the morphological description of species belonging to monophyletic Strongyloides-group. Species from this group were found from organic matter from an elephant seal molting ground, marine littoral zone, skin of a rodent, decaying bird, corm of diseased Saggitaria, head capsule of a termites, sewer of waste water and feces (Sudhaus, 2011; Andrássy, 2005). P. strongyloides which is similar with our specimen on morphological and molecular basis (SSU rDNA sequence) was found in soil enriched with manure while, P. punctata was found in roots of aquatic plants, decaying matter on the shore of freshwater bodies, and sewage sludge (Sudhaus, 2011). Andrássy (2005) pointed out that among the species within the genus; P. punctata differs because of its habitat: it occurs in freshwater or semi-freshwater habitat. However the occurrence of P. strongyloides in phytotelma might have been introduced by vertebrates since larvae of this species was found associated with rodents (Sudhaus, 2011).

Tripylella sp nov. (Fig 14) MATERIAL STUDIED: Fourteen females

LOCALITY: Table 2

MEASUREMENTS: Table 8

MORPHOLOGICAL DESCRIPTION Body curved upon fixation. Cuticle with very fine annulations. Labial region rounded. Inner labial papillae fine. Cephalic setae 6 longer and 4 shorter arranged in single whorl, six setae 2.5 – 3.4 μm long, the four setae very small and thin. Amphid slit-like located near dorsal tooth. Dorsal tooth triangular, posterior to 2 sub-ventral teeth. Pharynx cylindrical. Cardia very large composed of 6 huge fused cells. Intestine thick-walled, posterior to female reproductive organ, intestine possesses a sac with unidentified circular structure inside. Rectum wide and almost as long as anal body diameter. Female genital organ didelphic, branches not similar in length, posterior branch less than half of the length of the anterior. Vulval lips weakly sclerotized, heart-shaped, vagina oblique or “S” pattern and short 25% of corresponding body diameter. Tail long occupying 17 - 19% of body length, ventrally bent, anterior half gradually tapering and posterior half narrowly cylindrical (with digitate prolongation), length of prolongation 37 - 45% of tail length. Caudal glands well developed. Spinneret present. Males not found.

Phylogenetic analysis

Phylogenetic analysis of D2-D3 expansion segment of LSU rDNA places Tripylella sp. nov. a sister taxon (97% support) to another Tripylella sp. obtained from GenBank both of which are sister taxa (moderate support) to a highly-supported monophyletic group that includes species of Tripyla (Fig. 15). The Tripylella sp. nov. differed by 119 nucleotide positions (17.58%) from unidentified GenBank sequence

RELATIONSHIP AND DIAGNOSIS: The annulated cuticle, the presence of digitate prolongation on tail, the arrangement of setae with 6+4 in single whorl and the range of its length and the cardia correspond well to the description of Tripylella Brzeski & Winiszewska- Ślipińska, 1933. However, the female reproductive system is not typical for the genus since all identified species of Tripylella has equal length of both branches while the specimen possessed a reproductive system with reduced posterior branch. The anterior branch is very long,

somewhat resembles that of Tripylina. The morphological difference of Tripylella to Tripyla is the 6+4 setae arranged in a single whorl and from Tripylina, longer outer labial and cephalic sensilla. Moreover, the dorsal tooth and two sub-ventral teeth are contained in separate stomatal chambers and large cardiac glands make it distinct from Tripyla and Tripylina.

Figure 14. Tripylella sp.nov. MH specimen. A: entire body; B-D: head at different focus (B: showing the setae, C: showing pouch of dorsal tooth, D: showing dorsal and subventral teeth); E: female reproductive organ, vulva; F-G: cardia; H-I: Tail region. Scale bars = 20 µm except A = 100 µm.

Table 8. Measurements (in µm) of Tripylella sp. nov. MH specimen, T. iucunda Andrássy, 2006 and T. intermedia (Bütschli, 1873) Brzeski & Winiszewska- Ślipińska. Tripylella sp. Tripylella iucunda. Tripylella intermedia. 14♀ 4♀ 6♀ L 685 ± 36 680 - 750 810 - 960 (654 - 721) - -

W 33.1 ± 2.4 35 - 38 - (31.2-36.6) - -

st.l 16.6 ± 1.1 - - (15.6 - 18.2) - -

l.c.s 2.9 ± 0.38 2.0 - 2.5 2.0 - 3.0 (2.5 - 3.4) -

ph.l 166 ± 5.6 150 - 158 190 - 202 (160 - 173) - -

anus ant 566 ± 34 - - (531 - 598) - -

abd 22.1 ± 1.4 - - (20.5 - 23.8) - -

t.l 119 ± 5 - 108 - 120 (111 - 123) - =

tdg.l 48.1 ± 5.2 - - (45.0 - 55.8) - -

a 21 ± 1.4 20 - 21 21 - 24 (20 - 23) - -

b 4.1 ± 0.26 4.5 - 4.7 4.3 - 4.6 (3.8 - 4.4) - -

c 5.8 ± 0.30 6.0 - 6.5 7.3 - 8.3 (5.3 - 6.0) - -

c' 5.4 ± 0.28 4.2 - 4.6 3.6 - 4.5 (5.0 - 5.7) - - V 47 ± 2.1 47 - 49 51 - 52 (44.0 - 48.6) - -

Figure 15. Bayesian inference 50% majority rule consensus of phylogeny of Tripylella sp. from MH sample and other Tripylidae sequences from GenBank based on D2-D3 expansion of LSU rDNA data. Paratrichodorus poros was designated as outgroup. Brach support values are indicated with posterior probability. Drawing represents morphological differences of head and tail region of Tripyla and Trischistoma (after Zullini, 1982), Tripylella and Tripylina (after Brzeski & Winiszewska-Slipinska, 1993 and Hernandez and Jordana, 1988).

Based on the arrangement of setae our specimen is not Tripyla. However, the female reproductive system might be confusing since it somewhat resembles Tripylina. The molecular analysis, however, strongly places the individual in a monophyletic group jointly with another Tripylella sp. obtained from GenBank and together it form a separate clade with the clades of Tripyla and Tripylina. Hence, the cuticle annulations, the distinctive tail shape, the arrangement of setae with 6+4 in single whorl, buccal cavity with separate chamber for dorsal and sub-ventral teeth and the large cardia composed of six fused cell are good characters to delineate this genus from other genera within Tripylidae and that the structure of female reproductive system could be considered inconsequential diagnostic characteristic. Our sequenced Tripylella did not match any in the GenBank and morphometrical data of our specimen is comparable to T. iucunda Andrassy, 2006. However, our species differs from four other identified species because of the unequal length of female reproductive system with anterior branch well developed, twice or more as long as posterior branch. Hence, we regard this as new species.

ECOLOGY OF TRIPYLELLA

All members of family Tripylidae de Man, 1876 occurs in both terrestrial and aquatic habitats found in every continent except in Antarctica and regarded as intermediate in the c-p scale with c-p value of 3 (Bongers, 1990). The genus Tripylella only has 4 species known to science. Tripylella intermedia (Bütschli, 1873) Brzeski & Winiszewska-Ślipińska, 1993 was the first species recorded. This widely distributed species dwell in wet, terrestrial habitat Andrássy (2006a). In Hungary, it was normally found in moss and other limnic habitat in the mountains Andrássy (1996) and Soós (1940). In 2006, Andrássy (2006b) identified two additional species to the genus however due to non-accessibility of the paper we did not obtain information on the habitat for those two species T. maiuscula and T. minuscula. However, in his 2006 book, he mentioned that this genus occurs in moss, soil and Sphagnum bogs. Hence we could associate the mentioned habitats with the two new species described. The most recent species, T. iucunda was collected by L. Hufnagel from fallen leaves and humus from a mossy forest and subsequently described by Andrássy (2008). The information above gives an idea that this nematode actually inhabit aquatic environment. Based on record, all habitats where it appeared are associated with wet biotopes and that its presence in phytotelmata is not impossible.

Tylocephalus auriculatus (Bütschli, 1873) Anderson, 1966 (Fig 16) MATERIAL STUDIED: Three females

LOCALITY: Table 2

MEASUREMENTS: Table 9

MORPHOLOGICAL DESCRIPTION

Body fusiform, cuticle smoothly annulated, with somatic setae. Lateral field 4.3-5.5 µm wide at mid-body. Nerve ring located mid pharyngeal region. ES pore posterior to nerve ring. Deirid posterior to ES pore, located within the lateral field. Anterior region with distinct bilateral and dorsoventral symmetry. Cornua flattened, without tines, leaf shape with very fine round tip. Flabella absent. Amphid located one lip diameter from anterior end. Pharyngeal bulb oval shaped with valves. Cardia embedded in the intestine. Female reproductive system didelphic, amphidelphic with reflex ovarian branches. Vulva located mid body, transverse. Vulval flaps present. Rectum short and plump. Tail conical with gradual narrowing, ventrally arcuate, swollen tip. Spinneret well developed. Males not found.

PHYLOGENETIC ANALYSIS

Phylogenetic analysis places Tylocephalus auriculatus MH specimen together with other T. auriculatus (AY284707) specimens with maximal support. The monophyletic Tylocephalus clade has a sister relation with Wilsonema schuurmansstekhoveni with moderate support (PP: 0.78). T. auriculatus MH specimen differed in 4 nucleotides (0.2%) from T. auriculatus (AY284707) and T. auriculatus (AF202155) both taken from GenBAnk. W. schuurmansstekhoveni obtained from GenBank differed in 34 (2.1%) nucleotide positions from our specimen (Fig.17).

Figure 16. Tylocephalus auriculatus MH female specimen. A: entire body; B: pharyngeal region; C. head region showing amphid and cuticle annulations; D: mid body showing lateral field; E: vulval region with matured oocyte; F-G: Tail region (F: showing spinneret, G: showing rectum). Scale bars = 20µm except A = 100µm. 48

Table 9. Measurements (in µm) of Anaplectus sp., Plectus sp., and Tylocephalus auriculatus; Plectidae of MH specimen. Anaplectus Plectus Tylocephalus 5♀ 3♀ 3♀ L 765 ± 7 701 ± 35.2 391 ± 6.8 (755 - 774) (661 - 728) (384 - 397) W 25.8 ± 3.4 41.0 ± 3.1 32.9 ± 1.1 (22.9 - 31.8) (37.4 - 43.2) (31.9-34.1) ph.l 165 ± 6 169 ± 12.3 103 ± 4.2 (158 - 174) (156 - 180) (98-107) t.l 57 ± 1.2 96 ± 5.6 32.6 ± 2.9 (55 - 58) (92 - 103) (29.6-33.4) abd 18.3 ± 0.6 24.4 ± 3.6 11.9 ± 1.2 (17.4 - 19.1) (21.0 - 28.2) (10.5-12.6) a 30 ± 3.3 17.2 ± 2.1 12 ± 0.58 (24 + 33) (15 - 20) (11-12) b 4.6 ± 0.1 4.2 ± 0.16 3.8 ± 0.09 (4.5 - 4.8) (4.0 - 4.2) (3.8-3.9) c 14 ± 0.3 7.3 ± 0.36 12 ± 0.93 (13 - 14) (7.0 - 7.7) (11-13) c' 3.1 ± 0.1 4.0 ± 0.46 2.8 ± 0.53 (2.9 - 3.2) (3.6 - 4.5) (2.4-3.4) lip cbd 9.2 ± 0.2 10.4 ± 0.70 9.1 ± 0.43 (8.8 - 9.4) (9.6 - 11.0) (8.8-9.6) amph ant 8.0 ± 0.30 13.6 ± 0.24 8.2 ± 1.8 (7.7 - 8.4) (13.3 - 13.8) (6.2-9.9) st.l 21.3 ± 0.38 19.8 ± 0.91 13.5 ± 1.3 (20.7 - 21.6) (19.2 - 20.9) (12.3 - 15.0) corp.l 75 ± 2.1 76 ± 5.6 - (72 - 78) (70 - 80) - isthm.l 49.7 ± 2.6 47.8 ± 4.6 - (46.5 - 53.7) (42.7 - 51.6) - blb.l 28.4 ± 0.47 26.2 ± 0.66 15.6 ± 1.2 (27.9 - 29.1) (25.5 - 26.8) (14.3 - 16.7) crd.l 11.7 ± 0.72 9.7 ± 1.3 6.4 ± 0.77 (11.0 - 12.9) (8.4 - 11.0) (5.8-7.3 NR/ph.l (%) 52± 1.2 50 ± 3.7 48 ± 0.61 (51 - 54) (48 - 55) (47-49) EP/ph.l (%) 56 ± 0.70 67 ± 14.1 52 ± 2.7 (55 - 57) (58 - 83) (50-55) DEI/ph.l (%) 61 ± 0.60 80 ± 9.2 59 ± 0.92 (60 - 62) (72 - 90) (58-60) V 49 ± 1.1 51 ± 6.5 50 ± 0.52 (48-51) ( 47 - 58) (49-50)

Figure 17. Bayesian inference 50% majority rule consensus of phylogeny of Tylocephalus auriculatus from MH sample and other Plectidae sequences from GenBank based on SSU rDNA data. Acrobeloides buetschlii was designated as outgroup. Branch support values are indicated with posterior probability. Drawing represents the differences of head structure of Plectus sp. (after Zell, 1993), Tylocephalus auriculatus (after Bütschli, 1873 modified by Anderson, 1966) and Wilsonema sp. (after de Man, 1880 modified by Cobb, 1913).

RELATIONSHIP AND DIAGNOSIS: The absence of tines on the cornua, the absence of flabella and the presence of median ridges are important diagnostic characters to assign this specimen into Tylocephalus Crossman, 1933. This specimen was thought to be T. longicornis since both were found in a phytotelmata, the latter was found in water held by bromeliads (Holovachov, 2004). However, it varies morphologically since the latter lacks of median ridges. It resembles most to T. auriculatus (Bütschli, 1873) Anderson, 1966 although, smaller in body size but ratios were considerably within range. Additional morphological characters must be examined

further to confirm the species status of this specimen. Exact numbers of somatic setae, number of spur at tail region and the presence of taps and flaps in the median ridges must all be taken into account. Although several morphological characters still have to be examined closely, molecular analysis suggests that out specimen was T. auriculatus given that it was grouped with two T. auriculatus sequences from GenBank with only 0.2% nucleotide differences. Therefore, on the basis of all the morphological characters mentioned above, comparable morphometrical ratios, integrated with molecular analysis we could classify our sample as T. auriculatus.

Plectus sp. (Fig 18) MATERIAL STUDIED: Three females

LOCALITY: Table 2

MEASUREMENTS: Table 9

MORPHOLOGICAL DESCRIPTION Labial region strongly offset from body contour, Lips well separated from each other. Labial papillae located at the base of sub-dorsal and sub-ventral lips. Cephalic setae 4.5-5µm long. Amphid at fourteenth annules from the base of lips. Lateral field composed of two alae showing four incisures under light microscope. Nerve ring at mid section of the pharynx. Excretory-secretory pore posterior to nerve ring. Deirid situated posterior (70-90% of pharynx length) located within the lateral field. Basal bulb oval with well developed grinder. Cardia and posterior part of basal bulb embedded in the intestine. Female reproductive system didelphic amphidelphic, ovaries reflexed. Position of specimen after mounted enabled to see vagina and vulva clearly. Wide rectum. Tail gradually narrowing, ventrally arcuate, undefined number of caudal setae under light microscope. Three caudal glands opens at tail terminus. Spinneret well-developed, cuticularized. Fringes of spine at tail tip not observed.

PHYLOGENETIC ANALYSIS

Phylogenetic analysis places Plectus sp. MH specimen in a not well supported group of other Plectus species (PP: 0.59). Our species is sister to clade (PP: 0.95) that contains both P. acuminatus and P. murrayi. MH specimen only differs 1 nucleotide (0.5%) from Plectus

sp. (AY652779) and differs from two isolates of Plectus murrayi (AB649029 & HQ270139) obtained from GenBank by 11 (5%) and 13 (5.9%) nucleotides respectively (Fig.19).

Figure 18. Plectus sp. MH specimen. A: female entire body; B-D: head region at different focus (B: showing the cephalic setae and offset labial region, C: showing amphid, D: dorsal view showing stoma); E: lateral field; F: rectum; G: pharyngeo-intestinal junction, showing bulbus with conspicuous grinder; H: Tail region showing spinneret. Scale bars = 20 µm except A = 100 µm.

Figure 49. Bayesian inference 50% majority rule consensus of phylogeny of Plectus sp. from MH sample and other Plectus sequences from GenBank based on D2-D3 expansion of LSU rDNA data. Acrobeloides nanus and Teratocephalus lirellus were designated as outgroups. Branch support values are indicated with posterior probability.

RELATIONSHIP AND DIAGNOSIS: The specimen resembles Plectus (formerly placed under Chiloplectus). It closely resembles Plectus cancellatus (Zullini, 1978) Holovachov, 2004 (formerly known as Chiloplectus loricatus Andrassy, 1985) by the presence of its lateral field. Furthermore, the ratios V, b, c, and c’ of our specimen is within the range of the respective ratios of P. cancellatus from Ukraine described by Holovachov et al. (2000). However, there are more morphological characteristics to be considered before designating our specimen as P. cancellatus: (1) number of annules directed anteriorly because of lateral ridges, (2) presence of epiptygma, (3) actual number of somatic setae and caudal setae, (4) appendages on the base of spinneret, (5) ultra structural observation on the labial region. Lack of SEM study on the above mentioned parts, expertise and time make further confirmation impossible. In addition, the phylogenetic analysis could not reveal relationship to a particular species due to few available sequences in the GenBank. The close relation of our specimen with isolate AY652777 being only one nucleotide difference, could not be supported since this was just based on 222 positions. Hence, using SSU sequences will still be performed.

Anaplectus sp. (Fig 20) MATERIAL STUDIED: Five females

LOCALITY: Table 2

MEASUREMENTS: Table 9

MORPHOLOGICAL DESCRIPTION Body cylindrical, tapering gradually from end of stoma towards anterior end and posteriorly on tail. Body pores arranged in four lines located dorso-lateral and ventro-lateral from head to tail region. Amphidial fovea transverse slit. Cuticle annulated. Somatic setae papilla formed. Lateral field composed of two wings, marked by four incisures; labial region truncate distinctly offset from body contour. Gymnostom cuticularized, length shorter than width, in shape of a single chamber. Pharynx conspicuously delineated into corpus, isthmus and bulbus. Basal bulb oval shape with butterfly valve. Nerve ring located 52% of the pharynx length. Excretory-secretory pore posterior to nerve ring. Deirid present, posterior to E-S pore located within the lateral field. Cardia embedded in the intestine. Female reproductive system didelphic amphidelphic, ovary antidromously reflexed. Vagina and vulva not clear (position of specimen after mounting). Rectum very distinct, one anal body diameter long. Tail conoid, ventrally arcuate with rounded terminus. Caudal gland and cuticularized spinneret present.

Figure 20. Anaplectus sp. MH specimen. A-C: head region at different focus [B: showing cuticularized gymnostom and distinct offset of labial region, C: slit-like amphid (arrow) and anterior body pore]; D: female reproductive organ; E. pharyngo-intestinal junction, showing bulbus with grinder (arrow); F: entire body; G-I: tail at different focus. Scale bars = 20 µm.

RELATIONSHIP AND DIAGNOSIS: The single chamber gymnostom, cuticularized spinneret and the body pores being present all throughout the body, makes this specimen very similar to A. porosus Allen & Noffsinger, 1968. However, hypodermal glands were not that distinct, if present and different from what was observed on male A. porosus in the nematode collection of UGent nematology research group with very clear glands and pores. Unfortunately, a male specimen was not present and identifying Anaplectus solely by female specimen is impossible.

ECOLOGY OF PLECTIDAE

Every subfamily within family Plectidae Örley, 1880 is well represented with our sample from water held by Nepenthes pitchers. Five individuals were identified as Anaplectus and three adult females and six juveniles of genus Plectus and three adult females and one juvenile of genus Tylocephalus. The c-p score designated to family Plectidae was 2 (Bongers, 1990), considered to be general opportunists but unlike rhabditids, they only colonize if food supply decreases (Schiemer, 1983). This family belongs to the most widespread nematofauna and occurs in a wide range of habitats (Andrássy, 2005). They inhabit freshwaters, soil, litter, moss, rotting wood and the like (Holovachov & De Ley, 2006). Anaplectus are amphimictic nematodes and generally, mostly terrestrial which inhabit soil, litter and moss. However, A. granulosus and A. grandepapillatus were habitually found in freshwaters. Unlike Anaplectus, Plectus are parthenogenetic and dwell in both terrestrial and freshwater habitat. Several members of the genus are typical aquatic species such as: P. aquatilis, P. indicus, P. palustris, P. sambessi and P. tenuis although other species maybe found in aquatic habitat as well. Hendriksen (1983) and unpublished data of Paul De Ley showed that some species are able to undergo anhydrobiosis. Basically, all genera within Wilsonimatinae are terrestrial and moss-inhabiting, and only members of Tylocephalus were found in freshwater biotopes. The type species of this genus T. auriculatus is often found from freshwaters, associated with aquatic plants, in mosses and rotting wood. In addition, De Ley and Coomans (1997) recovered T. auriculatus from dehydrated sand with shell fragments and also revealed that the labial region of all members of Wilsonematinae is specialized for sweeping up bacteria on the soil surface. Holovachov et al., (2004) identified T. longicornis which was recovered from water impounded in the bracts of bromeliads. Since our specimen and T. longicornis shares almost 56

the same habitat, we suspected that our sample might be T. longicornis also. However, morphology. Morphometry and molecular analysis suggest it is T. auriculatus.

Other nematode taxa

We also found two other taxa which were not included in the description. The sole sample of Actinonema was lost during the process. Dorylaimus was not included since it consists only of juveniles which make identification uncertain. However, DNA sequence and BI tree based on D2-D3 expansion of LSU rDNA data of this Dorylaimus specimen is available (Appendix I).

OCCURRENCE AND SURVIVAL OF NEMATODES FOUND IN PHYTOTELMATA OF

NEPENTHES HAMIGUITANENSIS AND N. PELTATA

The areas where these Nepenthes were growing are characterized by wet environment. Since it is in tropical area, it receives rain more frequently and the range itself is extremely humid, with thick piles of litters and mosses all over the area. This gives sufficient attribute of an environment to support aquatic nematodes. Based on biological and ecological records presented, there is no doubt that all taxa we found in the phytotelmata of Nepenthes are capable of dwelling in an aquatic environment (Hunt, 1978; Andrassy, 1996, 2002 & 2005; Fonseca et al., 2006; & Holovachov & De Ley 2006) and some were even recovered from phytotelmata in bromeliad tanks i.e. Tylocephalus (Holovachov et al., 2004), actinolaimids and plectids (Zullini, 1977 & Jacobs, 1984) and actinolaimids and plectids from phytotelmata of Nepenthes gymnamphora (Menzel, 1922). Assessing the profile of the nematodes we recovered from our samples, only the genus Pelodera is known to survive in an extreme condition and has the ability to enter dauer stage if conditions are unfavorable. The presence of nematodes that are nearly all known to be sensitive is distinct from the previously reported nepenthebiont such as Baujardia mirabilis (Bert 2003) and Panagrellus nepenthicola (Menzel, 1922; Micoletzky & Menzel, 1928) which are closely related taxa to P. redivivus inhabiting sour paste and stomach of a monkey

(Peters, 1927), P. silusiae associated with beer filters and Turbatrix aceti inhabit vinegar (Aubertot, 1925); taxa known to withstand extreme environment. On the common belief that Nepenthes produces enzymes to digest its prey, it is just fascinating how those sensitive nematodes survive if not live on the phytotelmata of Nepenthes knowing that there were no records to support that they are capable of living in a totally extreme conditions like the acidic fluid of Nepenthes pitchers. Although Menzel (1922) observed that Dorylaims were still alive after a three-day exposure in the fluid of N. gymnamphora there might be loopholes on his observation in our opinion. There is a possibility that the fluid he mentioned in his observation might be enzyme free. This was because of a recent study done in N. alata by Owen et al. (1999) and An et al. (2002) who showed that the amount of enzyme being released is based on the size of prey. Hence, without prey, pitchers won’t likely release these digestive enzymes (Plachno et al., 2006). In addition, a study conducted by Eilenberg et al. (2006) identified two endochitinase isoenzyme in N. khasiana after injection of chitin into unopened sterile pitchers suggesting a prey specificity of the enzyme being produced. However there are some exceptions, as other Nepenthes species possess complex relationships with other organisms. Examples are N. bicalcarata that is known to secrete digestive enzymes; but shelters Campanotus ants that are equipped with an ability to swim in the phytotelmata and legs that are capable of walking through the walls of the pitchers. The Campanotus ants feed on the cadaver of trapped insects and by crushing the prey’s cadaver, it hastens the digestive breakdown of the trapped prey where this N. bicalcarata can sequester its needed nutrients while the feces of the commensal ant compensates for the partial loss of prey supply (Bonhomme et al., 2011). Similar with N. bicalcarata, N. rajah produces digestive enzymes and does the same mechanism together with mosquito larvae (Tsukamoto, 1989). In reality, not all species of Nepenthes are enzyme-producing such as N. ampullaria (Moran et al., 2003). N. rafflesiana elongata pitchers provide a home and at the same time lavatory to Kerivoula hardwickki, a small bat (Grafe et al., 2011). Clarke et al. (2009; 2010) studied the mutualistic relationship of N. lowii with tree shrews Tupaia montana which feeds on accumulated exudates from Nepenthes lid and in return they defecate into the pitcher which will be used as nitrogen source. The same was observed in N. rajah, although documented to produce enzyme, it also makes use of feces of tree shrew and a nocturnal rodent Rattus baluensis (Greenwood et al., 2011; Wells et al., 2011). Therefore, the nutrient absorption of Nepenthes comes from a complex system of carnivory and it is a reasonable

assumption that when there are symbiotic organisms that play a vital role in the prey- digestion, enzyme production is unnecessary. When enzyme production ceases, the content of phytotelmata will be pure water collected from rain which enables for normal aquatic nematodes to dwell. To date, no study had been conducted specifically on N. hamiguitanensis and N. peltata where we found our samples so we could not conclude with certainty if these two species particularly produced enzymes or had special relationship with their commensals. Therefore, we could hypothesize that the nematodes were there as component of foodweb in the phytotelmata feeding on bacteria, fungi and other minute organism that proliferate when decomposition of the prey’s cadaver starts, they are either not harmed by the enzymes released by Nepenthes or the Nepenthes were not enzyme-producing species. Phoretic association is one important factor on how these nematodes were able to reach the water held by pitchers of Nepenthes. But this is not limited to insects, since Sudhaus (2011) shows that some species of Pelodera in Strongyloides-group are found in skin of rodents. Moreover, we sampled hanging and ground level pitcher and we guess that pitchers which have nematodes are those pitchers lying on the ground (Appendix II). Similar to what was observed in 1922 by Menzel where no nematode was recorded in the hanging pitcher. Remarkably, all the nematodes we recovered were natural inhabitant of wet biotopes including mosses. However, it is not impossible to find these nematodes inside the phytotelmata since it is apparent that some of the pitchers we sampled were close to mosses on the ground and even hanging pitchers were close to epiphytic mosses and because of the presence of nepenthebiont and nepenthephile organisms such as frogs (Dover, 1928), flies (Kurahashi & Beaver, 1979), ants (Rembold, 2009), spiders (Beaver, 1983) (Appendix III) within the sampling site which could serve as vector of these nematodes. However, the first discovery of Molgolaimus is exceptional since this is a typical marine nematode. It remains a mystery on how this marine-inhabiting nematode reached the pitcher of Nepenthes located 639 to 1188 m a.s.l We could rule out the notion that this was a contamination from other samples since there were also several marine nematodes found (Appendix IV) in the same site five years ago. However, it was also peculiar that all marine taxa inside the pitchers of Nepenthes from current sampling were different compared to the sampling in 2010. Hence, it can be argued that it was just passively carried by insects whose part of the life cycle is within the marine sediments or an insect that has constant ecological interaction with other marine organism. This is possible since the mountain itself is surrounded with Pacific Ocean and the Davao Gulf. However, a thorough investigation is

reccommended. We need to consider all the nepenthebiont and nepenthephile organisms associated with N. hamiguitanensis and N. peltata whether one has an actual relationship with the marine environment. Also a great avenue to understand whether these marine nematodes were nepenthebiont or just a nepenthexenes is to observe if they are alive in the phytotelmata which we didn’t do because of lack of equipment and the accessibility of the area. It would be of help also to get information on pH and salinity of the fluid in the pitchers individually. At present, we are not able to provide solid conclusions about the occurrence of marine nematodes in the phytotelmata of pitcher plants. Hence, further study must be done to unravel the mystery behind these extraordinary findings.

Acknowledgment

A word seems insufficient to express the researcher’s appreciation for the success of this study. Truly, this simple work could not have been made possible without the help of people who, in various ways, unselfishly dedicated their time, effort and expertise. To his advisers, Prof. dr. Wim Bert and Dr. Irma Tandingan De Ley, for patiently checking his manuscript, giving valuable advice, sharing their knowledge, and for their trust. Sincere thanks are extended to the faculty members of the Nematology Section: Prof. dr. Wilfrida Decraemer and Nic Smol for their help in identification and constructive corrections. Profound appreciation is extended to Marjolien Couvreur, Dennis Vlaeminck, Andy Vierstraete, Martin Schellink, Dieter Slos, Xue Qing and Toon Janssens for sharing their knowledge, skills and their assistance to make this study done. Special thanks are also extended to the University of Southeastern Philippines thru Dr. Lourdes Generelao and Cecirly Gonzales; to Davao Oriental State College of Science and Technology thru Dr. Lea Angsinco Jimenez for accommodating the research team during the sampling and to Lapanday Foods Corporation for their extraction facilities. Greatest gratitude is also expressed to Flemish Inter-University Council (VLIR-UOS) for the scholarship and financial assistance.

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Appendices

Appendix I. Bayesian inference 50% majority rule consensus of phylogeny of Dorylaimidae specimen from MH sample and other Dorylaimidae sequences from GenBank based on D2-D3 expansion of LSU rDNA data. Xiphinema rivesi was designated as outgroup. Branch support values are indicated with posterior probability.

Appendix II. Different growth habitats of Nepenthes pitchers in Mount Hamiguitan. A, F, H: hanging few meters away from the ground; B, E: lying on neighbouring mosses; C, D, H: ground level pitchers; I: Flower of Nepenthes.

Appendix III. Organisms found in Mt. Hamiguitan which might have vectored nematodes into the phytotelmata of pitcher plants Nepenthes hamiguitanensis and N. peltata. A: curculionid beetle; B-C: flies; D: beetle; E: dragonfly; F: dipterans; G: spider; H: small red crab; I: millipede; J: mollusc; K: frog.

Appendix IV. Marine nematodes discovered in phytotelmata of Nepenthes from 2010 exploration. A-D: Desmodora sp.; E-G: Oncholaimus sp.; H-J: Cyartonema sp.; K-M: Axonolaimus sp.; N-P: Richtersia sp. (Scale bar not possible, the picture were captured from video compilation.) Videos and identification was based from Dr. Irma Tandingan De Ley.