BRI LL
Nematology 17 (2015) 377-400
brill.com/nemy
Deceptive morphological variation in Hirschmanniella mucronata (Nematoda: Pratylenchidae) and a polytomous key to the genus Kimkhuy K hun 1’2, Wilfrida D ecr aem er 2’3, Marjolein C o u v r e u r 2, Gerrit K a r s s e n 2’4, Hanne S teel 2 and Wim B ert 2’* 1Faculty o f Agronomy, Royal University o f Agriculture, Chamkar Daung, Dangkor District, Phnom Penh, Cambodia 2Nematology Research Unit, Department o f Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium 2 Royal Belgian Institute o f Natural Sciences, Department o f Invertebrates, Vautierstraat 29, B-1000 Brussels, Belgium 4National Plant Protection Organization, Netherlands Food and Consumer Product Safety Authority, Geertjesweg 15, 6700 HC Wageningen, The Netherlands Received: 8 September 2014; revised: 18 December 2014 Accepted for publication: 18 December 2014; available online: 18 February 2015
Summary - Hirschmanniella mucronata populations isolated from two Cambodian provinces were characterised using morphological, morphometric and molecular criteria. Examination of 1024 specimens from 60 different paddy fields revealed high intraspecific variation in morphology and morphometries, especially in tail terminus shape and stylet length. Sequence results confirmed that morphologically divergent individuals represent a single species, suggesting that neglecting morphological variation has led to an overestimation of Hirschmanniella diversity in former studies. Phylogenetic analysis of the SSU, D2-D3, LSU and ITS1-5.8S-ITS2 regions revealed three concordant clades, H. mucronata having a sister relationship with H. kwazuna and H. loofi. Plotting the diagnostic features, including tail terminus shape, stylet length and lip region morphology on the phylogenetic framework, revealed that none of them supported the clades and represented convergent features. All three molecular markers were able to discriminate all Hirschmanniella species, but the D2-D3 region was the easiest, fastest and most successful region to be amplified. Species delimitation and the diagnostic features of Hirschmanniella were re-evaluated. Hirschmanniella abnormalis and H. exacta are considered to be junior synonyms of H. oryzae and H. mannai a species inquirenda. Finally, a list of valid species with indication of synonyms and a polytomous key are provided. Keywords - Cambodia, D2-D3 LSU, Hirschmanniella abnormalis n. syn., Hirschmanniella exacta n. syn., Hirschmanniella mannai sp. inq., Hirschmanniella oryzae, identification, intraspecific variation, ITS1-5.8S-ITS2, molecular, morphology, morphometries, new synonym, plant-parasitic nematodes, species inquirenda, SSU.
The migratory endoparasitic genus Hirschmanniella Luc & Goodey, 1964 is known to be one of the most important nematode genera on rice. Several species are a serious economic threat to lowland rice fields in South and Southeast Asia (Bridge et al., 1990; Prot et al., 1994; Karssen, 2009; Maung et al., 2010; Win et al., 2013). For example, when H. oryzae (van Breda de Haan, 1902) Luc & Goodey, 1964 was present in the field before rice seedlings were transplanted, tiller growth was reduced by up to 50-60% and yield production decreased by at least 25% (Ou, 1985; Jairajpuri & Baqri, 1991). The pest species may also delay rice flowering stage by up to 12 days (van der Vecht & Bergman, 1952). More than half of the valid Hirschmanniella species have been recorded
as pests of rice while several other species are reported as pests of crops such as taro (Bridge et al., 1983), cabbage (Duan et al., 1996), maize, tomato and sugarcane (Bridge et al., 1990). Some Hirschmanniella species have been recorded as able to survive in marine environments and damaged aquatic plants such as Diplanthera wrightii (Sher, 1968) and Ceratophyllum demersum (Gerber & Smart, 1987). Although several species of this genus are known from natural habitats, Hirschmanniella is considered a harmful pest and, except for H. gracilis (de Man, 1880) Luc & Goodey, 1964, is a quarantine taxon for the European Union (Karssen, 2009). Sher (1968) made a comprehensive revision of the genus with a key to 23 species, this revision later be-
* Corresponding author, e-mail:
[email protected] Koninklijke Brill NV, Leiden, 2015
DOl 10.1163/15685411-00002867
K. Khun et al.
ing updated, adapted and modified by Razjivin et al. (1981) , Ebsary & Anderson (1982), Sivakumar & Khan (1982) , Loof (1991), Karssen (2009) and Geraert (2013). Loof (1991) and Siddiqi (2000) listed 24 and 34 nom inal species, respectively. Siddiqi (2000) included three species described after Loof (1991), but also seven species that were considered junior synonyms by Loof (1991). In this paper we accept the synonymisation by Loof (1991). In general, Hirschmanniella species are delineated based on morphological and morphometric features, es pecially body length, areolation of the lateral field, lip re gion profile, number of head annules, stylet length, shape of the stylet knobs, position of the secretory-excretory pore, presence/absence of intestine-rectum overlap, c' ra tio, position of the phasmids, presence/absence of the mucro, number of mucro/projections and their position, pres ence/absence of subterminal notch, presence/absence of male, spicule length, and presence/absence of sperm in the spermatheca (Sher, 1968; Razjivin et al., 1981; Eb sary & Anderson, 1982; Sivakumar & Khan, 1982; Loof, 1991; Karssen, 2009). However, earlier observations on several Hirschmanniella species illustrated that they may show intraspecific variation, e.g., in presence/absence of sperm in spermatheca, shape of the lip region, tail ter minus, body length (Ebsary & Pharoah, 1982; Van den Berg & Queneherve, 2000; Sturhan & Hallmann, 2010) and shape of the stylet knobs (Tandingan De Ley et al., 2007; Van den Berg et al., 2009), which makes correct identification difficult, particularly when a limited num ber of specimens is available. Therefore, morphological data needs to be substantiated by molecular data. The main aims of this study were: i) to identify and describe in detail the Hirschmanniella species collected from rice fields in Cambodia, based on both morpho logical and molecular data (sequences of small subunit (SSU), D2-D3 expansion segments of the large subunit (LSU) and internal transcribed spacer (ITS1-5.8S-ITS2) regions of the rDNA); ii) to analyse the phylogenetic re lationship between the species from Cambodia and other Hirschmanniella species; and Hi) to provide a polytomous key to the species of this genus.
Materials and methods Sampling and extraction Rice (Oryza sativa L.) tillers and rhizosphere soil were collected in lowland rain fed-rice fields from two provinces, i.e., Takeo and Kampot, in southern Cambodia. 378
Samples were collected from 30 randomly selected fields in each province in July 2011 during the tillering stage (2-3 weeks after transplanting). In each paddy field, five rice tillers (consisting of 20-25 rice plants each) and their rhizosphere soil were collected along a diagonal pattern. Each tiller was carefully uprooted with a shovel, placed in a transparent plastic bag and stored in an insulated container for transportation. Nematodes were extracted from soil using Cobb’s decanting and sieving method (Cobb, 1917) and from roots, which were cut in small pieces, using the tray extraction method (Whitehead & Hemming, 1965). The nematode suspensions were split into two, one part being preserved in 4% formalin for the morphological analyses and the other part being preserved in DESS (DMSO-EDTA salt-saturated solution) at room temperature for molecular analyses in combination with digital morphological vouchers (Yoder et al., 2006).
Morphological and morphometric characterisation Formalin-preserved specimens were transferred into pure glycerin according to the glycerin-ethanol method (De Grisse, 1969). All 1024 Hirschmanniella speci mens were morphologically checked and three nema tode populations were selected for morphometric ana lysis: from Angk Ta Saom district, Takeo province (11°01'09.12"N, 104°38'19.46"E); from Srae Ronoung district, Takeo province (10°57/ 14.39'/N, 104°39' 51.01"E); and from Kampong Trach district, Kampot province (10°51,01.08"N, 104°39'33.24"E). Measure ments, pictures and drawings were made with an Olympus BX50 microscope with differential interference contrast (DIC) equipped with a drawing tube and Olympus C5060 digital camera. The original measurements are available online at http://www.nematodes.ugent.be/vce.html. Vouchers of non-type populations from the Zoology Museum and the Nematology Research Unit of Ghent University were studied, including H. imamuri Sher, 1968 from Korea, H. mucronata (Das, 1960) Luc & Goodey, 1964 from the Philippines, H. spinicaudata (Schuurmans Stekhoven, 1944) Luc & Goodey, 1964 from Niger and H. oryzae from the Philippines. To analyse which morphometric characters could dis criminate between our three populations, a canonical dis criminant analysis (CDA) was performed including only those morphometric characters that had no significant cor relation with each other (significant correlation at P < 0.05, r > 0.8) using the CANDISC procedure in SAS® Nematology
Hirschmanniella mucronata variation and key to genus
9.3 based on the pooled within variance-covariance ma trix. For scanning electron microscopy (SEM) studies, spec imens from slides were dehydrated in a seven-step graded series of ethanol solutions, critical-point-dried with liq uid CO2 , mounted on stubs with carbon discs, coated with gold (Steel et al., 2011), and photographed with a JSM840 EM (JEOL) at 12 kV.
Molecular characterisation Each DNA sequence was obtained from a single DESSpreserved specimen after microscopic identification and archiving of LM pictures. They were then washed with distilled water for 10 min in a staining block, transferred into a 500 /xl Eppendorf tube with 20 /jl1worm lysis buffer (50 mM KC1; 10 mM Tris pH 8.3; 2.5 mM MgCl2; 0.45% NP 40 (Tergitol Sigma); 0.45% Tween 20) and frozen for at least 10 min at —20°C. 1 /ul proteinase K (1.2 mg ml-1) was added before incubation in a PCR machine for 1 h at 65°C and 10 min at 95°C. After centrifugation (1 min at 20 800 g), 2 /u.1 of the DNA template was added to 25 /i\ PCR mixture {Taq DNA Polymerase, Qiagen) with 0.5 /zl of each primer, i.e., G18S4, 18P (Blaxter et al., 1998), 4F and 4R (Tandingan De Ley et al., 2002) for small subunit (SSU) rDNA gene; D2Ab and D3B (De Ley et al., 1999) for the D2-D3 expansion segments of the large subunit (LSU) rDNA; Vrain2F and Vrain2R (Vrain et al., 1992; Elbadri et al., 2002) for the ITS1-5.8S-ITS2 rDNA. The thermal cycler program for PCR was, according to the manufacturer’s protocol, as follows: denaturation at 94°C for 4 min, followed by 50 cycles of 94°C for 30 s, 54°C for 30 s 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. Additional sequences of Hirschmanniella species for phylogenetic analyses were obtained from GenBank. Multiple sequence alignments of SSU, D2-D3 LSU, ITS 1-5.8S-ITS2 rDNA sequences were made using MUS CLE (Edgard, 2004) followed by post-alignment trim ming with G-Blocks as implemented in SeaView Ver sion 4 (Gouy et al., 2010). Other alignment methods did not influence the tree topology. Bayesian phylogenetic analysis was carried out in MrBayes v. 3.2.1 (Ronquist & Huelsenbeck, 2003) using the GTR 4 -1 + G model. Anal yses were run under default settings for 20 x 106 genera tions, 25% of the converged runs were regarded as burnin. Vol. 17(4), 2015
Results Hirschmanniella mucronata (Das, 1960) Luc & Goodey, 1964 (Figs 1-4) Although showing some intraspecific variation in the shape of tail terminus and the stylet length, all 1024 Hirschmanniella specimens from 60 different paddy fields of two provinces were identified as H. mucronata.
Measurements See Tables 1, 2.
Description Female Largely agreeing with information from literature but showing some new details as well as some variation in morphological and morphometric features. Body slender, slightly curved to irregularly shaped, 1260-2296 /zm long, max. body diam. 23-36 /rm. Lateral field with four in cisures, width 22-41% of max. body diam., with irregular and incomplete areolation along body, mostly distinct areolation in tail region. Annulus width 1.4-2 /zm. Lip region hemispherical, 9-12.5 /j.m diam., 2.8-4.6 /zm high, with 36 transverse annules lacking longitudinal lines. Cephalic framework moderately sclerotised. Stylet 1.8-2.5 lip re gion diam. long, stylet knobs round, clearly offset, stylet cone 0.8-1.5 times as long as shaft. Hemizonid 1-3 an nules long, located 1-3 annules anterior to secretoryexcretory (SE) pore. Position of SE pore highly variable, ranging from 6 /im posterior to median bulb valve, i.e. 47.5 /im anterior to pharyngo-intestinal junction (PIJ) to about level of PIJ or 55 /zm posterior to median bulb valve. Pharyngeal glands elongated, tapering posteriorly, overlapping intestine ventrally. Female genital branches opposed, outstretched. Anterior genital branch (52% of total length) longer than posterior genital branch (48% of total length). Spermatheca oval, filled with rounded sperm; anterior spermatheca often larger than posterior spermatheca. Vagina occupying 48-62% of corresponding body diam., 5% of specimens showing either copulatory plug-like structures in vagina or similar male secretory in clusions within uterus of specimens (4-10 x 2.2-4.6 /zm). Intestine not overlapping rectum. Tail terminus remark ably irregular in size and shape with pointed terminus to a projection, occasionally with a mucron (4% of speci mens). Phasmids distinct, located at ca 23-50% of tail 379
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Fig. 1. Hirschmanniella mucronata. A: Male, anterior region; B: Female, head region; C: Male, tail region; D: Hypoptygma in cloacal region; E: Variation in proximal end of gubernaculum; F: Female, tail region with irregular incomplete areolation; G: Female, variation in tail terminus shape; H: Vulval region with copulatory plug-like structures; I: Anterior female genital branch, from vulva to spermatheca; J: Female, lateral view near mid-body showing irregular incomplete areolation.
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0.02mm Fig. 2. Hirschmanniella mucronata. A: Female, head region; B: Male, head region; C: Female, lateral view near centre of body showing irregular incomplete areolation; D: Hypoptygma at cloacal region; E: Variation in proximal end of gubernaculum; F: Male secretions resembling copulatory plugs in vagina and/or vagina; G: Variation in female tail terminus shape. Vol. 17(4), 2015
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Fig. 3. SEM photographs of HirschmannieUa mucronata. A-E: Female; F-H: Male; I-L; Juvenile. A: Enface view; B: Lateral field in vulval region; C: Vulva; D: Lateral field at mid-body female; E: Ventral view tail; F: Enface view; G: Ventral view cloacal region; H: Lateral field at mid-body; I: Enface view; J-L: Variation in tail tip.
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A .S S U rD N A -Pratylenchus vulnus (EU130885 )
1 00
Hirschmanniella pomponiensis (DQ077795, California, USA)
1 .0 0 1
\Praty'enchusbolivianus (FR692329) *Pratylenchus bolivianus (FR692328)
------------------- Pratylenchus thomei (EU 130879) '
Hirschmanniella halophila (EU620474. Germany) 1.001
C la d e I
1
LHfn
Hirschmanniella santarosae (EF029859. California. USA)
Hirschmanniella halophila (EU620475, Germany)
1 00 r~Hirsctimann'ella halophila (EU620465, Germany) ■Hirschmanniella halophila (EU620464, Germany)
■Hirschmanniella mucronata (DQ309589, Taiwan) "
C la d e I C la d e I
Hirschmanniella mucronata (Cambodia, T3) KP179328
1 oo r hirschmanniella loofi (EU620469, Belgium)
■Hirschmanniella mucronata (Cambodia, T4) -
C la d e I l Hirschmanniella loofi (EU620468, Belgium)
Hirschmanniella mucronata (Cambodia. T1) K P179331
Hirschmanniella kwazuna (EU620466. South Africa)
Hirschmanniella mucronata (Cambodia, T2) -K P179329 Hirschmanniella mucronata (KF201166, the Philippines) 1 ootHirschmanniella loofi (EU620472, Belgium)
-Hirschmanniella kwazuna (EU620467, South Africa) ■Hirschmanniella mucronata (Cambodia. T1)......)
KP179333
j
■Hirschmanniella mucronata (Cambodia. T3) = i
■Hirschmanniella loofi (EU620473, Belgium)
w Hir. Hirschmanniella kwazuna (EU620470, South Africa)
Hirschmanniella mucronata (Cambodia, T4)... J -Hirschmanniella mucronata (Cambodia, T 2 )^ ^ -
’ •“ L Hirschmanniella
KP179327 irschmanniella mucronata (KF2011167, the "• Philippines, Bataan) =; irschmanniella mucronata (the Philippines, j Bulacan) -----Hirschmanniella sp. (EF029861, Arizona, USA)
C la d e III Hirschmanniella oryzae (the Philippines)
C
0.93 |||
Hirschmanniella oryzae (the Philippines, Pampanga) Hirschmanniella oryzae (the Philippines, Bataan)
■Hirschmanniella belli (EF029860, California. USA)
•Hirschmanniella oryzae (KF201164, the Philippines, Bulacan)
f
Hirschmanniella sp. (DQ328686, Vietnam)
■Hirschmanniella oryzae (DQ309588, Taiwan)
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h LHirschmanniella oryzae (KF201161, the Philippines) .oo B. D 2 -D 3 LSU rD N A
kwazuna (EU620471, South Africa)
0.791)■Hirschmanniella oryzae (KF201163, the Philippines. Batangas)
C, ITS 1 -5.8S-ITS 2 rD N A
Fig. 4. Phylogenetic relationships within Hirschmanniella as inferred from Bayesian Inference (BI) analyses of the sequences: A: SSU rDNA with Pratylenchus thomei and P. vulnus as outgroup; B: D2-D3 LSU rDNA with P. thomei and P. vulnus as outgroup; C: ITS15.8S-ITS2 rDNA with P bolivianus as outgroup. Schematic drawing of the shape of the Up region and tail terminus, traits that were considered as important diagnostic features, were plotted on the SSU rDNA tree (A). Vol. 17(4), 2015
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