CC BY
23Russian Journal of Nematology, 2011, 19 (1), 83 - 92
Molecular and morphological characterization of Longidorus henanus Xu & Cheng, 1992 (Nematoda: Dorylaimida) with all four juvenile developmental stages
Kai Guo, Hongli Shi, Matafeo Angelika, Hongli Shi and Jingwu Zheng
Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University,
Hangzhou 310029, China e-mail: [email protected]
Accepted for publication 16 December 2010
Summary. Two populations of Longidorus henanus Xu & Cheng, 1992 with all four developmental stages were found. Description and morphometries of all these stages are provided. The morphological and molecular variations among intraspecies of L. henanus are discussed and given. The polytomous key codes of Chen et al. (1997) for L. henanus are modified in parentheses as follows: A34, B23, C34, D2, E3, F(2)3, G(1)23, H12, I1. Furthermore, the phylogenetic trees of 18S rRNA gene and D2-D3 expansion region of 28S rRNA gene supported L. henanus as a unique and valid species with respect to previously sequenced Longidorus species.
Key words: juvenile, Longidorus henanus, morphometrics, phylogeny, taxonomy, 18S rRNA, 28S rRNA.
Longidorus henanus Xu & Cheng, 1992 was originally described from specimens recovered from the rhizosphere of grapevine in Linbao County, Henan Province, northern China. Later on, this species has been recorded from the rhizosphere of bamboo from Beijing and of poplar from Yuanqu County, Shanxi Province, China (Zheng et al., 2001).
Currently, many reports revealed that some Longidorus species have only three and not the usual four juvenile developmental stages (JDS). When describing L. henanus, Xu & Cheng (1992) reported only three juvenile developmental stages of this species. However, in a review of juvenile stages in the genus Longidorus Micoletzky, 1922, Robbins et al. (1995) found that data for a second-stage juvenile (J2) of L. henanus were probably missing based on the morphometrics and concluded that the species had four juvenile stages.
Recently, the phylogenetic relationship based on molecular sequence data of the ribosomal DNA (rDNA) of various Longidorus species has been widely used, and this can serve as a complement to morphological identification (He et al., 2005; Pedram, et al., 2008; De Luca et al., 2009; Kumari et al., 2009; Robbins et al., 2009; Sirca et al., 2009;
Niknam et al., 2010). However, these samples were mainly collected from Europe and America, and few were collected from Asia.
During an investigation on Longidoridae in various regions in China, several populations of L. henanus from different geographical regions were recovered. In this study, two populations of L. henanus, in which juveniles were numerous, were found to have four JDS. One population with all four juvenile stages was collected from the rhizosphere of Juglans regia in Fenyang County, Shanxi Province, northern China and the GPS coordinates 37o18'445''N; 111o46'192''E. Another population was collected from the rhizosphere of Cinnamomum camphora in Hangzhou, Zhejiang Province, eastern China and the GPS coordinates 37o27'074''N; 112o35'298''E. The presence of the J2 unequivocally demonstrated that this species has four juvenile stages.
The objectives of the present study were i) to determine the number of juvenile developmental stages based on morphology and morphometrics of L. henanus; and ii) to study the intraspecies morphological variation of L. henanus from different geographical regions in China; and iii) to investigate the phylogenetic relationships of L.
henanus along with other previously sequenced Longidorus species using 28S and 18S rRNA sequence data.
MATERIAL AND METHODS
Morphological characterization. Four populations were used to study the intraspecies variation of L. henanus; the Fenyang and Hangzhou populations were used for determining the number of juvenile stages; other two populations were collected from the rhizosphere of Populus sp. in Xinzheng and Zhengzhou, Henan Province. Nematodes were extracted from soil by a decanting and sieving method (Brown & Boag, 1988). Specimens were killed by heating, fixed in hot FG (formalin:glycerol, 4:1) for a minimum of 7 d, and processed and mounted in glycerin on slides by a modification of Seinhorst's (1959) method. All observations, measurements and photomicrographs were made using a Leica CTR 5000 compound microscope equipped with differential interference contrast (DIC). The four JDS were distinguished according to the method of Robbins et al. (1995). Measurements are expressed as a mean ± standard deviation (range).
DNA extraction, PCR protocols and sequencing. One juvenile or adult specimen of L. henanus was transferred into 20 ^l ddH2O and cut into 2-5 pieces with a sterilised scalpel. The following steps were prepared as described by Wu et al. (2007). Briefly, the nematode fragments were pipetted in 10 ^l ddH2O and transferred into a 0.2 ml Eppendorf tube containing 8 ^l worm lysis buffer (125 mM KCl, 25 mM Tris-Cl pH 8.3, 3.75 mM MgCl2, 2.5 mM DTT 1.1215, Tween 20) and 2 ^l of proteinase K (600 ^g ml-1), which was stored at -70oC for at least 10 min. Subsequently, the Eppendorf tube was incubated at 65oC for 60 min and the proteinase K was denatured at 95oC for 10 min. Finally, the DNA suspension was stored at -20oC until use. The D2-D3 expansion segments of 28S rRNA was amplified using the forward D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and the reverse D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') primers (De Ley et al., 1999; Gu et al., 2010). PCR protocols of 28S rRNA were described by Zheng et al. (2003). The near-full length of the 18S rRNA gene was amplified using SSU_F_04 (5'-GCT TGT CTC AAA GAT TAA GCC-3') and SSU_R_81 (5'-TGA TCC WKC YGC AGG TTC AC-3') primers (Griffiths et al., 2006). The cycling profile for 18S rRNA gene was as follows: 94oC for 2 min 45 s, then 40 cycles of 94oC for 1 min, 57oC for 45 s, and 72oC for 2 min. The extension phase was 72oC for 10 min. PCR products
were separated on 1% agarose gel and visualised by staining with ethidium bromide. Detailed protocols for purified DNA fragments, cloning and automated sequencing are described by Wu et al. (2007). The internal primer SSU_R_23 (5'-TCT CGC TCG TTA TCG GAA T-3') was used as the walking primer for sequencing the 18S gene (Palomares-Rius et al., 2008). Sequences were deposited in GenBank and accession numbers are as follows: HM749322, HQ634969, HQ822266, HQ822267.
Phylogeny inference. DNA sequences were aligned by ClustalX2 (Larkin et al., 2007). A complex general time reversible (GTR) model with a proportion of invariable site (I) and a gamma distribution (G) of rate heterogeneity (a complex GTR+I+G model) was evaluated using JModeltest (Posada, 2008) with the Akaike Information Criterion (AIC). A Bayesian inference analysis was performed using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001) running four chains for 1.0 x 106 generations and setting the "burn-in" at 1,000. Sampling the Markov chains was made at intervals of 100 generations. The trees were analysed using TreeView 1.6.6 (Page, 1996). Posterior probabilities are given on appropriate clades. The corresponding sequences of Xiphinema index were used as outgroup.
RESULTS
Morphological characters and morpho-metrics. Four populations of L. henanus were examined. Morphometrics of females and juveniles and the key morphological characters are presented in Tables 1, 2 and Figure 1. Two populations reported by Zheng et al. (2001) were compared with our four populations in Table 2. Males were absent in these populations.
All females from populations used in this study had the general characteristics of L. henanus, i.e., the female habitus varied from J-shaped to C-shaped when relaxed by gentle heat, lip region broad anteriorly, slightly set off or continuous with the body profile, tail bluntly conoid to almost hemispherical, amphids asymmetrically bilobed.
The L. henanus first-stage juvenile (J1) is differentiated from the subsequent stages by the position of the replacement odontostyle, which is embedded in the basal wall of the odontophore rather than posterior to the base of the odontophore in the other stages (Fig. 2). The J2 is identified when the functional odontostyle is in the same length range as the replacement odontostyle of J1. Similarly third- and fourth-stage juveniles (J3 and J4) were readily distinguished by a series of measurements
Fig.1. Photomicrographs of Longidorus henanus. A-C: Head region, vulval region, tail region of female. D-E: Head region, tail region of J4. F-G: Head region, tail region of J3. H-I: Head region, tail region of J2. J-K: Head region, tail region of J1.
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Fig. 2. The Bayesian tree inferred from 18S under GTR+I+G model (lnL=-7616.3971; freqA=0.2746; freqC=0.2339; freqG=0.2365; freqT=0.2656; R(a)=1.0462; R(b)=4.3864; R(c)=2.2069; R(d)=0.8060; R(e)=6.3199; R(f)=1.0; Pinva=0.092; Shape=0.134). Posterior probability values exceeding 50% are indicated on appropriate nodes.
Table. 1. Morphometries of Longidorus henanus juveniles from different localities and hosts in China.
Locality Fenyang County, Shanxi Province Hangzhou, Zhejiang Province Paratypes, Linbao County, Henan
Province
Host Jugl ans regia Cinnamomum camphora Vitis vinifera
Stages J1 J2 J3 J4 J1 J2 J3 J4 J1 J3 J4
n 3 6 14 9 4 21 9 11 7 2 6
L 1.0±0.1 1.7±0.1 2.6±0.3 3.4±0.3 l.OiO.l 1.7±0.2 2.3±0.3 3.4±0.4 1.0 2.5-3.4 3.8
(0.9-1.0) (1.5-1.9) (2.0-3.0) (3.0-3.9) (1.0-1.1) (1.3-2.0) (1.9-2.7) (2.8-4.4) (0.9-1.0) (3.3-4.3)
a 43.7±5.2 58.1±5.5 62.5±10.5 72.3± 10.3 38.7±1.8 49.5±4.9 54.9±6.3 65.5±8.4 51 76-77 91
(37.746.9) (52.2-67.6) (44.5-75.6) (57.8-91.2) (36.841.0) (41.2-61.0) (46.7-64.8) (53.6-79.7) (49-53) (86-95)
b 4.6±0.2 6.9±0.8 8.7±1.2 10.6±1.6 5.8±1.0 5.9t0.8 7.6±1.1 9.5±0.7 4.6 8.6-9.2 9.5
(4.44.9) (6.2-8.4) (6.3-10.6) (8.9-13.9) (4.3-6.6) (4.7-7.7) (6.2-9.0) (8.7-11.2) (4.1-5.2) (8.6-10.3)
c 31.2±2.6 49.6±4.0 76.0±7.5 114.2±13.2 31.4±2.0 49.1±6.3 71.6±13.1 106.6±16.5 29 66-88 102 (97-
(29.6-34.2) (43.4-53.9) (64.8-90.5) (95.7-135.1) (29.6-34.2) (40.8-67.1) (56.8-94.0) (87.7-139.2) (27-31) 112)
c1 2.3±0.3 1.6±0.2 1.1±0.1 0.9t0.1 2.0±0.2 1.5±0.2 1.2±0.1 0.9±0.1 2.47 1.1-1.5 1.15 (1.1-
(2.1-2.6) (1.4-1.9) (0.9-1.3) (0.8-1.0) (1.8-2.3) (1.2-1.8) (1.0-1.3) (0.7-1.1) (2.3-2.6) 1.2)
Odontostyle 43.1±6.6 54.8±2.4 69.9±3.8 81.6±2.2 50.2±3.2 58.5±3.2 72.3±2.9 85.9±2.5 49 65-70 82
(37.3-50.3) (52.7-57.8) (61.6-75.8) (78.7-86.3) (46.7-54.5) (54.2-67.4) (69.7-78.6) (82.6-90.8) (48-50) (78-88)
Odontophore 34.8±4.9 51.5±2.6 55.1±5.5 60.8±4.0 33.4±2.1 47.6±4.4 53.6±6.3 63.4±4.0 36 50-64 63
(30.440.1) (48.8-55.2) (43.6-63.0) (55.0-65.3) (30.3-34.6) (37.8-56.0) (45.5-63.1) (57.5-71.1) (34-37) (60-65)
Total stylet 77.9±11.6 106.3±3.7 125.0±6.4 142.4±5.9 83.6±4.0 106.1±6.0 125.8±7.8 149.3±5.1 85 115-134 145
(67.7-90.5) (102.2-112.5) (110.1-136.1) (135.0-151.6) (79.9-88.8) (92.9-115.2) (115.2-141.7) (142.3-157.7) (84-87) (141-149)
Replacement 52.5±4.4 70.1±2.5 81.0±3.9 95.4±2.7 55.6±1.9 68.1±4.5 82.3±2.1 95.5±1.8 54 74-83 98
odontostyle (48.3-57.0) (66.3-73.2) (73.1-86.8) (91.2-98.6) (53.6-57.8) (54.2-75.8) (79.8-86.4) (93.0-98.6) (53-55) (95-102)
Anterior to 20.6±1.2 27.4±0.6 31.9±1.3 36.6±1.9 22.7±0.5 30.2±1.7 34.2±1.2 40.6±1.6 21.0 29.0-33.5 36.5
guide ring (19.4-21.6) (26.5-28.3) (30.2-34.2) (34.0-39.0) (22.3-23.2) (27.4-35.0) (32.06-35.6) (37.3-43.7) (20.5-21.5) (35.0-38.0)
Width at 7.9±0.5 10.0±0.4 12.2±0.9 13.5±0.7 8.5±0.3 11.5±0.8 12.7±0.5 14.7±1.2 8.0 11.5-12.5 13.5
lip region (7.5-8.6) (9.5-10.6) (10.3-13.3) (12.8-14.7) (8.2-8.7) (10.2-12.9) (11.9-13.7) (13.4-17.4) (7.5-8.5) (13.5-14.0)
Width at 14.7±0.9 18.3±1.2 23.6±1.4 27.3±2.5 15.6±0.2 20.2±1.8 22.3±1.5 27.9±1.1 14.0 21.0-23.5 24.5
guide ring (13.7-15.4) (16.7-19.9) (21.1-25.7) (24.5-33.3) (15.4-15.8) (17.2-24.9) (20.4-25.1) (25.8-29.5) (13.5-14.5) (24.0-25.5)
Width at base of 19.7±1.2 27.2±2.4 37.0±3.1 43.9±5.2 22.7±2.4 30.9±3.8 37.6±4.4 47.0±6.1 19.0 33.044.0 41.5
pharynx (18.6-21.0) (23.8-30.1) (32.2-41.6) (38.1-51.9) (19.7-25.4) (25.441.5) (31.846.2) (38.4-58.8) (18.5-190) (37.0-45.5)
Width at 19.8±1.1 27.5±3.0 40.2±5.6 46.0±6.8 25.5±1.7 32.2±4.4 39.5±5.7 50.1±7.3 19.0 33.044.0 41.5
mid-body (18.6-20.6) (22.8-32.1) (32.1-48.5) (34.0-54.4) (24.6-28.0) (26.744.4) (32.7-52.2) (37.7-60.5) (18.5-19.0) (37.0-45.5)
Width 13.8±0.9 20.8±0.1 30.7±3.2 33.6±3.4 16.1±1.1 23.3±3.0 28.0±5.1 38.1±5.6 13.0 25.5-33.5 32.5
at anus (13.09-14.8) (18.9-24.6) (24.0-35.6) (28.6-39.4) (14.8-17.2) (18.8-31.6) (21.1-38.2) (29.5-47.0) (12.5-14.0) (30.0-35.5)
Tail length 31.6±1.7 33.4±2.0 34.3±2.6 30.2±3.3 32.2±1.2 34.7±2.7 32.6±5.5 32.0±4.2 32.7 38.0-38.5 37.0
(30.6-33.5) (30.9-36.3) (30.1-38.6) (25.7-34.7) (31.1-33.3) (29.241.5) (24.8-39.5) (25.0-39.2) (31.0-34.5) (33.0-39.5)
* All measurements in urn. except for L in mm.
Table 2. Morphometries of Longidorus henanus females from different localities and hosts in China.
Locality Fenyang Hangzhou Xinzheng Zhengzhou Yuanqu Shanxi Province Beijing Paratypes Henan
Shanxi Province Zhejiang Province Henan Province Henan Province Zheng et.al, 2001 Zheng et.al, 2001 Province
Host Juglans regia Cinnamomum Populus sp Populus sp Populus sp. Bamboo Vitis vinifera
camphora
n 19 14 6 5 8 4 11
L 5.0±0.5 (4.1-5.8) 4.5±0.3 (4.0-5.1) 5 .2±0.9 (4.1-6.8) 5.0±0.4 (4.6-5.5) 4.9±0.6 (3.8-5.4) 5.4±0.7 (4.6-6.3) 6.1±0.6 (5.1-7.0)
a 80.5±6.4 (66.0-98.1) 73.2±8.2 (51.0-82.0) 96.9±8.7 (88.4-111.7) 75.4±1.6 (73.1-76.7) 87.6±10.2 (75-103) 95.6±6.8 (88-104) 112±9.0(99-127)
b 12.6±1.7 (10.1-18.1) 12.5±2.1 (10.6-17.8) 11.7±2.2 (9.3-14.5) 13 4±1 8 (11.9-15 8) 13.1±0.7 (11.7-13.7) 12±1.5 (11.8-13.1) 13.2±1.0(12.2-15.4)
c 170.2±27.5 (120.0- 145.7±13.0(130.4- 166.5±29.4 (130.1- 149.9±21.7 (124.0- 142.3±9.8 (130-157) 168.1±22.2 (147- 168±13.4 (154-194)
221.7) 178.4) 215.9) 178.53) 198)
c1 0.7±0.1 (0.6-0.8) 0.8±0.1 (0.7-0.8) 0.8±0.1 (0.7-1.0) 0.8±0.1 (0.6-0.9) 0.9±0.1 (0.7-1.0) 0.8±0.1 (0.7-0.8) 0.9±0.1 (0.9-1.0)
V% 49.9±2.8 (44.7-56.2) 49.5±1.0 (47.0-51.3) 50.5±2.4 (48.4-54.5) 48.7±2.1 (45.4-50.6) 48.5±1.6 (46-52) 48.9±1.1 (48-50) 49±1.2 (47-51)
Odontostyle 97.6±2.8 (92.2-103.9) 94.7±2.6 (90.5-98.1) 96.6±2.3 (93.2-98.7) 100.0±0.9 (98.4-100.8) 98.3±3.0 (94-104) 98.8±4.4 (93-102) 98±2.1 (97-103)
Odontophore 71.1±4.1 (62.7-79.4) 66.0±3 .4 (60.1-71.4) 70.7±2.6 (68.0-75.0) 72.2±7.5 (64.3-84.5) 63.2±4.9 (57-68) 67.5±4.2 (63-71) 70±2.0 (66-74)
Total stylet 168.7±5.9 (159.2- 160.7±4.3 (153.2- 167.4±2.4 (164.4- 172.3±7.7 (164.9- - - 168±2.6 (165-173)
179.7) 167.3) 170.2) 184.7)
Anterior to 40.8±1.0 (38.7-41.9) 44.4±1.8 (42.1-47.6) 40.7±1.9 (38.2-43.0) 41.6±1.5 (40.0-43.5) 38.8±2.0(36-42) 40.8±0.9 (40-42) 42.0±1.9 (38.5-45.5)
guide ring
Width at 15.5±0.6 (14.5-16.0) 15.7±1.2 (13.9-18.0) 15.6±1.3 (13.4-16.7) 14.9±0.5 (14.3-15.4) 14.6±0.8 (13.2-15.7) 14.0±0.8 (13 .2- 16.0±0.5 (15.0-16.5)
lip region 15.1)
Width at 29.7±1.4 (27.4-32.5) 30.9±1.1 (29.5-33.1) 27.6±1.3 (25.7-29.5) 30.0±1.4 (28.6-32.1) 30.3±2.8 ( 28-35 ) 29.8±0.8 (29-31) 29.0±1.0 (27.5-30.5)
guide ring
Width at base 50.7±3.7 (44.6-58.3) 49.2±5.5 (40.3-57.0) 45.2±6.5 (38.2-54.1) 55.6±6.3 (49.4-62.7) 46.3±4.2 (40-54) 47.2±3.1 (45-52) 46.5±2.5 (43.0-49.5)
of pharynx
Width at mid- 61.0±4.2 (51.8-66.6) 59.2±8.4 (47.8-73.4) 52.8±7.9 (44.6-67.6) 65.4±4.0 (60.4-71.1) 56.1±6.4 (47-68) 56.5±3.3 (52-60) 54.0±3.61 (47.5-59.0)
body/vulva
Width at anus 41.1±3.0 (36.5-47.7) 41. 1±3.1 (35.3-46.4) 38.0±4.2 (34.2-46.1) 42.5±3.7 (37.9-47.9) 49.4±3.9 (35-41) 42.5±3.7 (37-45) 39.0±2.9 (35.5-43.0)
Tail length 29.7±3.5 (24.6-36.1) 30.8±2.4 (28.2-35.9) 31.5±3.4 (27.8-37.4) 33.7±3.0 (30.1-38.1) 34.3±3 (29-38) 32.4±4.1 (29-38) 36.5±4.3 (31.0-42.0)
* All measurements in urn. except for L in mm.
Fig. 3. The Bayesian tree inferred from D2-D3 expansion fragments of 28S rRNA under GTR+I+G model (lnL=-8637.327; freqA=0.2525; freqC=0.2187; freqG=0.2926; freqT=0.2363; R(a)=0.7704; R(b)=2.0135; R(c)=1.4718; R(d)=0.5899; R(e)=4.1832; R(f)=1.0; Pinva=0.0; Shape=0.518). Posterior probability values exceeding 50% are indicated on appropriate nodes.
of both functional and replacement odontostyles. Body shape of juveniles arcuates to J-shaped, smaller than adult females. Lip region shape and amphids in all juvenile stages are similar to that of adults. Total body length and distance from anterior to guide ring of JDS show a stepwise increase from J1 to J4. Tail length of the Fenyang population increases from J1 to J3 and then decreases in J4; however, tail length of the Hangzhou population increases from J1 to J2 and then decreases though J3 to J4 (Table 1). Tail shape progressively changes from elongate conoid in J1 to bluntly conoid in J4, with decreases in c' value.
Several populations reported from China and these new populations in this study were analysed (Table 2). The ranges of morphometric variation are: body length (3,790-6,990 pm), a (51-127), b (9.3-18.1), c (120-221.7), c' (0.6-1.0), v (44.7-56.2), odontostyle length (90.5-103.9 pm), odontophore length (57-79.4 pm), total stylet length (153.2-184.7 pm), anterior to guide ring distance (36-47.6 pm), body width at lip region (13.2-18 pm), body width at guide ring (25.7-35 pm), body width at anus (34.2-47.9 pm) and tail length (24.6-42 pm). The identification codes for L. henanus in the polytomous identification key (Chen et al., 1997) are A34, B23, C34, D2, E3, F3, G23, H12, I1. Comparing with morphometrics in the polytomous key, only body length is shorter than in the type specimens, and this affects the code F and code G. The polytomous key codes are modified in parentheses as follows: A34, B23, C34, D2, E3, F(2)3, G(1)23, H12, I1.
Molecular characterisation and relationship with other species. Amplification of the 18S and D2-D3 expansion segments of 28S rRNA from L. henanus yielded a single fragment of 1779 and 851 bp (including primers), respectively. Average composition of nucleotides of the 18S was 27.5% (A), 25.5% (T), 21.1% (C), 25.9% (G). Interspecific pairwise sequence variation for the 18S rRNA gene among populations of L. henanus was 1.1%. The phylogenetic tree constructed by the Bayesian inference analysis method for the 18S rRNA is presented in Figure 2. The results revealed that two populations of L. henanus clustered together as a sister clade with L. litchii Xu & Cheng, 1992 and L. diadecturus Eveleigh & Allen,1982 but the sister clade does not have high support (only 58%). These three species formed a distinct cluster separated from other available Longidorus 18S sequences from GenBank. The average nucleotide composition of D2-D3 expansion of 28S was as follows: 24.7% (A), 22.7% (T), 22.7% (C), 29.8 (G). Sequences of D2-D3 region of two populations of L. henanus
showed no sequence variation. The phylogenetic tree generated from the D2-D3 expansion sequences alignment formed a separate cluster, which formed a clade clearly differentiated from the other Longidorus spp. (Figure 3). Combining with a Blastn search of the sequences on GenBank, results supported L. henanus as a valid species with respect to sequenced Longidorus species.
DISCUSSION
Robbins et al. (1995) reviewed descriptions and selected re-descriptions of the 98 putative species in the genus Longidorus Micoletzky,1922. Among the 98 species, ten species had three juvenile stages and 39 species had four juvenile stages. Longidorus henanus was reported with three juvenile stages by Xu & Cheng (1992) but Robbins et al. (1995) noted that the J2 of L. henanus was probably missed and concluded that the species had four juvenile stages. In this study, morphometric and morphological data for juvenile development stages revealed L. henanus with four juvenile stages. Our results were largely in agreement with morphometrics and morphological characters of all juvenile stages described by Xu & Cheng (1992), with the addition of J2 data. In addition, the present work also studied more specimens and expanded the range of morphometric data of L. henanus juveniles from China, only two juveniles of J3 were studied in the type species.
Examination of the Longidoridae with three JDS provided some insight into the evolution and possible dispersal pathways of the family (Halbrendt et al., 1997). The shift from four to three juvenile stages was considered to be a derived state which may be a result of hormonally mediated heterochrony, possibly expressed as a single gene change (Robbins et al., 1995). Such a relatively simple genetic mutation could occur independently and randomly providing a shortened life-cycle, by reducing the number of JDS, which may confer a reproductive and ecological advantage (Halbrendt et al, 1997).
Morphometrics and morphological characters of L. henanus populations from Fenyang, Hangzhou, Xinzheng and Zhengzhou were compared with Yuanqu population, Beijing population reported by Zheng et al. (2001), and type specimens. Mean body length of six populations is shorter than that of the type specimens (4.98, 4.47, 5.21, 5.00, 4.89, 5.41 mm vs 6.08 mm) resulting in higher a and c values. In comparison with type specimens, the Hangzhou population had a more posteriorly located guide ring (42.1-47.6 vs 38.5-45.5 pm), a shorter mean odontostyle length (94.7 vs 98 pm), a shorter mean
tail length (30.8 vs 36.5 pm) and a lower c' (0.7 vs 0.9); the Zhengzhou population had a larger mean width at mid-body/vulva (65.4 vs 54 pm); the Yuanqu population had a shorter mean distance from anterior end to guide ring (38.8 vs 42 pm) and a larger mean width at anus (49.4 vs 39 pm). However, the differences observed between different populations of L. henanus are in good agreement with the polytomous identification key by Chen et al. (1997), except for body length and a value. These differences may be due to geographical variation and reflect inter-population variation. Most notably, this is the first report about the occurrence of L. henanus in Zhejiang province, southern China; the previous study has documented its occurrence in northern China. Longidorus henanus from different geographical regions suggests that this species may be widespread in China.
In the original species description, L. henanus most closely resembles L. apulus Lamberti & Bleve-Zacheo, 1977, L. iranicus Sturhan & Barooti, 1983, L. goodeyi Hooper, 1961 and L. crassus Thorne, 1974. A hierarchical cluster analysis of all published Longidorus species based on mean morphometric values of female characters suggested that the most closely related species to L. henanus were as follows: L. arthensis Brown, Grunder, Hooper, Kingler & Kunz, 1994, L. athesinus Lamberti, Coiro & Agostinelli, 1991, L. crassus Thorne, 1974, L. pseudoelongatus Altherr, 1976, L. igoris Krnjaic, Lamberti, Agostinelli & Radicci, 2000, L. iranicus Sturhan & Barooti, 1983, L. trapezoides Nasira & Maqbool, 1995. Chen et al. (1997) in a note of the revised polytomous key for Longidorus species revealed that L. henanus is much closer to L. athesinus, L. goodeyi, L. elongatus, L. dunensis, L. cohni and L. apulus. Unfortunately, few molecular data of these very similar species were available in GenBank, except for D2-D3 expansion segments of 28S rRNA from L. apulus, L. goodeyi, L. elongatus and L. athesinus and near full-length sequences of 18S rRNA from L. crassus, L. elongates and L. dunensis. Compared with the molecular data of the above similar species from GenBank, the sequence data from 18S gene or D2-D3 expansion segments of 28S rRNA gene revealed that L. henanus is distinct from these similar species. In contrast to D2-D3 expansion region of 28S, rRNA gene sequences of other Longidorus species deposited in GenBank, L. henanus was paraphyletic to all other Longidorus. The phylogenetic tree of the 18S rRNA constructed by the Bayesian phylogenetc analysis shows that the 18S sequences of L. henanus obtained from two populations (Fenyang and Hangzhou) formed a sister clade with L. litchii Xu
& Cheng, 1992 and L. diadecturus Eveleigh & Allen, 1982. Compared with the morphological characters, L. litchii and L. diadecturus have the posterior guide ring locations, which easily differentiated them from L. henanus. However, two phylogenetic trees in this study supported the position of L. henanus as a unique species in relation to all sequenced Longidorus species. The present study demonstrates that the successful identification of L. henanus can be performed combining morphometrical and phylogenetic analyses.
ACKNOWLEDGEMENT
The research was supported by National Natural Science Foundation of the People's Republic of China (No.30870274). The authors thank Dr Sergei A. Subbotin, Centre of Parasitology, A. N. Severtsov Institute of Ecology and Evolution, Russia, for reviewing the manuscript.
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Резюме. Обнаружены две популяции Longidorus henanus Xu & Cheng, 1992 и выявлены все 4 личиночные стадии. Приводится описание и результаты измерений. Обсуждается морфологическая изменчивость и внутривидовая вариабельность рибосомальных последовательностей у L. henanus. Политомический ключ, предложенный Chen et al. (1997), для L. henanus изменен на A34, B23, C34, D2, E3, F(2)3, G(1)23, H12, I1. Филогенетический анализ последовательностей 18S rRNA и D2-D3 участка 28S rRNA показывают, что L. henanus представляет собой валидный вид, обособленный от других видов Longidorus.
