RESEARCH/INVESTIGACIÓN
CANONICAL DISCRIMINANT ANALYSIS OF
ROTYLENCHULUS RENIFORMIS IN ALABAMA
Seloame T. Nyaku1,2*, Ramesh V. Kantety2, Kathy S. Lawrence3, Edzard van Santen4 and Govind C. Sharma2
1Department of Crop Science, College of Agriculture and Consumer Science, University of Ghana, P. O. Box LG 44
Legon-Accra, Ghana; 2Department of Natural Resources and Environmental Sciences, Alabama A&M University, 
Normal, AL 35762; 3Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849; 
4Department of Agronomy and Soils, Auburn University, Auburn, AL 36849; *Corresponding Author: seloame.nyaku@
aamu.edu or stnyaku@ug.edu.gh
ABSTRACT
Nyaku, S. T, R. V. Kantety, K. S. Lawrence, E. van Santen and G. C. Sharma. 2013. Canonical discriminant analysis of 
Rotylenchulus reniformis in Alabama. Nematropica 43:171-181.
The reniform nematode, Rotylenchulus reniformis, infests over 300 plant species worldwide and over the last two 
decades it has emerged as a major cotton pest in the southeastern United States. Nine locations in Alabama and one 
location in Mississippi were selected for study of R. reniformis.  Thirteen morphometric measurements were made on 20 
male and 20 female nematodes from each population. The sex and location interaction was significant (P < 0.005) for 
all traits except total body length (P = 0.29) and the derived ratio trait a (body length / maximum body width, P = 0.06). 
Canonical discriminant analysis effectively separated the 10 sampling locations into three distinct groups; among them, 
Group 1 and 3 were distinct with an intermediate group (Group 2) differentiating in the middle. Furthermore, both female 
and male R. reniformis based on the morphometrics measured here adhered to this metrics-based grouping. Belle Mina 
(Limestone County, AL), Huxford (Escambia County, AL), and Mississippi State University, MSU (Oktibbeha County, 
MS) locations were separated from the remaining seven locations based on the large positive CAN1 centroid means. 
Eight out of 13 traits had high phenotypic correlations (r > 0.80) with CAN 1 for both sexes. Anal width and length of 
the hyaline portion of the tail measurement accounted for a significant amount (r > 80%) of the variation in total and 
sex-based canonical structure. Occurrence of the three non-overlapping morphometric groups in cotton-growing fields 
in close proximity (250 mile radius) suggests a greater biological variation in this species than expected. Cotton cultivars 
with differential resistance and soil types are among the major factors to be tested for further delineating the causes of 
morphometric variation in R. reniformis. 
Key words: Reniform Nematode, Morphological Variation, Canonical Discriminant Analysis, Mahalonobis distance
RESUMEN
Nyaku, S. T, R. V. Kantety, K. S. Lawrence, E. van Santen and G. C. Sharma. 2013. Análisis discriminante 
canónico de Rotylenchulus reniformis en Alabama.  Nematropica 43:171-181.
El nematodo reniforme, Rotylenchulus reniformis, parasita más de 300 especies de plantas en todo el mundo 
y durante las dos últimas décadas se ha convertido en una de las principales plagas del algodón en el sureste de 
Estados Unidos. Para este estudio con R. reniformis fueron seleccionadas nueve localidades en Alabama y una 
en Mississippi. Trece medidas morfométricas fueron tomadas en 20 machos y 20 hembras de cada población. 
La interacción con el sexo y la ubicación fue significativa (P < 0.005) para todos los caracteres, excepto para 
la longitud total del cuerpo (P = 0.29) y su derivado índice a (longitud total del cuerpo/ancho máximo del 
cuerpo, P = 0.06). El análisis discriminante canónico separó efectivamente los 10 lugares de muestreo en tres 
grupos; el Grupo 1 y 3 fueron diferentes, con un grupo intermedio (Grupo 2). Además, tanto hembras como 
machos de R. reniformis se adhirieron a este agrupamiento basado en mediciones. Las localidades Belle Mina 
(Limestone County, AL), Huxford (Escambia County, AL) y Mississippi State University, MSU (Oktibbeha 
County, MS) fueron separadas de las 7 localidades restantes en base a los centroides en el eje positivo CAN1. 
Ocho de los 13 caracteres tuvieron alta correlación fenotípica (r > 0.80) con CAN 1 en ambos sexos. El ancho 
del ano y la longitud de la porción hialina de la cola representaron una cantidad significativa (r > 80%) de la 
variación en la estructura canónica total y basada en el sexo. La existencia de tres grupos morfométricos que 
no se superponen en campos de algodón muy próximos (250 millas de radio) sugiere una variación biológica 
mayor a la esperada en esta especie. Los cultivares de algodón con resistencia diferencial y los tipos de suelo 
están entre los principales factores para ser analizados a fin de evaluar las causas de la variación morfométrica 
de R. reniformis. 171
Palabras clave: nematodo reniforme, variación morfológica, análisis discriminante canónico, distancia de 
Mahalonobis. 
172 NEMATROPICA   Vol. 43, No. 2, 2013
INTRODUCTION  Nematicides are being eliminated due to health 
and environmental issues (Noling and Becker, 1994), 
The reniform nematode, Rotylenchulus reniformis despite positive impacts of nematicides on cotton 
Linford and Oliveira 1940, is distributed widely in yields. This concern therefore makes host resistance the 
tropical and subtropical regions of the world, and most efficient and economical method of RN control 
is found throughout the southern U.S. (Heald and (Robinson et al., 1999; Robinson et al., 2007). There 
Robinson, 1990; Kinloch and Sprenkel, 1994). This have been increased efforts in recent years to develop 
devastating pest has a wide host range that includes commercial upland cotton cultivars with resistance to 
cotton and a broad range of vegetable and field crops RN (Cook and Robinson, 2005; Robinson et al., 2007; 
(Robinson et al., 1997). This nematode is considered an Weaver et al., 2007). Plant breeders introgress this 
important pest in upland cotton (Gossypium hirsutum resistance from the wild relatives into the cultivated 
L.), especially in the southeastern United States  (Davis species, a time-consuming process. In upland cotton, 
et al., 2003; Koenning et al., 2004). Cotton yields Gossypium hirsutum (2n=52), there has been some 
are greatly affected by R. reniformis (RN) damage in success in this regard, through introgression of RN 
Alabama (Gazaway et al., 2001), Louisiana (Overstreet, resistance from G. longicalyx (Robinson et al., 2007, 
1999), and Mississippi (Lawrence and McLean, 1999) Dighe et al., 2009), and from G. arboreum and a G. 
and nine other states (Georgia, Florida, Texas, South hirsutum/G. aridum bridging line (Sacks and Robinson, 
Carolina, North Carolina, Arkansas) (Heald and 2009). Due to inefficiencies in chromosome paring, 
Robinson, 1990), Missouri (Wrather et al., 1992), interspecific crossing among related plant species is 
Tennessee (Newman, 2005), and Virginia (Eisenback always not successful, and therefore limits the transfer 
and Hopkins, 2004). This nematode was first discovered of agronomically important traits among species 
in cotton fields in east-central region of Alabama in (Beasly, 1940, 1942). Novel and specific strategies are 
1958 (Minton and Hopper, 1959), and was identified thus needed for controlling RN.
as a serious pest of cotton in 1986 (Gazaway and Our objectives were to i) Identify and measure the 
McLean, 2003).  The RN is spread from farm to farm variation in morphological attributes of female and 
through soil particles on farm implements and vehicles, male RN, ii) Locate the most useful morphometric 
and can survive for more than two years in stored soil characters in discriminating among the populations 
(Lawrence et al., 2005). Nematode populations in through Canonical Discriminate Analysis. 
Alabama are well established on croplands because  
over 90% of cotton is monocultured (Gazaway and  
McLean, 2003).  Cotton yield loss due to RN usually MATERIALS AND METHODS
range from 10% to 25% but may be as high as 50% in 
heavily infested and drought-stressed fields (Gazaway Soil Sample Collection, Extraction and Establishment 
et al., 2001; Kirkpatrick and Robbins 1998). of RN Populations
While increasing cotton yield is of importance   
(Ribera and Landivar, 1999), all upland cotton cultivars Samples of RN-infested soil were obtained from nine 
presently being marketed are susceptible to RN (Weaver cotton farms located in four counties in Alabama and 
et al., 2007). one location in Mississippi (Table 1). Each sample was 
Traditional management of plant-parasitic thoroughly mixed and a 150 cm3 subsample was used 
nematodes has been mainly through crop rotation, for the extraction, identification, and quantification of 
nematicides, and host resistance. Common resistance the nematodes. The infested soil was placed in a bucket 
strategies can be applied to minimize populations of running water until the soil was covered by at least 
of RN if these pests are found to be homogeneously two times its volume. The solution was then mixed 
spread in a field. However, if these populations are vigorously until the soil was sufficiently dispersed and 
heterogeneous, then divergent host-plant resistance then allowed to settle for 3 min. The liquid supernatant 
strategies for RN management may be required; was then poured through an 841 µm sieve nested onto 
hence more specific studies will need to be developed. a 44 µm sieve. The 44 µm sieve with was washed 
Nematode management in cotton is through non-host thoroughly with water until as much clay and other fine 
crop rotations and the use of chemical nematicides, particles were washed out of the sieve. The remaining 
among these are abamectin (Avicta), oxamyl (Vydate), sample with the nematodes was then washed into a 250 
thiodicarb (Aeris), and 1,3dichloropropene (Telone). ml beaker and allowed to settle for 5 min, afterwards the 
Other nematicides are applied as seed treatments such solution was transferred into a 50 ml centrifuge tube. 
as abamectin and thiodicarb and these are effective Centrifugation was carried out at 1,000 x g for 3 min in 
in improving yield increases in cotton (Lawrence and a Marathon 21 k benchtop centrifuge (Fisher Scientific, 
Lawrence, 2007).  New investigations into relationships Suwanee, GA). The supernatant was discarded and 
existing between environmental variables (e.g., water if necessary, successive samples centrifuged until a 
and temperature) and their response to nematicides final pellet obtained from the collective population of 
for yield improvements are underway (Wheeler et al., nematodes.  Two mL of OptiprepTM (Axis-Shield PoS 
2013). AS, Oslo, Norway) solution was gently added to a 15 mL 
Reniform Nematode Morphometric Variation in Alabama: Nyaku et al. 173
Table 1. GIS information for one Mississippi (MS) site and nine Alabama (AL) sites that were sampled for the 
morphometric analysis of female and male reniform nematodes on cotton farms. Locations are listed in descending 
order of the CAN 1 centroid mean for females.
Group Abbreviation Location County / State Latitude Longitude Infested since*
I M MSU Oktibbeha, MS 88.78 W 33.48 N Early 1980s
I B Belle Mina Limestone, AL 86.89 W 34.66 N Early 1990s
I X Huxford Escambia, AL 87.46 W 31.22 N Early 1980s
II S Shaw Limestone, AL 86.94 W 34.64 N Early 1990s
II R Murphy Limestone, AL 86.75 W 34.59 N Late 1980s
II L Lamons Lawrence, AL 87.12 W 34.63 N Early 1980s
III H Hargrave Limestone, AL 86.85 W 34.62 N Late 1980s
III A Hamilton Lawrence, AL 87.18 W 34.61 N Early 1980s
III T Thornton Lawrence, AL 87.37 W 34.73 N Early 1980s
III W Whitehead Fayette, AL 87.73 W 33.84 N Late 1980s
*Personal communication with farmers
Table 2. Phenotypic correlation between original response variables and canonical variates (between canonical 
structure), Eigenvalues, and percent total variance accounted for by the first two canonical variates for female and 
male reniform nematodes collected from nine locations in Alabama and one location in Mississippi. The discriminant 
analysis was performed separately for females and males.
Females Males
Morphometric and derived ratio traits CAN1* CAN2 CAN1* CAN2
Anal Width (AW) 0.93 0.35 0.88 0.37
Length of Hyaline Portion of Tail (TL) 0.92 -0.02 0.99 -0.07
Body Length (BL) 0.91 0.04 0.86 -0.03
Position of Vulva in females / spicule length in males 0.89 0.29 0.89 -0.02
Position of Excretory Pore (EP) -0.87 0.38 -0.67 0.28
c’ =  (TL/AW) -0.86 -0.38 0.92 -0.30
Maximum Body Width (MW) 0.85 0.10 0.34 0.47
Position of Dorsal Esophageal Gland Orifice (DEGO) 0.80 -0.55 0.90 0.39
Stylet Length (SL) 0.77 -0.58 0.92 0.30
c  = (BL/TL) -0.67 0.09 -0.99 0.03
Esophageal Length (EL) 0.47 -0.74 0.18 0.55
a  = (BL/MW) -0.28 0.04 0.30 -0.41
b  = (BL/EL) 0.10 0.87 0.32 -0.42
Eigenvalue 4.98 1.45 7.26 0.68
% of total variance 0.66 0.19 0.82 0.08
*Traits are listed in descending order of absolute values for the between CAN 1 structure in females. Absolute 
correlations ≥ 0.92, 0.85, 0.72, and 0.55 are significant at P ≤ 0.0001, 0.001, 0.01, and 0.05, respectively.
174 NEMATROPICA   Vol. 43, No. 2, 2013
conical centrifuge tube, and the nematode suspension also calculated (Dasgupta et al., 1968).
added to the Optiprep solution and centrifuged at 1,000 Statistical Analysis
x g for 1 min. Immediately after centrifugation, half the 
supernatant without any nematodes was discarded. The  A two-step approach was used to investigate the 
solution layer with RN just above the OptiprepTM – pattern of morphometric variability among the locations 
water interface was collected into a new 15 mL conical sampled. First, canonical discriminant analysis (CDA) 
centrifuge tube. The efficiency of the OptiprepTM as implemented in SAS® PROC CANDISC, SAS 8.0 
method for the extraction of nematodes has recently (SAS, Cary, NC) was used to collectively observe all 
been determined to be 85% or higher using this method morphometric traits with sampling location as the class 
compared to the sucrose gradient method (Deng et al., variable. The P-values for pairwise differences among 
2008). The remaining infested soil samples were place locations were adjusted for multiple comparisons by 
in the greenhouse and RN populations were maintained the Bonferroni method as implemented in SAS® PROC 
on cotton cultivar ‘Delta and Pineland 425 BG/RR’ MULTTEST. Individual morphometric traits were then 
(DPL 425) for RN multiplication. evaluated by ANOVA using location, sex, and location 
x sex as fixed effects. Residual variation was modeled 
Morphometric Measurements with the repeated statement in SAS® PROC MIXED 
using the group = sex option to allow for heterogeneity 
Extracted RN from soil samples were placed on of variances among sexes. If the likelihood ratio test 
slides which were briefly passed over a flame prior was significant, then separate residual variances were 
to measurements. Morphometric measurements were used for females and males.
determined on 20 individual vermiform female (F) 
and male (M) nematodes from each field sample using RESULTS
an IMT-2 microscope (Olympus Optical Co. Ltd, 
Japan). Nine morphometric variables were measured Canonical Discriminant Analysis (CDA)
separately for each sex: body length (BL), stylet length 
(SL), position of vulva (PV), spicule length (PS), The first two canonical variates (CAN 1 and 2) 
length of hyaline portion of tail (TL), position of dorsal accounted for 85% and 90% of the multi-variance among 
esophageal gland orifice (DEGO), position of excretory the nine measured and four derived traits for female and 
pore (EP), maximum width (MW), esophageal length male RN, respectively (Table 2). The contributions of 
(EL), and anal width (AW). In addition, de Man’s the first canonical variates (0.66 and 0.82) were 4 and 
formula ratios a = body length / maximum body width, 10-fold higher than the second variates (0.19 and 0.08) 
b = body length / esophageal length, c = body length / in females and males respectively, resulting in a strong 
tail length, and, c’ = tail length / anal body width were differentiation among locations along the first axis. 
The magnitude of the phenotypic correlation 
between class means for original response 
variables and canonical variate means (between 
canonical structure in SAS parlance) gives an 
indication which original variables drive the 
separation among classes along a particular axis 
(Table 2). For females, CAN 1 had the highest 
correlation with anal width (r = 0.93), followed 
by length of the hyaline portion of tail (r = 0.92) 
and total body length (r = 0.91). Canonical variate 
2 had the greatest correlation with the ratio trait 
b (r = 0.87), followed by esophageal length (r = 
-0.74), and stylet length (r = -0.58). For males, 
CAN 1 had a near perfect negative correlation 
with length of hyaline portion of tail (r = 0.99) 
and the derived ratio trait c (r = -0.99), followed 
by stylet length (r = 0.92) and the derived ratio 
trait c’ (r = 0.92). Canonical variate 2 had the 
greatest correlation with esophageal length (r = 
0.55), followed by maximum body length (r = 
Fig. 1. Centroid means from canonical discriminant analysis 0.47).
of female and male reniform nematodes collected from nine Canonical discriminant analysis visually 
sites in Alabama and one site in Mississippi. The axes in separated the 10 locations into three groups 
each panel were scaled to reflect the relative contributions to for both females and males (Fig. 1). The three 
the multi-variance among nine morphometric traits and four locations Belle Mina (Limestone County, AL), 
derived ratio traits. Huxford (Escambia County, AL), and MSU 
Reniform Nematode Morphometric Variation in Alabama: Nyaku et al. 175
Table 3. Mahalonobis pairwise distances (D) among locations (above the diagonal) and Bonferroni adjusted P-values (below the diagonal) for female reniform 
nematodes. The shaded rectangles indicate the distances and P-values within groups (see Fig. 1 and Table 1). Locations are listed in descending order of the CAN 
1 centroid mean for females.
Mahalonobis pairwise distances (D)
Group Location MSU Belle Mina Huxford Shaw Murphy Lamons Hargrave Hamilton Thornton Whitehead
I MSU 3.5 5.0 4.1 4.5 5.1 6.3 6.4 6.8 6.8
I Belle Mina < 0.0001 3.2 2.5 3.1 3.4 4.6 4.8 4.8 5.1
I Huxford < 0.0001 < 0.0001 4.4 4.6 4.6 5.1 5.3 5.3 5.5
II Shaw < 0.0001 0.0002 < 0.0001 1.3 2.2 3.7 3.7 4.1 4.1
II Murphy < 0.0001 < 0.0001 < 0.0001 1.0000 1.6 2.8 2.8 3.3 3.3
II Lamons < 0.0001 < 0.0001 < 0.0001 0.0060 1.0000 1.7 1.9 2.2 2.3
III Hargrave < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 1.0000 1.1 1.1 1.6
III Hamilton < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.2643 1.0000 1.4 1.6
III Thornton < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0068 1.0000 1.0000 1.7
III Whitehead < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0044 1.0000 1.0000 1.0000  
Table 4. Mahalonobis pairwise distances (D) among locations (above the diagonal) and Bonferroni adjusted P-values (below the diagonal) for male reniform 
nematodes. The shaded rectangles indicate the distances and P-values within groups (see Fig. 1 and Table 1). Locations are listed in descending order of the CAN1 
centroid mean for females.
Mahalonobis pairwise distances (D)
Group Location MSU Belle Mina Huxford Shaw Murphy Lamons Hargrave Hamilton Thornton Whitehead
I MSU 2.1 1.2 4.9 6.3 5.4 6.3 6.2 6.2 6.6
I Belle Mina 0.0461 2.0 4.4 6.0 5.2 6.0 5.8 6.0 6.3
I Huxford 1.0000 0.1527 4.7 6.2 5.2 6.3 6.1 6.2 6.6
II Shaw < 0.0001 < 0.0001 < 0.0001 2.8 2.1 3.0 2.9 3.0 3.2
II Murphy < 0.0001 < 0.0001 < 0.0001 < 0.0001 1.7 2.7 2.7 2.6 2.6
II Lamons < 0.0001 < 0.0001 < 0.0001 0.0169 1.0000 2.3 2.1 2.1 2.3
III Hargrave < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0013 1.5 0.6 1.4
III Hamilton < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0339 1.0000 1.5 1.5
III Thornton < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0329 1.0000 1.0000 1.4
III Whitehead < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.0034 1.0000 1.0000 1.0000  
176 NEMATROPICA   Vol. 43, No. 2, 2013
Table 5. Location ranges for female and male reniform nematodes for the morphometric  and derived ratio traits with phenotypic correlation (between structure in Table 2) ≤  |0.85| 
with the first canonical variate for females.
Group I I I II II II III III III III  
Location MSU Belle Mina Huxford Shaw Murphy Lamons Hargrave Hamilton Thornton Whitehead SEDwithin SEDbetween
Females 14.3-19.0 14.3-21.4 11.9-19.0 14.3-16.7 14.3-16.7 14.3-16.7 14.3-16.7 11.9-16.7 14.3-16.7 11.9-16.7 0.5
 MW 0.30
Males 14.3-16.7 11.9-14.3 11.9-16.7 14.3-16.7 14.3-16.7 11.9-16.7 14.3-14.3 11.9-14.3 14.3-16.7 11.9-14.3 0.4
Females 30.9 -47.6 30.9-38.1 23.8-40.5 28.6-45.2 26.2-47.6 28.6-38.1 23.8-33.3 28.6-35.7 26.2-35.7 26.2-35.7 1.17
DEGO 1.12
Males 28.6-47.6 28.6-40.5 28.6-50 28.6-38.1 26.2-38.1 26.2-38.1 23.8-30.9 26.2-35.7 26.2-31.0 26.2-33.3 1.14
Females 16.7-23.8 14.3-19 11.9-23.8 14.3-19 14.3-19 14.3-19 14.3-16.7 14.3-16.7 14.3-14.3 11.9-16.7 0.53
SL 0.43
Males 11.9-16.7 11.9-16.7 11.9-21.4 11.9-14.3 11.9-16.7 11.9-16.6 11.9-11.9 11.9-14.3 11.9-11.9 11.9-11.9 0.48
Females 100.0-200.0 80.0-160.0 81.2-147.6 110.0-150.0 100.0-140.0 100.0-160.0 100.0-140.0 100-130 100.0-140.0 90.0-140.0 4.96
EL 3.07
Males 100.0-130.0 100.0-130.0 90.0-120.0 90.0-120.0 100.0-140.0 100.0-140.0 100.0-120.0 100.0-120.0 100.0-120.0 80.0-130.0 4.12
Females 20.0-28.0 17.7-30.1 15.8-32 21.0-28.7 19.8-28.7 21.0-28.0 20.4-28.0 22.4-29.4 18.6-27.3 18.6-28.6 0.81
a  0.79
Males 22.2-30.8 25.2-35.3 18.9-31.1 21.0-30.1 20.4-28.0 19.8-33.6 23.8-28.0 23.1-34.5 20.4-28.7 23.1-31.1 0.8
Females 1.1-3.7 2.5-4.2 0.6-1.2 2.3-3.3 2.7-3.4 2.5-3.6 2.3-3.8 2.4-3.5 2.4-3.5 2.6-3.8 0.12
b 0.11
Males 3.1-4.4 2.9-3.9 3.0-4.0 3.2-4.2 2.6-3.6 2.5-4 3.2-4.0 2.9-4.0 2.9-4.1 2.9-4.4 0.12
Females 8.9-16.4 8.0-15.1 9.5-17 9.8-14.4 10.7-15.4 8.9-15.3 9.7-15.6 11.0-14.7 11-14.9 10.7-17.3 0.53
c 0.45
Males 6-10.1 6.9-10.9 6.9-10.1 9.8-15.9 11.9-16.8 10.1-15.3 12.3-17.7 11.2-18.1 11.6-16.8 12.3-17.2 0.49
Females 7.1-14.3 9.5-14.3 7.1-16.6 7.1-11.9 7.14-9.52 7.1-9.5 7.1-7.1 7.1-7.1 7.1-9.5 7.1-9.5 0.43
AW 0.31
Males 7.1-11.9 7.1-11.9 7.1-11.9 7.1-11.9 7.1-9.5 7.1-9.5 7.1-9.5 7.14-7.14 7.1-7.1 7.1-7.1 0.37
Females 23.8- 42.8 28.6-47.6 24.7-44.5 26.2-35.7 23.8-35.7 23.8-35.7 23.8-33.3 23.8-30.9 26.2-30.9 21.4-30.9 1.2
TL 1.40
Males 35.7-61.9 35.7-54.7 33.3-57.1 23.8-35.7 23.8-30.9 23.8-35.7 21.4-28.6 23.8-30.9 21.4-31.0 21.4-28.6 1.3
Females 350.0- 450.0 320.0- 430.0 300.0-430.0 350.0-410.0 320.0-440.0 320.0- 400.0 300.0-400.0 310.0-410.0 310.0-420.0 310.0-390.0 8.44
BL 8.29
Males 350.0-440.0 350.0-440.0 270.0-420.0 340.0-430.0 310.0-410.0 300.0-420.0 340.0-400.0 320.0-430.0 340.0-410.0 330.0-410.0 8.37
PV Females 240.0-380.0 250.0-310.0 261.8-345.8 250.0-300.0 240.0-320.0 220.0-300.0 220.0-300.0 210.0-300.0 210.0-300.0 220.0-280.0 7.2
N/A
PS Males 14.3-21.4 14.3-21.4 14.3-23.8 11.9-19.0 14.3-21.4 11.9-19.0 14.3-19.0 14.3-16.7 14.8-19.0 14.3-19.0 5.11
Females 47.6-71.4 47.6-76.2 47.6-83.3 59.5-83.3 47.6-83.3 59.5-83.3 66.6-80.9 64.3-80.9 64.3-83.3 59.5-83.3 2.1
EP 2.0
Males 54.7-71.4 47.6-76.2 50-83.3 52.4-73.8 59.5-83.3 59.5-76.2 59.5-80.9 59.5-78.5 59.5-71.4 59.5-71.4 2.1
Females 1.3-3.0 1.4-3.3 1.3-2.9 1.6-2.5 1.6-2.2 1.6-2.5 3.3-4.7 3.3-4.3 2.8-4.3 2.5-4.3 0.11
c' 0.2
Males 3.8-7.7 3.0- 7.0 3.4-7.3 2.5-5 2.5-4 2.5-5 2.8-4.0 3.3-4.3 3.0-4.3 3.0-4.0 0.16
Reniform Nematode Morphometric Variation in Alabama: Nyaku et al. 177
(Oktibbeha County, MS) in Group I were separated highest mean value for MW (16.9 μm) was observed 
from the remaining seven populations based on large from group I from the female Belle Mina population, 
positive CAN1 values. Members of this group were their range fell within (14.3-21.4). The lowest mean 
significantly (P < 0.0001) different from the remaining value for DEGO (27.7 μm) was in group III from the 
seven locations (Tables 2 and 3). Whereas the centroid male Hargrave population, their measurements were 
means for males in this group are tightly clustered (P in the range of (23.8-30.9), while the highest mean 
> 0.05), there was a greater spread for females with value for DEGO (39.9 μm) was in group I from the 
Mahalanobis’ distances of up to 5.0 (Table 3 and 4). female MSU population, with ranges of (30.9-47.6). 
The second location group (Group II), consisting of The highest value for stylet length SL (18.9 μm) was 
Shaw (Limestone County, AL), Murphy (Limestone in group I from the MSU female population, these 
County, AL), and Lamons (Fayette County, AL), had a group had ranges of (16.7-23.8), and the lowest value 
maximum within group distance of D = 2.8 for males (11.9 μm) was observed in three male populations 
and 2.2 for females (Tables 3 and 4). The last group within group III. The female population from MSU, 
(Group III) consisting of the North Alabama locations in group I had the highest esophageal length of 150.5 
Hargrave (Limestone County), Hamilton (Lawrence μm, measurements ranged from (100.0-200.0). The 
County), Thornton (Lawrence County), and Whitehead standard error of a difference (SED) within and between 
(Fayette County) was very homogeneous with a the male and female populations were less than 1 with 
maximum distance among locations of 1.7 and a unity the exception of values for DEGO and Esophageal 
P-value following the Bonferroni adjustment to account length. Female populations from MSU could easily be 
for the increase in Type I error due to the 45 pairwise differentiated from the other populations within group I 
comparisons that were made. because of the length of their esophagus. Similarly, the 
location means for female and male RN for additional 
Analysis of Variance (ANOVA) and Individual Traits 6 morphometric traits (AW, TL, BL, PV, PS, EP) and 
the derived ratio trait c’  (Table 5) differed similarly 
The sex x location interaction was significant at P < among the locations and thus contributed to CDA and 
0.001 for most morphometric traits (data not shown), groupings.
except for total body length (P = 0.293), position of 
the excretory pore (P = 0.014), maximum body width DISCUSSION
(P = 0.005) and the derived ratio trait a = body length 
/ maximum body width (P = 0.059). Trait means, 
therefore, were presented separately for females and Our study utilized nine morphometric characters 
males. and four derived ratio traits for canonical discriminant 
The strong separation of Group III from Group analysis providing clear evidence that morphological 
I and II for females along CAN 1 can be seen in the differences exist at various locations based on genotypic 
morphometric traits that had the highest absolute or environmental variables. The CDA greatly aided in 
phenotypic correlation with CAN 1 centroid means progressively pooling linear combination of variables 
(Table 2). The class means for the first four traits – anal having the highest multiple correlations with data from 
width, length of hyaline portion of tail, body length each location. Such an approach has also been utilized 
and position of vulva – generally followed the ranking in discriminant analysis of nine species of Longidorus 
obtained by CDA, with a few rank changes involving including five new species in Arkansas (Ye and Robbins, 
adjacent classes (Table 2).  The correlation between 2004). This study is unique because it covers a finite 
the independent variable and the canonical variate can geographic area, where seven of 10 sampling sites were 
also be interpreted geometrically as the cosine between located within a 25-mile radius spanning two counties 
the two vectors (Anderson, 2003); a high correlation (Lawrence and Limestone). The remaining sites were 
thus is equivalent to an acute angle between these approximately 80 miles (Whitehead, AL), 125 miles 
vectors. As shown in Table 2 for males, six of the nine (MSU, MS) and 250 miles (Huxford AL) away. Our 
morphometric traits and two out of four derived traits findings and those of Agudelo et al. (2005) report high 
had r ≥ 0.85 underscoring the stringency of most traits variability in morphometrics within populations across 
selected for measurements in this study and affirmed the southern United States. An interesting observation 
by CDA. This value < 0.85 thus became an important in our study was the specific groups for male and female 
determinant of significance. populations which were not observed in the study by 
The location ranges for female and male populations Agudelo et al. 2005.  Morphometric variability among 
for four morphometric traits and three derived ratio RN populations has been mentioned by a number of 
traits with phenotypic correlation (between structure in authors (Dasgupta et al., 1968; Linford and Oliveira, 
Table 2) ≤ |0.85| with the first canonical variate, CAN1 1940; Nakasono, 1983; Robbins, 1994; Sivakumar 
for female populations are shown in (Table 5). The and Seshadri, 1971; Soares et al., 2003; Van der Berg, 
lowest mean value for MW (13.4 μm) was observed 1978). Larger body lengths of RN than those observed 
in group III from the male Hamilton population, their in our study are reported from Hawaiian and Japanese 
measurements fell in the range of (11.9-14.3), and the populations (Nakasono, 1983). Anal width and length 
178 NEMATROPICA   Vol. 43, No. 2, 2013
of the hyaline portion of the tail measurement also 1, followed by length of hyaline portion of tail (0.92) 
accounted for a significant amount of the variation in under the between canonical structure. This shows 
total canonical structure as well as between canonical the importance of the length of the hyaline part of 
structures in both RN sexes in our study.  Agudelo et al. the tail in discriminating among the male and female 
(2005), found similar correlations.  RN populations.  Significant morphological variation 
Canonical discriminant analysis was used by Cho therefore exists for male and female RN populations 
and Robbins (1991), among 23 Xiphinema americanum across Alabama. This was also confirmed in the ANOVA 
mixed with seven additional species collected from documenting significant variation for the locations 
seven disparate states. In their study, three groups (P < 0.05) and sexes (P < 0.05). Single characters 
were generated that were closely related to geographic were insufficient in differentiating variability within 
origin of these populations. However, due to overlap populations and groups because of the high degree of 
among them, no clear distinction was observed among variability within and between populations for male 
their populations. Based on the analyses conducted in and female RN. The populations in group I which had 
this study, we grouped these ten populations into three the highest Mahalanobis distance values clustered 
distinct groups by centroid means. The first location distinctly from groups II and III. 
group (Fig 1: Group I), for female and males include Canonical discriminant analysis was therefore a 
populations from Belle Mina (Limestone County, AL), useful tool for discrimination of RN populations. This 
Huxford (Escambia County, AL), and MSU (Oktibbeha could be seen in the three major groups that were 
County, MS).  The Huxford and Belle Mina locations observed for the populations; again the important 
are believed to be some of the oldest populations in variables discriminating among these populations were 
Alabama.  These are comparable to the population identified through the first and second canonical axis. 
from MSU because, these populations are believed Our study should be considered a prelude to extensive 
to have spread laterally from the Mississippi river molecular analysis of the 18S and ITS1 rRNA regions in 
area and the MSU populations possibly spawned the the RN genome and further based on the morphometric 
Alabama region.  The second location group (Group groupings generated after CDA was performed, which 
II) for females and males, consistently supports high will potentially identify molecular signatures for 
populations of RN independent of varying soil types variation in RN groups. 
and crop rotation practices included populations from Representative populations from each of the three 
Shaw (Limestone County, AL), Murphy (Limestone groups may be tested for their pathogenicity using 
County, AL), and Lamons (Fayette County, AL). The genotypes of variable resistance to explore the presence 
third location group (Group III) made up of the North of biotypes of RN.
Alabama locations Hargrave (Limestone County), 
Hamilton (Lawrence County), Thornton (Lawrence 
County), and Whitehead (Fayette County), although ACKNOWLEDGEMENTS
dissimilar in soil morphology, has typically supported 
lower populations of RN. This work was supported by USDA-CSREES 
Subbotin et al. (1999) identified groups within Grant # 2004-38814-15160, USDA ALAX-011-706 and 
Heterodera avenae species using CDA, the first two NSF/PGRP award #0703470. The authors gratefully 
canonical variables accounted for 87% of variance. acknowledge the technical assistance provided by Dr. 
In our study, for the female populations, the first two Yonathan Tilahun.
canonical variables accounted for 85% of variance, 
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Received:                  Accepted for publication:  
            26/VI/2013                      4/IX/2013
Recibido:          Aceptado para publicación: