Differentiation of the Xiphinema americanum-group nematodes X. brevicollum, X. incognitum, X. diffusum and X. oxycaudatum in Taiwan by morphometrics and nuclear ribosomal DNA sequences

Abstract

Morphometrics and molecular variability of the X. americanum-group collected in Taiwan were studied. Morphometric data, derived from the females and their developing juveniles, revealed that the 27 populations comprised four taxa: X. brevicollum, X. incognitum, X. diffusum and X. oxycaudatum, the last species being found to have only three juvenile stages. Further identifications were conducted by analysing the nucleotide sequences of the first internal transcribed spacer (ITS-1), 5.8S gene and second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA). Pairwise comparisons revealed the sequence differences among these taxa ranging from 3.5 to 21.8% for the ITS-1, 2.9 to 28.6% for the ITS-2 and 0 to 3.1% for the 5.8S. Little or no intraspecific variation was detected in the four species for which multiple populations from different geographical regions or hosts were sequenced. Although X. incognitum is morphometrically closer to X. diffusum than it is to X. brevicollum in mean body length, total stylet length, and percentage position of vulva, there is a higher degree of genetic similarity between X. incognitum and X. brevicollum.In 1971, Xiphinema americanum Cobb, 1913 was first reported in Taiwan (Tsay, 1971), although the morphometric data were not recorded. Xiphinema americanum is the type species of the genus Xiphinema Cobb, 1913 but its brief original description, which was based on only a few specimens, has caused a long debate about the boundary of intraspecific or interspecific variation between X. americanum sensu stricto and a host of morphologically related species (Lima, 1965;Tarjan, 1969), all of which were ascribed to the collective X. americanum-group by Tarjan (1969). Tarjan (1969) considered that the morphometrics of populations of this species collected from different geographic locations clearly overlapped and to separate and define the different species seemed impossible. Subsequently, Lamberti and BleveZacheo (1979) redescribed the species of the X. americanum-group and proposed an additional 15 new species.

Molecular and morphological characterisation of species within the Xiphinema americanum-group (Dorylaimida: Longidoridae) from the central valley of Chile

“…(Hugall et al, 1999) and Heterodera zeae . At the same time, ITS diversity was much higher than the values considered as criteria for differentiating other species of the X. americanum-group (Chen et al, 2005) and other nematode groups, such as the Heterodera avenae complex, where a threshold value close to 1.6% has been accepted (Subbotin et al, 2003). Altogether, these data are (1.71-2.14) a 5 2 .4 ± 3.8 4 8 .5 ± 1.9 5 5 .4 ± 2.8 5 2 .3 ± 4.9 4 6 .3 ± 3.2 (46.9-58.7) (45.7-52.7) (49.6-63.7) (43.9-60.7) (40.9-54.1) b…”

“…These primers bind to the 3 portion of the 18S small ribosomal subunit (forward primer, named ITS1-S: 5 -TTGATTACGTCCCTGCCCTTT-3 ) and 5 end of the 28S subunit region (reverse primer, named P28S: 5 -TTTCACTCGCCGTTACTAAGG-3 ) (Vrain et al, 1992;Chen et al, 2005). The PCR reaction was done in a solution of 20 μl containing 1.5 mM MgCl 2 , dNTPs (0.1 mM each), 1 μl of each primer (10 pM), 0.2 μl of Taq DNA polymerase, 2 μl of 10× buffer (composed of 500 mM KCl plus 100 mM Tris-HCl adjusted to pH 8.3), 6 μl of DNA solution and 8.4 μl of sterile distilled water.…”

“…), with 2.5% in ITS1 and 4.7% in ITS2. There are almost no differences between the northern populations and X. oxycaudatum (Chen et al, 2005). When the northern populations were aligned with X. floridae (Gozel et al, 2006), the differences exceeded 13% in both regions (ITS1 and ITS2).…”

“…Species identification is very challenging and often unreliable by traditional methods (Luc et al, 1998;Chen et al, 2005). Although several keys have been published (Lamberti & Carone, 1991;Lamberti et al, 2000Lamberti et al, , 2004, ambiguities still persist due to poorly defined species boundaries and overlapping morphometrics (Luc & Baujard, 2001).…”

“…Despite these difficulties, there has been a strong interest in the study and classification of species in this group. Research performed in Brazil (Oliveira et al, 2004), Taiwan (Chen et al, 2005), the USA (Gozel et al, 2006) and Serbia (Barsi & De Luca, 2008) has demonstrated significant progress in this field. In these studies, molecular technologies have been used in combination with traditional methods.…”