On the species status of the root-knot nematode Meloidogyne mayaguensis Rammah & Hirschmann, 1988

Abstract Holo- and paratypes of the root-knot nematodes Meloidogyne mayaguensis Rammah & Hirschmann, 1988 and Meloidogyne enterolobii Yang & Eisenback, 1983 were morphometrically and morphologically compared. All observed female, male and second-stage juvenile morphometrical and morphological characters are identical for the two studied species. Additionally, contradictions between the original species descriptions were unravelled. The present study of holo- and paratypes confirms the taxonomical status of Meloidogyne mayaguensis as a junior synonym for Meloidogyne enterolobii.


Introduction
In 1983 Yang and Eisenback described the root-knot nematode Meloidogyne enterolobii from roots of pacara earpod trees (Enterolobium contortisiliquum (Vell.) Morong), on Hainan Island in China. The authors reported severe damage on these pacara earpod trees. In 1988 Rammah and Hirschmann described the root-knot nematode M. maya-guensis from eggplant (Solanum melongena L.) roots, from Puerto Rico. Meloidogyne mayaguensis was described by the authors as: "superficially resembles M. enterolobii", and reported at the same time "several distinct morphologically features and a unique malate dehydrogenase pattern (N3c)".
It was Fargette and Braaksma (1990) and Fargette et al. (1996) who reported for the first time on the resistance-breaking behaviour of M. mayaguensis in Africa and concluded that it is present in both continents of Africa and America. The authors reported (1996) on M. enterolobii: "M. enterolobii from China has been described as having the same esterase phenotype as M. mayaguensis. However it is not known whether their DNA are closely related". In 2000 Carneiro et al. published esterase and malate dehydrogenase patterns for a Brazilian population of M. mayaguensis, and detected a different (N1a) malate dehydrogenase pattern. Additionally Blok et al. (2002) published mtDNA results from different M. mayaguensis populations, including type material from Puerto Rico.
In their comprehensive studies on the characterisation of Meloidogyne species from China, with isozymes and mtDNA, Meng et al. (2004) and Xu et al. (2004) included two M. enterolobii populations from Hainan Island, isolated from the fruit tree Guava (Psidium guajava L.). They proved for the first time that M. enterolobii esterase (VS1-S1) and malate dehydrogenase (N1a) patterns and mtDNA results are identical to reported M. mayaguensis data, and concluded carefully: "the mtDNA sequence evidence presented here, suggests that M. mayaguensis could be conspecific with M. enterolobii".
In 2005-2006 we compared the available holo-and paratypes of M. enterolobii and M. mayaguensis. Meanwhile our Chinese co-authors collected live M. enterolobii material on Hainan Island at the type locality from the type host and we kindly received live M. mayaguensis type material from Dr. V. Blok (originating from Dr. M. Fargette). The preliminary isozyme and morphological results were presented by the first author during a Pest Risk Analysis meeting on M. enterolobii at EPPO in Paris (Anonymous, 2008). Additionally this type material of both species was compared at DNA level to Meloidogyne sp. from Switzerland and we identified the Swiss population as M. enterolobii (Kiewnick et al. 2008).
Although taxonomical not strictly necessary, we present herein a morphological and morphometrical comparison between the holo-and paratype slides of M. mayaguensis and M. enterolobii. Additionally we discuss anomalies between the descriptions of M. mayaguensis and M. enterolobii.

Material and methods
Holo-and paratype slides (Table 1) originating from USDA Nematode Collection (USDANC), Beltsville, USA were kindly provided by Dr. Z. Handoo. The type slides are in good condition and includes female holotypes, male allotypes, perineal patterns and second-stage juvenile paratypes. These slides were observed by compound light microscopy (Olympus BH-2 and Zeiss Axio Imager), including Differential Interference Contrast and photographed by Leica DMC-50 digital camera. For the overall morphological and morphometrical comparison between the types we focussed on the most differential and supplementary Meloidogyne characters, as described by Jepson (1987) and as previously applied by Karssen (2002). Live type material of both species was propagated and maintained on tomato at the greenhouse of the PPS the Netherlands. This material was studied morphologically (females, males and secondstage juveniles) and used for isozyme electrophoresis (Mdh; EC 1.1.1.37 and Est; EC 3.1.1.1). For details on the preparation of slides and applied electrophoresis method we respectively refer to Karssen (1996) and Karssen et al. (1995).

Results and discussion
See Figure 1 and 2 for LM photographs of female and second-stage juvenile morphological characteristics.
See Table 2-5 for respectively female, male and second-stage juvenile morphological and morphometrical observations.

Females
The important morphological characters, like female stylet knob and perineal pattern shape do not differ between the species, as can already be observed by comparing the original illustrations between M. mayaguensis and M. enterolobii (see original descriptions respectively Fig. 2 A-D & Fig. 3 A-D). This perineal pattern type is not species specific within the genus Meloidogyne and can best be marked as typical for many species within the M. incognita-group, including the observed variation within the dorsal part. Additionally we observed a relatively large tail remnant area, free of any striae, just above the covered anus ( Fig. 1 A-D). Also the observed stylet knob position variation, slightly sloping backward to set off from the shaft, is a common Meloidogyne feature. Strangely this variation is also clearly visible in the SEM photographs of excised female stylets of M. mayaguensis (see original description, Fig. 3 A-C), but not described. With the light microscope one can observe a weak longitudinal indention, for both species, in the female stylet knobs at the anterior side. The reported differences "not divided so conspicuously as those of M. enterolobii" as mentioned in the M. mayaguensis descrip- tion (see diagnosis original description), was not confirmed by our observations. Also the described position of one of the M. mayaguensis stylet knobs "the dorsal knob is slightly sloping posteriad in lateral view" was not observed by us.

Males
The male head shape for M. mayaguensis is described as "not set off", while a slightly set off head region was observed as described for M. enterolobii. Comparing the original SEM pictures of the head for M. mayaguensis and M. enterolobii (see original descrip- tions respectively Fig. 6 A-D & 5 A,B) shows clearly not any differences in head morphology. Also the male stylet knobs have been SEM studied for the original descriptions (Fig. 3 E, F & Fig. 6 B) of both species. Large oval to rounded shaped knobs, slightly sloping backwards are clearly visible. This was also observed by LM for both species, however described as "rounded and set off" for M. enterolobii and "set off from the shaft, rounded, sloping backward" for M. mayaguensis. The later description of the knobs is rather odd, i.e. set off and sloping backward at the same time! The same results were described and observed for the second-stage juvenile knobs for both species.

Second-stage juveniles
The second-stage juvenile stylet knob size is described as small for M. mayaguensis and large for M. enterolobii. We indeed observed a larger size variation for M. enterolobii stylet knob width (2.5 -4.0 µm) compared to M. mayaguensis (2.2 -2.9 µm). However when observing live second-stage juveniles, the same large stylet knob width variation was observed for both species.
As for the males, the published SEM second-stage juvenile head shape is absolute identical for M. mayaguensis and M. enterolobii (see original descriptions respectively Fig.  7 A-D & Fig. 8 A, B). The tail is distinctly tapering and in the posterior tail (roughly the hyaline tail part) nearly straight and running parallel for both second-stage juvenile paratypes. Also, for both species the hyaline tail part is described as "distinctly set off" or "clearly defined". We observed for both species however not a clearly anterior delimitated hyaline tail part, in fact the body content runs deep into the hyaline tail part (Fig 2 A, B), as comparable to M. hapla (Karssen, 2002). The second-stage juvenile drawings for both species descriptions (Fig. 4 E, F & Fig. 7 E-F) show a clearly delimitated anterior hyaline tail part, while the original photographs (Fig. 5 F, G & Fig. 9 B) do not show this at all. The fact that both descriptions did not include the hyaline tail measurements (a standard procedure), suggest strongly that the hyaline tail part is not clearly defined. Also in live second-stage juveniles we did not observe a clearly defined hyaline tail part (Table 2).

Morphometrics
The morphometrical characters between the types of M. mayaguensis and M. enterolobii (Table 3-5), are comparable for the described and observed data, i.e. all mean data are the same or at least within the calculated range. Body length and body width data are generally slightly smaller when comparing observed to described data, this is a well known effect due to a slight shrinking of the nematode body within permanent slides. For M. enterolobii males we noticed however an unusual difference in greatest body width between the described 42.3 µm (37-48 µm) and observed 32.0 µm (24-39) µm data. The differences can not only be explained due to a shrinking effect, particularly as the observed greatest body width data agrees with the observed data for M. mayaguensis. Also for the M. enterolobii female holotype unexplainable differences were noticed between described and observed data for the DGO (3.7 µm versus 4.8 µm) and stylet length (13.4 µm versus 14.7 µm). The described and discussed M. mayaguensis differences (see diagnosis original description) within the female perineal pattern for the interphasmidial distance, vulval slit length and vulva-anus distance is not confirmed by our observations. All these measurements are within the observed range. Perineal pattern measurements are generally highly variable and a logical reason for Jepson (1987) not to list this type of data when discussing differential characters for the genus Meloidogyne.

Reproduction and cytogenetics
The two species descriptions report also on the mode of reproduction and number of chromosomes, both reproduce by mitotic parthenogenesis (= apomixes) and have a somatic chromosome number of 2n = 44-45 for M. mayaguensis and 2n = 44-46 for M. enterolobii. In conclusion, both species have the same mode of reproduction and somatic chromosome number.

Host plants
Additionally, both species descriptions report in their introduction part some hosts, i.e. they both previously applied the North Carolina differential host test (Hartman and Sasser, 1985). Both species showed the same positive host response for tobacco, pepper, watermelon and tomato and no host response on peanut. Beside this, M. mayaguensis did not infest cotton, while M. enterolobii moderately infested cotton. As the details of the previously applied host tests have not been described in the material and method part of the species descriptions, we can not explain the reported host response differences on cotton for M. mayaguensis and M. enterolobii. Interesting is the M. mayaguensis study by Brito et al. (2004) with four isolates from Florida (USA). All four isolates, maintained on tomato, reproduced also on cotton, tobacco, pepper and watermelon but not on peanut, i.e. identical to the published results for M. enterolobii.

Isozymes
The observed esterase (VS1-S1 type) and malate dehydrogenase (N1a type) isozyme patters are identical for both species and agrees with previous results (Carneiro et al. 2000;Xu et al. 2004).

Conclusion
In conclusion, the holo-and paratype material of Meloidogyne mayaguensis and M. enterolobii is morphological and morphometrical identical and it confirms the taxonomical status of M. mayaguensis as a junior synonym for M. enterolobii.