Notobasis

Knio, Khouzama M., White, Ian M. & Al-Zein, Mohammad S., 2007, Host-race formation in Chaetostomella cylindrica (Diptera: Tephritidae): Morphological and morphometric evidence, Journal of Natural History 41 (25 - 28), pp. 1697-1715 : 1703-1713

Notobasis -associated host race of C. cylindrica

Males. Head yellow to yellow brown, sometimes yellow green, 0.89¡ 0.02 mm wide (n 530), 1.1¡ 0.02 mm long (n 530). Antennae yellow to yellow orange, 0.44¡ 0.01 mm (n 511) long. Thorax yellow or yellow brown; usually no spots or rarely very small faint brown spots at the base of dorsocentral setae. Black spots at base of prescutellar setae, but sometimes brown or faint. Shiny black spots on scutellum. Grey black or black pattern on mesonotum. Abdomen similar to the Onopordum -associated host race.

Females. Head yellow to yellow green, 0.97¡ 0.02 mm wide (n 530), 1.23¡ 0.003 mm long (n 530). Antennae yellow, yellow orange, or sometimes orange, 0.46¡ 0.02 mm long. Thorax yellow, sometimes yellow brown or yellow green, with no spots at base of dorsocentral setae. Black or sometimes brown spots at base of prescutellar setae. Shiny black or dark brown spots on scutellum. Grey black or black pattern on mesonotum. Abdomen similar to the Onopordum -associated host race.

Puparia . Brown; 4.4¡ 0.1 mm long (n 510, range 4–4.8); 1.81¡ 0.06 mm wide (n 510, range 1.4–2.04).

Third instar larvae. Same appearance as the larvae from Onopordum but smaller; 4.35¡ 0.13 mm long (n 517; range 3.48–5.4); 1.54¡ 0.1 mm wide (n 517; range 1.1– 2.29). Cephalopharyngeal skeleton similar in shape to that of the Onopordum -associated host race, sclerotized and reddish brown, 0.82 mm long ( Figure 4A ). Posterior spiracular plate and anterior spiracles similar to those of the Onopordum -associated host race ( Figure 4B, C ), but posterior spiracles slightly smaller, 0.048–0.05 mm long (n 56).

Eggs. White, smooth, cylindrical; 1.07¡ 0.029 mm long (n 529; range 0.85–1.26); 0.255¡ 0.006 mm wide (n 529; range 0.22–0.33).

Comparative morphometry of the immatures

The size of all the immature stages of the Onopordum- associated host race was significantly greater than those of the Notobasis- associated host race. The egg length and diameter of the two host races were statistically different (t 57.4, df579, P,0.001 and t 55.97, df579, P,0.001, respectively). The mean diameter and mean length of the third instar larvae were found to be significantly different between the two races (t 55.5, df573, P,0.001 and t 55.85, df573, P 50.027, respectively). The length and width of the puparia also showed significant differences between the two races (t 54.19, df543, P,0.001 and t 52.51, df543, P 50.016, respectively).

Comparative morphology and morphometry of the adults

Adult head and wings. Seven morphometric characteristics (five wing and two head measurements) were measured for a random sample of 60 flies of each race, 30 males and 30 females. Statistical analysis using two-tailed t test for independent samples showed that the means for the seven characters studied were significantly different ( P ǩ0.05) for males and females within and between the two races, as well as between the 60 Notobasis - associated and 60 Onopordum- associated flies regardless of the gender ( Table I). The variation within species reflects sexual dimorphism in the two races as males were usually smaller than females. Given the intraspecific variation, principal component analysis and canonical discriminant analysis were performed to determine whether the observed interspecific variation could be useful in differentiating between these two races .

Principal component analysis revealed that the first principal component which accounted for most of the observed variation between the specimens was the length of the preapical cross band measured as one entity (component loading (CL)50.965), followed by the wing width (CL50.947) (Table II). The first principal component also accounted almost equally for the variation due to any of the wing measurements. This suggests that the five wing measurements were highly correlated and including any one of them in any further analysis is sufficient. The second PC showed high loadings for the head width (CL50.579) followed by the head length (CL50.305).

Similar results were obtained when PC analysis was done for only female specimens and for only male specimens. Plots of PC1 versus PC2 done for male specimens only and for female specimens only showed that there are two separate clusters reflecting the two races and that there is some overlap between the two groups ( Figure 5 ). However, there was less overlap and better clustering for male specimens compared to female specimens.

To separate between the two races based on within- and between-group variations, two canonical discriminant analyses (CD) were performed, one using all the seven morphometric characters and the other using two characters, namely the head height and the wing width, as the canonical discriminant function based on these two variables gave good separation between the two races. The characteristics of the two CD functions used are summarized in Table III.

Based on the two CD functions, the flies were classified into two groups, in such a way that each fly has an equal probability of belonging to any of the two groups. Based on the first CD function, 79.2% of the flies were predicted to belong to the race they actually belonged to. However, 18.3% of the flies belonging to the Notobasis- associated host race were classified as Onopordum- associated and 23.3% of the flies belonging to the Onopordum- associated host race were classified as Notobasis- associated (Table IV). Based on the second CD function, in which only two variables were considered, 72.5% of the flies were predicted to belong to the race they originally belonged to. However, 26.7% of the flies belonging to the Notobasis- associated host race were classified as Onopordum- associated and 28.3% of the flies belonging to Onopordum- associated host race were classified as Notobasis- associated (Table IV). This shows that CD analysis can be useful in differentiating between these cryptic races with more than 70% accuracy (or 30% error).

Using the standardized CDF coefficients of the two CD functions (Table III), a score for each of the 120 flies analysed was calculated, and three scatter plots of these scores were constructed ( Figure 6 ). In the first plot, the scores obtained for each fly using the second standardized CD function were plotted as a function of the scores obtained using the first CD function. The same was done in the second and third plots, except that only male scores were used in the second plot and only female scores were used in the third. Analysis of the plots shows that there is, to some extent, clustering of the flies into two groups. The presence of some flies of the first species in the vicinity of the second race can be attributed to the fact that some small or less mature flower heads had been picked up and adult flies had been reared from these flower heads. These flies might have been smaller than normal due to the fact that their larvae did not have enough food and had to pupate ahead of time, before storing enough nutrients to be used in the pupal stages ( Tsitsipis 1989).

Male terminalia. The morphological and morphometric comparisons undertaken proved that the terminalia of adult males belonging to the two races are similar, with the exception of fine differences in the glans, which was also longer in the Onopordum host race ( Figure 7 ). The length of the epandrium was 0.48¡ 0.01 mm (n 56) and 0.5¡ 0.01 mm (n 59) while its width was 0.22¡ 0.02 mm (n 56) and 0.23¡ 0.01 mm (n 59), for the Notobasis - and Onopordumassociated host races, respectively. No significant differences were detected in the length (t 50.99, df513) and width of the epandrium (t 50.1, df513) between the two host races (P.0.05). On the other hand, the glans (the sclerotized part of the aedeagus together with the vesica) was significantly larger in the Onopordum host race. Its length was 0.58¡ 0.01 mm (n 58; range 0.54–0.62) and 0.64¡ 0.01 mm (n 58; range 0.57–0.68) (t 53.23, df514, P,0.001) while its greatest width was 0.19¡ 0.004 mm (n 59; range 0.15–0.2) and 0.20¡ 0.006 mm (n 59; range 0.19–0.24) (t 52.2, df516, P,0.05), for the Notobasis - and Onopordum- associated host races, respectively. The similarity in morphology of the male terminalia, however, does not necessarily mean that the two races can interbreed; taking into consideration that successful mating does not necessarily produce viable offspring.

Female terminalia. The oviscape of the Onopordum- associated host race was comparable to that of the Notobasis- associated host race. Its length was 1.26¡0.02 (1.18–1.36; n 518) in the Onopordum- associated host race and 1.25¡0.05 (0.89–1.48; n 512) in the Notobasisassociated host race. Its width was 0.92¡0.02 (0.71–1.06; n 518) in the Onopordumassociated host race and 0.85¡0.03 (0.65–1.06; n 512) in the Notobasis- associated host race. No significant differences in the means of the oviscape length (t 50.13, df528) and width (t 51.91, df528) between the two races were detected (P.0.05).

The size and shape of the aculeus of the two races differed greatly. Table V summarizes nine measurements taken on the aculeus of both races. Significant differences between the two races were detected in five of these measurements: mean length of the aculeus (t 52.9, df597, P 50.005), tip width of the aculeus (t 58.5, df597, P,0.001), ratio 1 (t 54.7, df597, P,0.001), ratio 2 (t 53.4, df597, P 50.001), and aculeus width 1 (t 53.8, df597, P,0.001). No significant difference was detected in aculeus width 2 (t 50.4, df597, P 50.7) ( Figure 2 ). A marginally significant difference was found in the mean maximum width of the aculeus (t 52.0, df597, P 50.05) of the two races ( Table V).

The aculeus of the Onopordum- associated host race seems to be longer, a little wider and slightly blunter than the more pointed aculeus of the Notobasis- associated host race ( Figures 8 , 9 ), as is reflected by its mean length and width and the two angle measurements taken and as is cited in the literature ( Knio et al. 2002). The aculeus apex angle differed by 1.2 ° between the two races; however, no statistical difference could be detected in this regard. The longer aculeus in the Onopordum- associated host race can be attributed to the difference in size between the hosts of the two races and can be considered a morphological adaptation of the females to their oviposition substrates. Since the flower heads of Onopordum illyricum are larger than flower heads of Notobasis syriaca , the Onopordum- associated females might need a longer aculeus to be able to penetrate deeper into the flower heads of their host and deposit their eggs between the bracts. Zwölfer (1972) suggested that a narrower and sharper aculeus was needed by a female fly in order to be able to deliver its eggs deeper into the larger flower heads it infests. However, the aculeus of the Onopordum- associated host race turned out to be a little wider and less pointed than that of the Notobasis- associated host races, although the flower heads of Onopordum illyricum are larger in size.

Another significant difference in the aculeus between the two races was in the location of the ventro-lateral grooves bearing three pairs of elongated sensilla as measured by ratio 1 and ratio 2 ( Table V). In the Onopordum- associated females, the sensory ventro-lateral groove was located closer to the aculeus tip although the aculeus was longer ( Figures 8 , 9 ). This could also reflect the larger size of the host exploited by this race. The sensilla on the ventro-lateral grooves of the aculeus tip have been identified as mechano-chemosensilla and these are used by female tephritids to locate suitable hosts and to access the host quality and suitability for oviposition ( Stoffolano 1989; Stoffolano and Yin 1987; Zacharuk et al. 1986).

Key to host races

This key allows the correct identification of at least 70% of specimens. The best way for identification is to check the plant host, examine sampled populations, and check the sample means for the diagnostic measurements mentioned in the following key.

Identifications (with 70% accuracy) could also be made by calculating the position of sampled specimens with respect to CV functions (or axes) I and II:

CDF1~8:04 HHz1:51 HW —2:17 WWz4:12 WLdm —2:92 WLR 4z5 —4:24 WLpband1z 2:54 WLpband2 —6:64:

Figures

Figure 2. Tephritid ovipositor showing measurements made on the aculeus tip. W1 and W2, width 1 and width 2 of the aculeus tip below the distal and proximal ends of the ventro-lateral groove respectively; W, aculeus tip width measured 0.014 mm from the very tip of the aculeus; ratio 15L/Lii; ratio 25L/Li.

Figure 4. Third instar larvae of Notobasis-associated host race of Chaetostomella cylindrica. (A) Typical cephalopharyngeal skeleton; (B) posterior spiracle; (C) anterior spiracle. SS, spiracular slit; IP, interspiracular process; P, one of the six papillae.

Figure 5. Plot of principal components 1 and 2 for (A) female specimens and (B) male specimens of Chaetostomella cylindrica from the Notobasis-associated race (race 1) and the Onopordum-associated race (race 2).

Figure 6. Scatter plots of: (A) the scores obtained for each fly using the second standardized CD function (CDF 2) as a function of the scores obtained using CDF 1; (B) for each male; and (C) for each female fly using CDF 2 as a function of the scores obtained using CDF1. Squares represent Chaetostomella cylindrica from the Onopordum host race; triangles represent flies from the Notobasis host race.

Figure 7. Typical glans of the male distiphallus of Chaetostomella cylindrica from the Onopordum-associated race (A) and the Notobasis-associated race (B). Scale bars: 0.1 mm.

Figure 8. Typical aculeus of Chaetostomella cylindrica females from the Notobasis-associated host race (A) and the Onopordum-associated host race (probably C. lurida) (B). Scale bar: 0.5 mm.

Figure 9. Aculeus tip showing the position of the sensory ventro-lateral groove in Chaetostomella cylindrica females from the Notobasis-associated host race (A) and the Onopordum-associated host race (B). Scale bar: 0.1 mm.