Materials and methods

A literature review of cynipid species associated with oaks was conducted, leading to the construction of a database to locate the species and establish them as starting points for the collection. Sampling was performed from March to August for four years (2008-2012) in 120 localities in total. For each oak species, at least five localities were visited. Geographic coordinates were taken at each location to define the distribution pattern of the cynipids associated with the different Quercus species (Figure 1).

Figure 1 Sampling localities of gall morphotypes of oaks in Mexico. 

Once the different oak species were located, an exhaustive search for galls was conducted at each site by checking each structure (i.e., apical buds, leaves, petioles, branches, stems, roots, catkins and acorns) of at least ten trees from each available Quercus species. In addition, herbarium specimens were collected to identify the oak species.

The collection of cynipids was based on gall morphology and was taken from across the country. A total of 80 oak species that were associated with this guild of herbivores was analyzed. For this study, each distinct gall morph is considered a potentially distinct species. We classified the galls according to the number of larval chambers as unilocular (a single chamber) or multilocular (with several chambers) and with or without ornaments (spines, hairs).

The collected galls were brought to the laboratory for growth and subsequent taxonomic determination. The taxonomy of the species is being determined in collaboration with Dr. José Luis Nieves-Aldrey from the National Museum of Natural Sciences in Madrid, Spain.

Results

A total of 224 distinct gall morphotypes induced by cynipids was found in 73 of the 80 oak species under analysis, of which 40 correspond to the Lobatae section (red oaks) and 33 to the Quercus section (white oaks). Galls were not found in the following seven oak species: Q. canbyi, Q. gentryi, Q. crispifolia and Q. hypoleucoides from the Lobatae section and Q. glabrescens, Q. glaucoides and Q. lancifolia from the Quercus section. The largest number of morphotypes was found in leaves (125), followed by branches (37), buds (31), petioles (20), catkins (5), acorns (3) and roots (3) (Figure 2). A total of 126 gall morphotypes were found in the Quercus section, and 113 were found in the Lobatae section.

Figure 2 Number of gall morphotypes in each plant organ of oaks. Total number of galls = 224. 1 = leaves (125 galls; 55.8%); 2 = branches (37; 16.5%); 3 = buds (31; 13.8%); 4 = petioles (20; 8.9%); 5 = catkins (5; 2.2%); 6 = acorns (3; 1.3%); and 7 = roots (3; 1.3%). 

The number of gall morphotypes associated with each oak species, their taxonomic section, and the amplitude of geographical distribution and whether they are endemic to Mexico is summarized in Table 1. In the Lobatae section (red oaks), the species with the greatest number of morphotypes are Quercus crassifolia (11), Q. castanea (10) and Q. mexicana (7). The remaining species have one to five distinct morphotypes (Figure 3). In the Quercus section (white oaks), the oak species with the greatest number of gall morphotypes were Q. microphylla (20 morphotypes), followed by Q. resinosa (17) and Q. magnoliifolia (14). The remaining species have one to five distinct associated morphotypes (Figure 4). As in the case of section Lobatae, the species with the greatest number of morphotypes are those with a wide geographical distribution.

Figure 3 Number of gall morphotypes according to the morphology in the two sections of oaks (Lobatae and Quercus). A) Unilocular without ornaments. B) Unilocular with ornaments. C) Multilocular without ornaments. D) Multilocular with ornamentes. 

Figure 4 Number of gall morphotypes associated to each oak species in the Section Lobatae. 

Only 31.5 % of the 73 oak species (13 red oaks and 10 white oaks) had only a single associated gall morphotype; 24.7 % (12 red and 6 white oaks) had two gall morphotypes, 16.4 % (6 red and 6 white oaks) had three gall morphotypes, and the remainder (27.4 %) had more than 4 morphs. Of the 13 species of red oaks with a single gall morphotype, 9 are endemic to Mexico, and of the 10 white oaks, 6 are endemic.

We found 63 galls with a single larval chamber (unilocular) with smooth surface (without ornaments) (36 in Lobatae and 27 in Quercus), 35 unilocular with ornaments (14 Lobatae and 21 Quercus), 109 multilocular without ornaments (56 Lobatae and 53 Quercus) and 32 multilocular with ornaments (7 Lobatae and 25 Quercus) (Figure 5).

Figure 5 Number of gall morphotypes associated to each oak species in the Section Quercus. 

Discussion

In this study, we found a great diversity of galls induced by wasps on oaks as host species (Figure 6). The assumption that gall-inducing wasps from the Cynipidae family exhibit a high degree of specialization to their host plants in the Quercus genus (Weld 1960, ^{Ronquist 1995}, ^{Liljeblad & Ronquist 1998}, ^{Nieves-Aldrey 2001}, ^{Stone et al. 2002}, ^{Csóka et al. 2005}) should be reviewed based on the results of this study. Most of the oak species studied here had more than one gall morphotype, and in some cases, they had an unexpectedly high number of gall morphotypes throughout their geographical distribution. In particular, three species of white oaks [Q. microphylla (20 morphotypes), Q. resinosa (17) and Q. magnoliifolia (14)] and three red oaks [Q. crassifolia (11), Q. coccolobifolia (11) and Q. castanea (10)] had a considerable number of galls collected from throughout their broad distribution in Mexico. These oak species have a wide range of geographic distribution occurring in different environmental conditions that could represent diverse ecological niches occupied by different gall wasps. It is also necessary to study the degree of host specialization of these galls through their entire geographic range in future studies.

Figure 6 Photos of gall morphotypes in different oak species in Mexico. 1. Quercus conzati (petiole) Jal. 2. Q. sideroxyla (branch) Chih. 3 y 4. Q.magnoliifolia (leaves) Gro. 5. Q. resinosa (catkin) Mich. 6. Q. crassifolia (bud) Chis. 7. Q. ocoteifolia (yema) Oax. 8. Q. uxoris (leaf) Oax. 9. Q. sp. (branch) Ver. 10. Q. castanea (branch) Mich. 11. Q. segoviensis (catkin) Chis. 12. Q. polymorpha (leaf) NL. 13. Q. arizonica (leaf) Coah. 14. Q. segoviensis (leaf) Chis. 15. Q. cupreata (root) NL. 16. Q. obtusata (leaf) Dgo. 17. Q. microphylla (acorn) Gto. 18. Q. laurina (branch) Hgo. 19. Q. gregii (leaf) Dgo. 20. Q. laeta (bud) Mich. 21. Q. rugosa (bud) Jal. 22. Q. conspersa (branch) NL. 23. Q. frutex (leaf) Pue. 24. Q. viminea (bud) Dgo. 25. Q. deserticola (leaf) Sin. Abbreviations of each locality (state) as indicated in Table 1. 

In these cases, in particular, these oak species could be considered super hosts, since they have galls throughout their entire distribution and because galls occur over the entire structure of the tree. They also have morphotypes that are specific to certain localities, which may indicate endemism.

In the case of the endemic oak species, many have only a single morphotype, and even similar morphotypes had variations in bark texture or the inside the gall itself. It can therefore be assumed that they are distinct species and probably endemic and a detailed ecological and taxonomic study would need to be conducted for each of them.

By contrast, very unique morphs were discovered, which were only found on the leaves of oaks in the Quercus section, for example, Q. magnoliifolia, Q. resinosa, Q. laeta, Q. deserticola, Q. rugosa and Q. obtusata. According to ^{Pujade-Villar et al. (2010)}, there is a new genus called Kinseyella, although confirming it would require a thorough review of both the gall and the gall inducers to know whether it is the same genus and a distinct species, or whether it is the same species of gall inducer in all the hosts listed here.

In the case of Q. magnoliifolia and Q. resinosa, recent studies showed that these oak species form hybrids in regions where their geographic distributions overlap (^{Albarrán-Lara et al. 2010}), generating new adaptive zones for the formation of new species of gall insects (^{Pérez-López et al. 2016}). These zones are also suggested by the Q. crassifolia × Q. crassipes complex (^{Tovar-Sánchez & Oyama 2004}) in which three gall morphotypes were exclusively associated with the hybrid individuals found there (^{Tovar-Sánchez & Oyama 2006}). However, this pattern cannot be generalized, since in another hybrid complex formed by Q. affinis × Q. laurina (^{González-Rodríguez et al. 2004}, ^{González-Rodríguez & Oyama 2005}, ^{González-Rodríguez et al. 2005}), only one gall morphotype was found in each of the species in this study (see Table 1).

The galls induced by cynipids can be classified according to the number of larval chambers (e.g., unilocular and multilocular) and their ornamentation (e.g., without ornamentation and a smooth surface, or with hair and spines). The highest numbers of galls for both sections are multilocular galls without ornamentation, and they are usually associated with the branch of the host. According to ^{Ronquist (1995}, ¹⁹⁹⁹⁾ and ^{Rokas et al. (2003)}, this gall morphotype is the most primitive, suggesting the colonization of the host by cynipids. Confirming this colonization would require a phylogenetic and comparative biological study to locate these species relative to European species and assess their degree of relationship.

There is, however, a peculiar morph in the Lobatae section that is unilocular, globular, with or without ornamentation, and similar to galls from the Amphibolips genus (^{Nieves et al. 2012}). This finding may indicate that there is a particular association between this endemic group of oaks with a genus of cynipid inducers, an interaction so specific that it could be considered a process of speciation that often occurs in the hosts of this section of oaks (^{Nieves et al. 2012}).

The collection effort is a key factor in this type of study. ^{Maldonado-López et al. (2015a}, ^b, ²⁰¹⁶⁾ recorded up to 40 morphologically distinct galls that were induced by wasps on Q. castanea throughout its geographic distribution. This finding suggests that studies with both greater temporal and spatial collection intensity, focused on a single species of oak throughout its entire distribution, can produce this type of result.

Conclusions and perspectives

Gall-forming wasps (Hymenoptera: Cynipidae: Cynipini) represent a very diverse group with ecologically interesting characteristics (^{Nieves-Aldrey 1998}, ²⁰⁰¹, ^{Ronquist & Liljeblad 2001}, ^{Hayward & Stone 2005}). In this study, an important effort to add new records to this guild of insects in Mexico was undertaken. However, it is necessary to continue this type of study to determine the total species richness of wasps in Mexico as well as their phylogenetic relationships, and finally, to establish their patterns of diversification. Mexico represents one of the two most important centers of oak diversification in the world, especially for red oaks (section Lobatae). These collection efforts must be accompanied by taxonomic studies that elucidate the discovery of new species (^{Nieves-Aldrey et al. 2012}) and even genera as observed in some of the collections made in this study.

One important and characteristic aspect of the cynipids associated with oaks is that they are cyclically parthenogenetic, which has been a source of confusion during their taxonomic classification. Thus, studies should focus on finding both morphological (^{Hernández-Soto et al. 2015}) and molecular tools to resolve this problem, in addition to elucidating which processes cause this phenomenon.

At the population level, it is important to determine the dispersal distances of individuals and their patterns of colonization in both themselves and their new hosts through either ecological studies (Schönrogge et al. 1994, ¹⁹⁹⁹) or genetic studies using molecular markers (^{Castillejos-Lemus 2016}).