TRITROPHIC ASSOCIATIONS AND TAXONOMIC NOTES ON LYSIPHLEBUS FABARUM (MARSHALL) (HYMENOPTERA: BRACONIDAE: APHIDIINAE), A KEYSTONE APHID PARASITOID IN IRAN

An investigation of host associations, distribution and types of reproduction (sexual, asexual) of Lysiphlebus fabarum (Marshall) across 20 provinces of Iran during 2006-2011 was undertaken. The parasitoid was reared from three groups of host aphids belonging to genera Aphis and Brachycaudus, and occasional host aphid genera. Aphis craccivora Koch was the most frequent host aphid for L. fabarum on various host plants, including economically important crops. The field sex ratio generally favored females, but in some cases, only thelytokous (uniparental) populations were found. In those cases, the host was always an Aphis species. Specimens reared from Brachycaudus aphids were all biparental, indicating the presence of a sibling biological species. Overall analysis of diagnostic morphological characters in the forewing indicated intra-specific variability in forewing marginal setae as well as variations in length of the R1 vein.

Lysiphlebus fabarum (Marshall) is an aphid parasitoid that has been known to manifest numerous taxonomic problems.It may represent a group of taxa with specific host associations or even a single taxon with a wide range of host aphids (Belshaw et al., 1999;Carver and Franzmann, 2001;Sandrock et al., 2007Sandrock et al., , 2011;;Starý, 1999Starý, , 2006)).Lysiphlebus fabarum is also a specialized parasitoid of ant-defended aphid colonies, avoiding aggression from ants through mimicry of aphid cuticular hydrocarbons (Völkl and Mackauer, 1993;Rasekh et al., 2010b).The females tend to parasitize aphids from the same colonies from which they themselves emerged (Rasekh et al., 2011).
L. fabarum is the most abundant parasitoid of some economically important aphids such as Aphis fabae Scopoli (Starý, 1986b;Starý et al., 2000) and Aphis craccivora Koch (Rakhshani et al., 2005a,b) on various different crops.Such parasitoid species need to be classified as "key-stone species" within an overall evaluation of parasitoids in agro-ecosystems (LaSalle and Gauld 1993;Starý et al., 2002).Knowledge about host range patterns and consequent relationships with type of reproduction within different populations of L. fabarum in Iran has remained insufficient; it has therefore been necessary to compile a summary and to cover the fundamental work on different aspects of host associations as well as to provide a background for further studies on the biological complexities of the diversity and distribution of the species.Host associations and distribution of the aphid parasitoid reflect the importance of the biosystematics and biodiversity of these parasitoids as potentially promising bio control agents.

MATERIALS AND METHODS
These studies were carried out across 20 provinces of Iran, extending from its eastern to its western borders, to represent the whole country.Sampling was done from 2006-2011.Colonies of different host aphids collected from fields were transferred to a laboratory where they were kept inside mesh-covered plastic boxes, together with parts from the host plants.A number of healthy adult aphids was simultaneously collected and preserved in 55% ethanol for later identification.The rearing boxes were kept at room temperature for 2-3 weeks and the emerged parasitoids were clipped daily using an aspirator, and dropped into ethanol (75%) for later examination.
A few specimens from each sample were dissected and slide-mounted in Hoyer media.The external morphology of the parasitoids was studied using a NIKON SMZ645 stereomicroscope and a NIKON Eclipse E200 microscope.Total numbers of emerged wasps (males and females) were counted for each sample to determine the sex ratio and the relationship with host associations.For evaluation of the field sex ratio and type of reproduction, those samples with fewer than ten specimens were removed from the analysis.In addition, host aphids that were found only in a single sample were not included in comparisons.The ant-attended colonies were recorded in the case of each aphid species with regards to presence of the parasitoids.Aphid nomenclature and classification follows that of Remaudière and Remaudière (1997).The specimens were deposited in the collection of the first author.

Host associations
Tritrophic parasitoid-aphid-plant associations for L. fabarum including 47 aphid hosts belong to 12 genera and over 99 host plant species were presented from 20 provinces (Table 1).Most of the host aphids belonged to the genus Aphis L. (78.84%) followed by Brachycaudus van der Goot (11.54%) and other genera (09.61%).Aphis craccivora Koch and Aphis fabae Scopoli were the most frequently encountered host aphids from the first group.The colonies of A. craccivora on its primary host plants were evidently heavily parasitized early in the season.The same situation was evident at the end of spring as well as on colonies of some other aphids, such as Aphis idaei van der Goot and A. fabae.The third most commonly encountered host aphid was Aphis gossypii Glover on a wide variety of host plants including vegetables and ornamentals.No association between L. fabarum and Brachycaudus spp., was evident in lowland regions such as northern Sistan and Baluchistan province (altitude less than 475m A.M.S.L), but Brachycaudus aphids occurred on respective host plants in the same area.Instead, some other species of aphid parasitoids, including Aphidius colemani Viereck and The association of L. fabarum with aphids on the collar regions of host plants was found in the case of Aphis (Protaphis) elongata (Nevsky) on Artemisia absinthium and Dysaphis radicola (Mordvilko) on Rheum palmatum.In both cases, aphids were protected by ants, but parasitism occurred in considerable numbers.The same relationship with ants was commonly evident in cases of many other aphid species that had colonized stems and leaves (Table 2).

Sex ratio and type of reproduction
The relationship between host aphid and sex ratio of L. fabarum is indicated in Table 3.There was an equal male:female ratio in association with Brachyunguis zygophylli (Nevsky) in only a few cases, but in the rest of samples there was a great bias toward females in the range of zero to 0.94.The occurrence of these completely uniparental specimens within the sampled material was evident in the case of Aphis (Protaphis) elongata (Nevsky) and Aphis euphorbicola Rezwani and Lampel.In both samples it can be assumed that males are rare because they were found on only two occasions, while the occurrence of uniparental populations ranged from zero to 50% in other host aphids.a sex ratio of 0.49-0.71:1(M:F).An exceptional sample was identified with only female specimens, reared from Brachycaudus (Appelia) tragopogonis.The host aphids that were only sampled on a single occasion were ignored, but it is noteworthy that for some of them, there was an insufficient number of specimens per sample to explain the overall type of reproduction.For example, all specimens of L. fabarum from Aphis epilobii were females.

Morphological variability
A few specimens from each sample were slidemounted and diagnostic characters were compared with descriptions and keys.There was a great variation in morphological characters, excluding color pattern, among different populations of L. fabarum.
The most important diagnostic character for separation of L. fabarum from allied species (Lysiphlebus confusus Tremblay and Eady and Lysiphlebus maris-mortui Mescheloff and Rosen) was the absence of long forewing marginal setae.Several specimens of L. fabarum were identified from various host aphids or even from a single sample with different patterns of forewing marginal setae .Furthermore, the length of the forewing R1 vein was variable in different populations (Figs.1-10).Geographically, there were different populations of L. fabarum in association with Aphis affinis del Guercio.These specimens from high southeastern mountains (2500-3000 m A.M.S.L) always had a set of forewing marginal setae and a medium-sized R1 vein (Fig. 9), but in specimens from central and western provinces the forewing marginal setae were absent and the R1 vein was commonly elongated.The number of antennal segments in all specimens ranged from 12-13 as a normal variability among the individuals even from the same sample.The hind femora in female wasps were always covered with short depressed setae, which were slightly erect toward the tip of the femur, except on the few occasions where there were a few erect setae on the hind femurs.

Biocontrol significance
Generally, host aphids could be divided into three groups, 1. Aphis spp., 2. Brachycaudus spp., 3. Oc-casional various aphids.L. fabarum showed a great tendency toward the genus Aphis that includes many important pest species.The more frequently occurring samples of host aphids were A. craccivora and A. fabae, followed by A. gossypii, which are the most common and important aphid pests on various cultivated plants.Starý (1981) indicated that the genus Lysiphlebus is prevalently in association the phylogenetically youngest group of aphids, the Aphidi-  nae.The common association of L. fabarum with Aphis species as a large genus of aphids with a fast process of diversification (Coeur d'acier et al., 2007), has been clearly confirmed.Furthermore, the establishment of this parasitoid on aphids with economically indifferent importance can act as a reservoir or a refugium (Starý, 1986a;Kavallieratos et al., 2002Kavallieratos et al., , 2004;;Havelka et al., 2012) from which the parasitoid can migrate short distances onto neighboring crops.
Both Aphis and Brachycaudus aphids are categorized as myrmecophilous aphids based on their poorly developed cornicles, reduced cauda, and a thin coating of wax filaments (Holldobler and Wilson, 1990).Lysiphlebus fabarum is known to have highly evolved adaptations to forage undisturbed within ant-attended aphid colonies (Rasekh et al., 2010a, b).The same behavior has also been recorded and investigated in some other Lysiphlebus species (Völkl and Mackauer, 1993;Kaneko, 2003;Vinson and Scarborough, 1991).While, ant-attendance is thought to protect aphid colonies against natural enemies (Kaneko, 2002;Vinson and Scarborough, 1991;Völkl, 1997), L. fabarum may benefit from ant-attendance and parasitize the host aphids even at higher rates compared to the antunattended aphid colonies (Rasekh et al., 2010b).Therefore, it can be concluded that in the case of the above-mentioned, of those aphids that are protected by ants, L. fabarum is the most important natural biological control agent.

Host range pattern, type of reproduction and biological species
Like other aphidiine parasitoids, L. fabarum also manifests a more or less definable oligophagy (Starý, 1988) that enables it to alternate between various host aphid species in the course of a season.This feature will increase its resistance to changes in population densities of the host aphid, that has been subjected as a significant reason for the speciation within this group (Starý, 1970).Lysiphlebus fabarum seems to be a complex species that includes several biological species.The correlation between the host range pattern, i.e.Aphis vs Brachycaudus, and shifting to asexual reproduction in the first group of host aphids (Aphis spp.) suggests a young group that is undergoing biological evolution that enables them to readily become specific to their host aphids.However, shifting to another host is a hypothesis that has already been discussed, as the thelytokous populations of L. fabarum were not affected by host alternation, as well as generation number (Starý, 1999;Sandrock et al., 2011).In cases where there is the presence of both thelytokous and biparental populations on the same host group (Aphis spp.), it can be an indication of sibling species that co-exist in the same area.Additionally, it is possible to elaborate a new species, Lysiphlebus brachycaudi (MS name), based on its specific host range which is restricted to Brachycaudus spp., with a biparental type of reproduction (Starý, 1999).An absence of any association between L. fabarum and Brachycaudus species in lowland areas may also be an indication of habitate preference for L. brachycaudi.Lysiphlebus melandriicola Starý is a very close taxon to L. fabarum, strictly attacking Brachycaudus lychnidis (L.) in Europe (Starý, 1961b).It can be a good example of sympathric speciation after selection of a specific host by an aphid parasitoid.
Generally, there was a great bias toward females, even in those cases in which the host aphids were Brachycaudus spp.Mackauer and Völkl (2002) has also analyzed seasonal variations in brood size and sex ratio in three aphid parasitoids and reported that the sex ratio at eclosion was biased toward females for all three species.The biased sex ratio in parasitoids can result either from the female's control of fertilization at oviposition or from the differential pre-imaginal mortalities (Charnov et al., 1981;Jarošík et al., 2003).In addition, mated females are able to regulate the sex ratio of their offspring in relation to the size of the host they attack (Godfray, 1994).Sex ratios of progeny emerging from small hosts tend to be malebiased and those from large hosts tend to be femalebiased.Pandey and Singh (1999) reported that females of Lysiphlebia mirzai Shuja-Uddin could adjust the progeny sex ratio according to the host size as host size affected the fitness of the females more than that of male progenies.However, the biased sex ratio may not be attributed to manipulation of the primary sex ratio at the time of oviposition, but on the contrary, arises directly from the higher mortality of female progenies in aphids with smaller size compared to males (Jarošík et al., 2003;Wellings et al., 1986).Generally, it can be a naturally selected trait, as the males of L. fabarum are polygamous (Bagheri Matin et al., 2005) and in this condition, fewer males manage larger numbers of females.However, if females oviposit without copulation, the progenies will all be males (Starý, 1988) to compensate for the deficiency of the male:female balance.The sex ratio of the aphid parasitoids naturally fluctuates along the life span of a reproductive female (Srivastava and Singh, 1995) and it is affected by environmental and genetic factors (Starý, 1988).Specimens of L. fabarum were collected mostly in early and mid spring, which is the normal time for reproduction and population growth.Temperature is assumed to be an important factor affecting the sex ratio (Bagheri Matin et al., 2005).These authors have recently found a positive correlation between temperature and percentage of males in L. fabarum.Generally, this phenomenon was attributed to a decline in the activity of parasitoids, including mating, that increases the number of male progenies (Tremblay, 1964).However, hot temperatures (in territories like Iran) are known to have an inhibitory effect on the activities of the microbes in association (Wolbachia) with sex determination (Bordenstein and Bordenstein, 2011;Stouthamer and Werren, 1993) that could account for the common occurrence of males in different populations.However, no evidence of bacterial symbiont Wolbachia was found in thelytokous populations of L. fabarum (Belshaw and Quicke, 2003); instead, they found the central fusion automixis as the mechanism and asexuality.Further evidence has demonstrated that thelytokous reproduction in L. fabarum manifests from only a simple genetic basis, which is consistently inherited as a single-locus recessive trait.This contagious system allows closely related individuals to reproduce with different modes (Sandrock and Vorburger, 2011).The occurrence of both thelytokous and biparental (arrhenotokous) populations of L. fabarum on the same host aphids (Table 3) was quite common when the host aphid was an Aphis species or even in the case of Myzus persicae (Sulzer).
Lysiphlebus cardui (Marshall), a closely related species to L. fabarum, is also a questionable taxon.Its host range is limited to species of Aphis, as the major host group for L. fabarum.Morphological separation of these two species is rather difficult.This species is a specialized parasitoid of Aphis fabae cirsiiacanthoidis (Schrank) on Cirsium arvense (Starý, 1986a, b), but often co-exists with L. fabarum on the same host (Starý, 1999).Molecular studies have mostly failed to separate these two species (Belshaw et al., 1999); sometimes L. cardui was classified as a sister taxon of L. fabarum (Kambhampathi et al., 2000).
Until now, the results of molecular studies have largely not elucidated species' boundaries and sibling species/biotypes in the case of L. fabarum.The important issue here is relations between molecular and morphological variability affected by asexuality.Molecular markers can help to detect some species but they create new problems that sometimes contradict morphological and biological approaches (Belshaw et al., 1999).One of the major reasons for the incomplete or controversial results of molecular studies is the aim of those specific researches that often target higher-level phylogeny (Smith et al., 1999;Kambhampathi et al., 2000;Sanchis et al., 2000).

Variability of the morphological characters
Lysiphlebus fabarum can typically be separated from the other congeneric species, L. confusus, by the absence of long marginal setae of the forewing (females).But there were various patterns of marginal hairs in position and length within the population of L. fabarum from differrent host aphids and localities.Lysiphlebus marismortui is a recently described species with marginal setae of different and irregular lengths together with an arch on the 3RSb vein (Mescheloff and Rosen, 1990).But even the latter character seems to be an intraspecific variation irregularly shifting in different individuals.Furthermore, this species was described from A. craccivora as the most common host aphid for L. fabarum.Thus, there is still not enough evidence to validate L. marismortui as a separate species.The number of maxillary and labial palpomers was constant in all specimens.However, measuring the length of the palpomers, as well as the characters of the flagellar segments (number of placods) as the major sensorial organs associated with with host selection (Quicke, 1997), should be considered in future research.
A recent morphometric analysis of populations of L. fabarum from different host aphids (Barahoei et al., 2011) surprisingly clarified signficant differences among so called "biotypes".However, this study was carried out on a restricted number of biotypes.They found that the major variable characters were on the forewing venations, as well as in the lengths of the ovipositors and hind legs.In spite of limitations on the scope of this essay to the local host aphids, which evolutionarily says nothing, it throws the significance of the traditional means of species identification into a big controversy.The same procedure should be effective on more justified biological species reared from phylogenetically distant aphids with different biology and type of reproduction.Limitations in the numbers of diagnostic characters, together with intra-specific variations cannot be justified for populations of L. fabarum and presents a complicated question both for known members of the L. fabarumcomplex and for the more recently identified groups (Starý, 1999).
The exact identification of a species or sibling species, sometimes termed as "biotypes" has created some problems not only in taxonomy, but also in terms of biological control (Carver and Franzmann, 2001).Consequently, a detailed morphological and biological investigation is needed to confirm the presence of some reliable differences between representative taxa and the relation between morphological studies with biological data.At the same time, selection of suitable molecular markers is also required as proof.

Table 1 .
Host associations and distributional data for Lysiphlebus fabarum at different provinces of Iran

Table 1 .
Continued Aphidius matricariae Haliday replaced L. fabarum in this area.However, the association of Brachycaudus species on the creeping thistles, Cirsium arvense, and goatsbeard, Tragopogon spp., were quite common in sub-mountainous areas of the various different provinces.

Table 1 .
The populations of L. fabarum reared from Brachycaudus spp.were almost completely biparental, with Continued

Table 2 .
The host aphids belonging to the genera Aphis and Brachycaudus on their host plants, which were commonly found in association with ants

Table 3 .
Sex ratio and Frequency of the asexual population of Lysiphlebus fabarum on different host aphids