Comparative morphology of the type-species of Isotes and Synbrotica ( Coleoptera , Chrysomelidae , Galerucinae ) , with a new synonymy of species

In order to solve the affinities of the species of Isotes Weise, 1922, a detailed morphological comparative study was carried out based on type-species of Isotes and its junior synonym, Synbrotica Bechyné, 1956. Isotes tetraspilota (Baly, 1865) and Isotes borrei (Baly, 1889) had their morphology of mouthparts, endosternites, wings and both male and female genitalia compared by the first time. A new synonymy is established between Isotes borrei (Baly, 1889) and Isotes crucigera (Weise, 1916) syn. nov. based on external and genitalia morphology. New structures for Section Diabroticites Chapuis, 1875 are presented and discussed.

The genus Isotes was proposed by Weise (1922) to include a single species, Isotes quadrimaculata Weise, 1922 -type-species by monotypy.The distribution of the genus is Neotropical, occurring in Central and South America.Aslam (1972) studied the holotype of Isotes quadrimaculata Weise, 1922, established synonymy with Synbrotica tetraspilota (Baly, 1865), and considered Synbrotica Bechyné as junior synonym of Isotes.The synonymy was proposed in a single note, without remarks of any features that led to genera synonymy.
This fact increased a problem because a great number of species were assigned to Synbrotica since the genus was proposed by Bechyné (1956).Additionally, subsequent authors never provided diagnostic features for the genus.Bechyné (1956) only designated Diabrotica borrei Baly, 1889, from Brazil, as type-species of Synbrotica.Bechyné & Bechyné (1969) provided only six diagnostic features for Synbrotica, but also they affirmed that the genus included many not congeneric species, forming an artificial group, and that they should be transferred.Despite this, even after the synonymy with the genus Isotes, none other study has been conducted to investigate this problem.
Since that the previous authors synonymized these genera and did not compare both type-species, the aim of this work was to perform a detailed study of the morphology of the type-species Isotes tetraspilota (Baly) and the typespecies of Synbrotica, I. borrei (Baly), including external morphology, mouthparts, wing venation, endosternites, and female and male genitalia.In parallel, a comparative study of Isotes borrei (Baly) and Isotes crucigera (Weise) is performed by first time to determinate if they should be synonymized.
Taxonomic history.Diabrotica tetraspilota was described by Baly (1865) based on a single male from Mexico.Later, Smith & Lawrence (1967) noticed another male from Guatemala with a Baly's label, but could not be considered a syntype because Guatemala was not originally included in the description.This species was assigned to the genus Synbrotica by Smith & Lawrence (1967).After that, it was considered as senior synonym of Isotes quadrimaculata Weise by Aslam (1972).
Another species, Diabrotica cruciata, was proposed by Baly (1889) in the same paper and page of D. borrei, based on a male from Santa Catarina (Brazil).Both species had short descriptions with a minor variation between them.Gahan (1891a) finished the uncompleted Baly's work (1890), transcribing the descriptions for D. borrei and noticed that "D.cruciata, Baly (Ent. Mo. Mag.,xxv.,p. 253) is merely a variety of this species".Finally, Gahan (1891b) and Weise (1916) provided the replacement name crucigera, pointing out D. cruciata Baly as junior homonym of D. cruciata Jacoby, 1887. Weise (1924) considered D. crucigera as an aberration of D. borrei in the catalogue, and Bechyné (1958) placed D. crucigera in the genus Synbrotica.Despite of the overall similarity they were treated as distinct species.

Morphological comparison between Isotes borrei, I. crucigera and I. tetraspilota
Integument, general coloration (Figs 1-5).In Galerucinae the pattern of the integument color is highly variable, often being the only feature used to separate species when lacking studies with genitalia.By other hand, the species also exhibit interspecific variation, while other  species morphologically distinct show the same color pattern.Both I. borrei (Figs 1-2), I. crucigera (Fig. 3) and I. tetraspilota (Figs 4-5) have elytra with integument yellow-brownish, with two large black or blue-black spots.
Head .The width across the eye is narrower than anterior margin of pronotum.Vertex convex (Figs 8,11), with variable punctuation, and a weak depression near frontal tubercles.Antennal calli transverse and subtriangular, slightly larger than antennal cavities (Figs 6,9).Eyes  small (by relation between the maximum eye diameter in lateral view, against the interocular distance), and well-marked orbital lines near the outer margin.Antennal cavities rounded, inserted just below the midline of the eye.Frons with frontal ridge and fronto-clypeal suture, surface variably punctate and rugose.Isotes borrei and I. crucigera exhibit elongated genal area (length subequal to half or feebly more than the maximum diameter of the eye), and front as wide as long .Isotes tetraspilota has genal area and front evidently wider than long .
Mandibles (Figs 14,15) symmetrical, subtriangular, outer margin distinctly curved, inner cutting edge with five apical teeth, with short and dense setae on the outer surface.First tooth small, attenuated at the apex; second tooth twice the length of the first; third narrowed and twice the length of the second and fourth teeth; fourth variable at apex, slightly longer than the fifth, this short and blunt; prostheca wide, with abundant setae, extending almost from the basal margin of the mandible to the first apical tooth; mola longer than wide and sculptured with rows of transverse elevations.Both species have similar shape and length of mandibles, except: 1) inner cutting edge concave in I. tetraspilota and distinctly straight in I. borrei; 2) mandibular teeth, in external view: tooth three with inner margin shaped and more projected in I. borrei, and inner margin with slight sinuosity and less projected in I. tetraspilota.While that in ventral view, it is robust and little curved in the last species and narrower and bent to the side in the first one; 3) tooth two and four, ventral view, blunt in I. tetraspilota and pointed in I. borrei; and 4) mola wider in I. borrei than in I. tetraspilota.Savini & Joly (2006) observed that the presence or absence of mola is not an informative character for the classification when they studied the mandibles of 22 genera of Galerucinae.Thirteen of the 22 genera studied does not have mandibular mola, but when the mola exist, it is variable in the shape, can be formed by parallel blades, either toothed or fused with the body of mandible, and separated by a well-defined suture, an indication that the structure and shape of the mola could be contribute together with other characters to definition of genera or groups of genera in the subfamily.The shape of the mandible, prostheca and number of teeth of the incisive area are other valuable characters that may contribute to the classification of the group.
Maxilla (Figs 16,17).In all species examined, the maxilla present cardo narrowed at proximal portion, wider apically, and with long setae; basistipe with long setae at the latero-external edge, and dististipe well developed with short setae at the inner margin, and short setae near the lacinia, with apical pubescence fringed and strongly dense; galea subcylindrical and slightly longer than lacinia, with long and dense setae at the apex.Maxillary palps well developed, with four palpomere, exceeding the galea by apical palpomere; palpomere one short, subrectangular, subequal as half the second; palpomere two subconical; palpomere three subcylindrical, twice longer than the apical; apically acute.Distal part of the galea twice the length of the basal, even more curve in I. borrei that in I. tetraspilota.Palpomeres clearly shorter and robust in I. tetraspilota and more elongated and narrower at the base in I. borrei.
Labium (Figs 18,19).Labium poorly developed, with ligula membranous and not lobated.Labial palps with three palpomeres, basal short and strongly transverse; palpomere two slightly longer than the apical, subcylindrical, and slightly longer than wide; apical narrow to apex.Prementum subrectangular in both species.Ligula reaching the base of the apical palpomere in both species, but it is distinguished in the shape, apex narrowed in I. tetraspilota, and broadly rounded in I. borrei.Mentum (= postmentum Cabrera & Durante, 2003) transverse and subparallel at sides in I. tetraspilota and subtrapezoidal in I. borrei.
Antennae.Antennae with 11 antennomeres, exceeding the middle of elytra.Scape cylindrical, slightly thickened to apex, one third longer than the pedicel and subequal to the antennomere III; antennomere IV longer than the III; V-VI subequal in length, both slightly shorter than the IV; antennomeres VII-X lightly shorter; XI slightly longer than the X, acute at apex.
Thorax.wider than long and narrower than the elytral base.Pronotum (Figs 20-21) slightly convex, with variable depressions in the basal half.Isotes borrei exhibits well marked lateral margins, subparallel and slightly sinuous in the basal third (Fig. 21); anterior and posterior angles rounded; each angle with a long setae inserted in a coarse and deep punctuation; and other short setae between angles, unstable in number.In I. tetraspilota the lateral margins of pronotum is convex, slightly rounded, and wider in the middle (Fig. 20).
The species studied have the pronotum wider than long, but the proportion varies from 1.6 to 1.7 times wider in I. tetraspilota (Fig. 20), while in I. borrei and I. crucigera from 1.8 to 2.0 times (Fig. 21).The type-species Isotes tetraspilota has no depressions on pronotum (Fig. 20).On the other hand, I. borrei has a transverse depression in the posterior half (closer to the posterior margin), reaching the sides, where it is evidently deeper (Fig. 21).Prosternum (Fig. 22) transverse, with a short and laminar prosternal process between the procoxae.Procoxal cavities contiguous and opened behind.Proendosternite (Figs 23,24) with divergent projections towards posteriorly, slightly curved in I. tetraspilota and linear in I. borrei.
Legs.All legs are similar, slightly increasing in length.Coxae of pro-and mesolegs rounded, and mesocoxae with a glandular opening near the anterolateral area (Fig. 25).Metacoxae transverse.Trochanters subtriangular and reduced.Femora fusiform, tibiae slender, with apical spur -except protibiae of males.
Tarsi with tarsomeres I and II subrectangular (males with adhesive disc on the ventral surface of proand or mesotarsomeres); tarsomere III bilobed, with dense pubescence; tarsomere IV reduced on the basis of V, this elongated, with bifid claw at its end.
Abdomen .Abdomen with urosternites I-V subequal in length, wider than long, gradually decreasing in width until the urosternite V; this subtrapezoidal, with variable apex between male and female species of Isotes.Apex of the urosternite V of males weakly notched (Fig. 37) in I. tetraspilota and truncated (Fig. 39) in I. borrei and I. crucigera.In females, in turn, both species exhibit the apex of the urosternite V rounded (Fig. 38).41).Median lobe, ventral view, placed laterally in the abdomen, with curvature toward the left side, and subequal in length to the last four urosternites in I. borrei and I. crucigera.In I. tetraspilota it reaches the first urosternite.Both species have variable curvature in the median lobe, as follows: 1) basal third strongly curved, 90° from the rest of the body of the median lobe at the height of the basal constriction, well-marked in I. borrei and I. crucigera (Fig. 40); and 2) basal third slightly curved, less than 45° relative to the apical two thirds of the median lobe, with well-marked constriction; apical third slightly curved in I. tetraspilota (Fig. 41).Isotes borrei and I. crucigera have a basal crest, slightly sclerotized with length equal to half of the basal third of the median lobe; I. tetraspilota also have this crest, but much shorter and narrowed.
According to Moura (2009), the basal orifice (= median foramen Matsumura & Suzuki, 2008) in Luperini is protected by a hood-shaped process, with rounded border.The basis of this process, in ventral view, is similar in both species, being truncated and notched at apex.In lateral view, all have rounded base, being sinuous in I. borrei and I. crucigera (Fig. 40) and straight in I. tetraspilota (Fig. 41).None of them have basal hooks, corroborating the results found for the tribe Luperini by Moura (2009).
The apex of the median lobe is variable in Luperini (Moura, 2009).However the conformation of the apical flap together with the ventral lobe (Figs 42, 43) indicated importance for the determination of species.In this study we found the following relation of forms to the apex of the median lobe: 1) apical flap rounded, with crenulated margin, and ventral lobe narrowed at the apex, which is indented in I. borrei and I. crucigera (Fig. 42), and 2) apical flap rounded, with smooth margin, and ventral lobe with parallel sides that narrow at the apical margin in a rounded projection in I. tetraspilota (Fig. 43).
The lateral margins of the apical third of aedeagus, in ventral view, may or may not present a pair of lateral lobes (stuck to the sides of the ostium, according to Cabrera & Cabrera Walsh, 2004a,b) that vary in shape.Similarly to the apex in dorsal view, this feature is specific.The studied species show the following shapes: 1) subtriangular in I. tetraspilota (Fig. 43), and 2) sinuous in I. borrei and I. crucigera (Fig. 42).Isotes tetraspilota exhibit a highly sclerotized structure at apex surrounded by a moderately thick and dense punctuation (Fig. 43), not observed in the other species.
The internal sac is a membranous structure supported by the median lobe; during mating it is everted through the median orifice (= ostium Moura, 2009) under pressure from the hemolymph, forming the endophallus (Matsumura & Suzuki, 2008).
The internal sac in these two species varies in the number and shape of the sclerites (Figs 40,41): one in I. borrei and I. crucigera, in the middle region of the median lobe, dorsally with the anterior half subretangular, rounded edges, and posterior half bifid -with two spiny projections directed towards the apex, the right greater than left; and two in I. tetraspilota, median, highly curved, pointed at the apex.
Female genitalia .Both species present tergite VIII trapezoid shape, with sides slightly rounded, anterior angles rounded and pointed posteriorly.The sides fold toward the structures of the female genitalia, visible in ventral view.
Tergite IX (Figs 48,49).Structure situated between the tergite VIII and sternite VIII, above the opening of the gut.It is characterized by a small plate, usually little sclerotized (Lingafelter & Konstantinov, 1999).The tergite IX is present in all species, with little observed variation in conformation and the length/width ratio: 1) longer than wide, inner margin sub truncated in I. borrei and I. crucigera (Fig. 48), and 2) as wide as long, robust with narrow sclerotized region in I. tetraspilota (Fig. 49).All tergites IX exhibit basal setae.
Despite being a structure present in most taxa of Chrysomelidae, including most Alticinae, Galerucinae and Chrysomelinae species (Lingafelter & Konstantinov, 1999), it is neglected in descriptions of female genitalia.
Spiculum ventrale .The spiculum ventrale (= tignum Konstantinov, 1998) is a rod-shaped sclerite of the endoskeleton, begin the attachment of muscles (Torre-Bueno, 1989).It is present in the two species, in connection with the sternite VIII, varying in the degree of sclerotization, size -compared with sternite VIII -and within reach of the same in the sternite.Regarding the sternite VIII connection can reach the area: distal in I. borrei and I. crucigera, or proximal in I. tetraspilota.
Vaginal palpi (Figs 52,53) are formed by invagination of the dorsal side of the vagina (Konstantinov, 2002).There are two opinions about the function of these structures: the first considers the palpi as adaptations for posture, with sensorial function in selecting the best substrate to lay eggs, and the second believes that they act in mating (Konstantinov, 1998;Moura, 2008).In all species studied, the palpi are subcylindrical (Cabrera & Cabrera Walsh, 2004b), approximately two times shorter than the spiculum ventrale, strongly punctuated in the apical half and with setae apically.The palpi are united at the basis on all species and vary according to it.At the apex they can be united or separated, a characteristic that varies among specimens of the same species.Moreover, they can be entirely sclerotized or not.Separated palpi apex were observed in I. tetraspilota (Fig. 53).There was variation in I. borrei and I. crucigera, from being completely united to moderately separated (Fig. 52).The shape of the sclerotization of the apex varies inter-and intraspecifically.Additionally, the sides of the palpi are sclerotized in all species, except I. borrei and I. crucigera.
Bursa copulatrix .In female insects, it works as a purse, being the genital chamber or part of it.In Coleoptera, it is the blind proximal end of the vagina, with which it is connected widely or closely (Torre-Bueno, 1989).In the studied species, the bursa copulatrix varied in relation to form and the presence or absence of sclerotization.The species present different types and forms of sclerotization: 1) I. borrei (Fig. 56) with median region slightly more sclerotized, quadrangular aspect, and with small bands more sclerotized anteriorly; 2) I. crucigera syn.nov.(Fig. 57) with small oval sclerotized area in the median region; 3) I. tetraspilota (Fig. 58) generally differentiated: median membrane very dense, sharply rounded, without sclerotization.
In this paper, the first terminology was adopted for the comparison of the morphology of the spermatheca of Isotes (Figs 54,55).Both species have elongated spermathecae with curved cornus and rounded apex.The shape and width from nodulus varies from elongated, with the same width in the median part in I. borrei and I. crucigera (Fig. 54) to strongly enlarged, cylindrically shaped in I. tetraspilota (Fig. 55).The morphology of the spermatheca has been used to separate genera (Cryptocephalinae in Chamorro-Lacayo et al., 2006), species groups (Stolas, Cassidinae in Borowiec & Pomorska, 2009) and species (Alticini, by Leonardi, 1970), however it was clearly neglected in Galerucinae studies that lacking information terminalia.This study showed the importance of using such characteristics and the real possibly can be tested in further phylogenies.
Spermathecal duct and gland.Both species have the duct length slightly greater than the spermatheca, narrower and slightly sclerotized at the base, and extending until the nodulus.The opening of the spermathecal gland is lateral and proximal to it, with a moderately dilated region after the same.I. tetraspilota (Fig. 55) shows a strongly sclerotized duct, similar to the spermatheca, and unlike other species where it is less sclerotized.The spermathecal gland is poorly sclerotized, narrow and elongated.

DISCUSSION
The comparison of I. tetraspilota (Baly, 1865) and I. borrei (Baly, 1889), type-species of the genera Isotes and Synbrotica, showed that the species have a very distinct morphology, including the frons (I.borrei as long as wide and gena elongated; I. tetraspilota wider than long and short gena), clypeus (I.borrei with six setae; and, I. tetraspilota with eight setae), mandibles (inner margin concave in I. tetraspilota and substraight in I. borrei; mola wider in I. borrei that in I. tetraspilota), maxillae (distal part of the galea more curved in I. borrei than in I. tetraspilota; with the articles of palp shorter and robust in I. tetraspilota), labium (ligula narrowed at the apex in I. tetraspilota and broadly rounded in I. borrei; mentum subretangular, transverse, with subparallel sides in I. tetraspilota and subtrapezoidal in I. borrei), pronotum (I.borrei twice wider than long and with depression; I. tetraspilota 1.63 to 1.74 times wider than long and without depression), proendosternite (projections posteriorly directed slightly curved in I. tetraspilota and linear in I. borrei), mesonotum (mesoscutellum more broadly rounded in I. tetraspilota, with sides strongly convergent in I. borrei), metanotum (wider than long in I. borrei and as wide as long in I. tetraspilota), postnotum (lateral projected distally in I. borrei and not projected in I. tetraspilota), metendosternite (angular at the apex in I. tetraspilota and straight in I. borrei), epipleura (I.borrei large to the basal half; I. tetraspilota large to the basal third), and hindwings (Costa short, less than half of Subcosta in I. tetraspilota and elongated in I. borrei, at least two thirds of Sc; Posterior Radial and Anterior Anal thinned in I. borrei).
Furthermore, regarding the genitalia: 1) basal third of aedeagus strongly curved in I. borrei and slightly curved in I. tetraspilota, 2) tegmen V-shaped, with rounded base with a projection in I. borrei, and U-shaped, truncated base with a projection in I. tetraspilota, 3) vaginal palpi slightly to moderately separated from the apical third in I. borrei and widely separated in the apical half I. tetraspilota and 4) spermatheca nodulus with the same width as the rest of spermatheca and duct ending in the base in I. borrei, and nodulus strongly broader, cylindrical, and the spermathecal duct reaching the cornus in I. tetraspilota, providing information to revalidate Synbrotica after a further test with cladistical methods.
Morphological characters presented by the type species showed that the synonymy of genera performed by Aslam (1972) was premature, since he did not observe the type species of Synbrotica (subjective synonymy).The characteristics cited by Weise (1922) in original description were confirmed to Isotes, but cannot be considered diagnostic since they include other genera.As initially discussed, Bechyné & Bechyné (1969) reported six diagnostic features for Synbrotica, which did not include all species of the genus.Comparative and more comprehensive studies can reinstatement of Synbrotica, only those species with the characteristics listed above remain, as pointed out by Bechyné & Bechyné.
In the original descriptions, these species were considered as different species by the following characteristics (I.borrei versus I. crucigera): (1) body shape narrowly oblong-oval vs. oblong oval, (2) body slightly posteriorly enlarged vs. posteriorly enlarged, (3) apex of antenna yellow vs. articles 9 and 10 and base of the 11 whitish, (4) thorax yellow vs. thorax (this tinged dark) yellow, (5) thorax wide, with two foveae vs. thorax subarcuate excavated, groove more deeply impressed on both sides, (6) elytra punctured, margins, apex dilated, wide band near the middle, suture and apex yellow vs. yellow elytra with two large black stripes.