The female genitalia of the genus Zabrus ( Coleóptera : Carabidae : Zabrini ) . I . The general structure and the subgenera Zabrus , Euryzabrus , Platyzabrus and Epomidozabrus

The systematics of the genus Zabrus Clairville, 1806 is currently based on morphological characters that show a high degree of parallelism. The aim of this study is to find new characters in the female genitalia, which give a better understanding of the phylogeny of the genus and result in a new classification based on monophyletic taxa (subgenera and species groups). For this pur­ pose slides of the whole female reproductive tract were studied under light microscopy and the gonocoxa IX under scanning micro­ scopy. The study of species belonging to eight subgenera of Zabrus, and of twelve subgenera of its sister taxon Amara, shows that Zabrus is characterised by the lack of a spermatheca. This apomorphy is reported for the first time in carabids, and corroborates the monophyly of the genus in comparison to Amara. A villous canal that is intimatelyjoined to the distal bursa copulatrix was found in both genera and in species of related tribes. This feature could be an apomorphy of the Harpalidae sensu Deuve (1988). However, only in Zabrus does the villous canal end in a long falciform head, which is probably another autapomorphy of the genus. It is postu­ lated that the primitive bursa copulatrix of the genus was made up of a moderately inflated basal half and an elongated and simple distal half. This pattern is found in species of the subgenus Zabrus, which are widely distributed, and the subgenera Euryzabrus, P!atyzabrus, and Epomidozabrus, which are endemic to the Iberian Peninsula. Extra lobules arising in the region where the two parts of the bursa meet, and cup-like infoldings of the distal bursa are possible apomorphic states that characterise the subgenera Pe!or and Iberozabrus. Gonocoxite 2 does not have the strong and short spines found in species of related taxa. Differences in size and shape of both gonocoxites, distribution of setation and microtrichia, and presence of furrow “pegs” are characters of potential phylogenetic interest, which should be thoroughly investigated in other subgenera.


INTRODUCTION
The genus Zabrus Clairville, 1806 is included in the subfamily Pterostichinae and the tribe Zabrini, and com prises about 105 species mostly distributed in the Medi terranean Basin.The current classification of the subgenera is mainly based on external characters of the adult and the geographic distribution of taxa (Ganglbauer, 1915).Most of these subgenera are endemic to particular areas and are probably monophyletic groups, which may be characterised by one or more apomorphies.However, the large subgenus Pelor Bonelli, 1810 is a polyphyletic assemblage of more than 60 species, a major systematic problem that remains to be solved.Freude (1986Freude ( , 1988Freude ( , 1989Freude ( , 1990) ) revised the genus Zabrus but used the same criteria as Ganglbauer, which contributed little to a better understanding of the nature of the species groups pro posed by Ganglbauer (1915) for Pelor.The subgenus Iberozabrus Ganglbauer, 1915 with 22 species also pre sents many taxonomic difficulties (Andujar & Serrano, 2001).As noted by these authors parallel evolutionary changes in external morphology are common among and within the subgenera of Zabrus, thus giving rise to homoplasies that make it difficult to analyse phylogenetic rela tionships.Characters other than those found in the external morphology of the imago are needed for assessing the phylogenetic relationships between the sub genera and establishing a new taxonomy based on these relationships.
The female genitalia of Zabrus are a possible source of taxonomic and phylogenetic characters (e.g., Holdhaus 1912;Liebherr & Will 1998;Ortuno 1994Ortuno , 1998;;Ortuno et al. 1992), which have not been adequately investigated.To date only Schuler (1968) has reported a few observa tions on Z. tenebrioides (Goeze, 1777) and Z. obesus Audinet-Serville, 1821.We have started a detailed analysis of the female genitalia of Zabrus, looking for characters that could have a phylogenetic and a taxo nomic value.In this paper we aimed to establish a general pattern for the genus and to study all species included in the nominal subgenus and three subgenera endemic to the Iberian Peninsula.We also studied species belonging to the genus Amara Bonelli 1810, the sister taxon of Zabrus, to help with the phylogenetic analysis.
The last abdominal segments of the female were gently squeezed with the forceps and introduced in an aqueous satu rated solution of KOH for 24-48 h.Then they were washed in Scheerpeltz's solution, opened dorsally to check alkaline diges tion, and stained with Chlorazol black E® in aqueous solution for 1-3 min under visual control.The excess of dye was removed by washing in hot KOH and the structures washed again in Scheerpeltz's solution.The dissected structures were placed in a watchglass with Scheerpeltz's solution and observed and drawn using a ZEISS stereomicroscope.Permanent slide mounts were made of selected dissections.A routine procedure was followed in preparing the female reproductive appendages for scanning microscopy.Observations and photographs were made using a JEOL JSM-6100.

The general structure of the female genitalia of Zabrus
The gonocoxa IX is well sclerotised and separated medially by a membranous articulation into gonocoxite 1 and gonocoxite 2 (Fig. 1).This last segment has on the ventral surface a subapical groove with two large senso rial setae (Fig. 7) and 1-3 short sensorial setae (Fig. 4; "furrow pegs" sensu Liebherr, 1989a, b), and some addi tional setae of varying size scattered over the segment (Figs 1b, 7).The dorsal surface has one conspicuous seta on each side of the basal half of the segment, but the strong and lateral spines we found in most of the species of Amara are absent.In some species there are additional setae dispersed over gonocoxite 2 (Fig. 5).
Gonocoxite 1 is more elongate and parallel sided (Fig. 1).On the ventral surface there are many setae, usually arranged in a triangular field close to the articulating membrane with gonocoxite 2, and many evenly distrib uted small setae (microtrichia).In some species this seg ment is ventrally grooved.In SEM images there are microtrichia grouped in sets of 2-6 small spines on the articulating membrane of both segments (Fig. 2); in the subgenus Platyzabrus these microtrichia are single toothed (Fig. 3).
The laterotergite IX is triangular in shape, weakly sclerotised and almost devoid of setae (Fig. 1a).
The basal half of the bursa copulatrix is usually inflated.It is joined to the distal tubular part via an invaginated area, where some additional lobules may be found (Figs 14,17).The excretory duct of the single spermathecal gland opens where the two regions of the bursa meet (Fig. 8).The distal bursa is more or less elongated and may show a variable number of cup-like infoldings (Figs 9,19).A villous canal arises at the beginning of the distal bursa and extends towards the common oviduct (Fig. 8).This canal ends in a well-developed glandular falciform head, in the region where the bursa and the common oviduct meet .The villous canal is also found in related species of Amara but lacks the falci form head. Species of other tribes also have this canal, which only becomes apparent after prolonged staining with Chlorazol black® (data not shown).
The most distinctive feature of the genus is the lack of a spermatheca, a structure that is present in species of Amara and other more distantly related taxa of the tribes Pterostichini, Platynini and Sphodrini.

The subgenus Zabrus
Gonocoxite 2 is short and has a rounded apex.The ven tral groove has one to three furrow pegs.Gonocoxite 1 is ventrally grooved in Z. ignavus, and smooth in Z. tenebrioides and Z. morio.The bursa copulatrix in the three species of this subgenus has a relatively simple tubular region, which is very long in Z. ignavus (Fig. 10), short in Z. morio (Fig. 8), and with three rudimentary infoldings in Z. tenebrioides (Fig. 9).

The subgenus Euryzabrus
Both gonocoxites are elongate and of about the same length, gonocoxite 2 has a large number of setae on the ventral surface and an unusual row of setae on each margin of the dorsal surface.The articulating membrane between these gonocoxites varies from complete to par tially incomplete (Fig. 6).The bursa copulatrix is among the simplest in the genus, with a short tubular region.The villous canal has a long glandular head (Fig. 11).

The subgenus Platyzabrus
Gonocoxite 2 is relatively long and may have up to 10 setae.The tubular part of the bursa lacks the infoldings seen in other species but is somewhat dilated close to the junction with the common oviduct.The basal half of the bursa has two clear infoldings (Figs 12,13).

Other subgenera of Zabrus
The species of other subgenera usually have an inflated basal portion to the bursa copulatrix , fol lowed by an elongated distal part with a variable number of infoldings.In the case of Z. (Pelor) corpulentus, there are 7-8 of these cup-like infoldings (Fig. 19).In the sub genus Iberozabrus there may be additional features such as some extra lobules arising from the region where the two parts of the bursa meet (Z.seidlitzi, Fig. 17).

The subgenus Epomidozabrus
Gonocoxite 2 is relatively short (slender in Z. mateui) and has a deep apical groove without furrow pegs in Z. flavangulus.This needs to be confirmed by studying more individuals.The dorsal surface of this gonocoxite has a few setae in Z. mateui.Gonocoxite 1 has a welldeveloped field of setae close to the articulating mem brane between the gonocoxites.It is clearly grooved in Z. mateui, and almost smooth in Z. humeralis and Z. flavangulus.The tubular part of the bursa is relatively simple, without clear infoldings.An extra lobule arises where the two parts of the bursa met in Z. flavangulus (Fig. 14) and (to a lesser extent) Z. humeralis (Fig. 15), but is almost wanting in Z. mateui (Fig. 16).

The general structure of the female genitalia in Zabrus
The most remarkable feature of the genus is the lack of a spermatheca, an organ that is present in all the carabids studied to date.Only in Loxandrus celeris (Dejean, 1828) (tribe Loxandrini) is the spermatheca markedly reduced (Liebherr & Will 1998).The presence of the spermatheca in species of the closely related genus Amara was shown by Ali (1967) and corroborated by this study.Schuler (1968) described a spermatheca in Zabrus tenebrioides, but his drawings indicate he is referring to the villous canal and its falciform head.In the case of Z. (Pelor) obesus he correctly noted the lack of a spermatheca.This character state is thus considered an autapomorphy indi cating the monophyly of the genus.Freude (1986) ques tioned the separation of Zabrus from its sister taxon, the genus Amara, because some species of the latter show one or two of the diagnostic traits of a typical adult Zabrus (e.g., the single periorbital seta found in A. (Polysitamara) luppovae Kryzhanovskij, 1962).However, the lack of a spermatheca plus the high chromosome number and some larval characters of Zabrus (Andujar & Serrano, 2000) support the monophyly of Zabrus.
Both Zabrus and Amara have a villous canal similar to that described for the tribes Licinini and Panagaenini among others (Liebherr & Will, 1998), all of which belong to the phyletic lineage of "modern" carabids (the Conchifera stock of Jeannel, 1941, or the Harpalidae of Deuve, 1988).This villous canal may be a general feature of this large lineage, as Ortuno described it in species of the tribes Chlaenini (1994) and Oodini (1998), and we have found it in species of the tribe Harpalini (data not shown).However, only in Zabrus does the villous canal end in a well developed falciform shaped head (perhaps of glandular nature), which appears to be another autapomorphy of the genus.The differentiation of the bursa copulatrix into two regions, a distal one that has either a simple tubular shape or from 1 to 12 cup-like infoldings and a basal that has a more or less inflated shape, is also remarkable.Schuler (1968) reported the extraordinary number of infoldings in the distal bursa of Zabrus (Pelor) obesus.Both regions of the bursa may serve to store sperm, although as Liebherr & Will (1998) noted, the function of the various components of the female repro ductive tract in Carabidae is unclear.
Gonocoxite 2 is variable in size, shape, and number and distribution of setae, both within and between the tribes of Carabidae (see for example Liebherr & Will, 1998).It has not the strong and short spines that are found in spe cies of Amara (although the presence of these spines varies within and between its subgenera), and in the related tribes Pterostichini, Platynini, and Sphodrini.

Variation in the general structure in the subgenera of Zabrus
The structure of the female genitalia of the subgenus Zabrus is relatively simple, which accords with the primi tiveness of the three species of the subgenus, which can fly, have a large distribution area, and are not limited to particular mountains (Andujar & Serrano, 2001).The species of this subgenus are considered to be closely related to the ancestors of the genus (Andujar & Serrano, 2001).The three species can be identified by the differ ences described above.The gonocoxites of the single spe cies of the subgenus Euryzabrus, Z. pinguis, are unusually elongate and of about the same length, a char acter perhaps related to the preference of the species for sandy places on the NW coast of the Iberian Peninsula.On the other hand, the bursa is very simple.These fea tures plus other morphological peculiarities described by Andujar & Serrano (2001), indicate the phylogenetic iso lation of Euryzabrus.
In the subgenus Platyzabrus gonocoxite 2 has a remarkably high number of setae.Both species of the subgenus, Z. constrictus (Sierra de Bejar) and Z. pecoudi (Sierra de Gredos), have a very similar female genitalia.Their overall similarity suggests they are a result of recent allopatric speciation in the mountains of the Sistema Cen tral (middle Iberian Peninsula).Minor but consistent dif ferences are found in the female genitalia of the three species of the subgenus Epomidozabrus.The most remarkable feature is the variously developed extra lobule where the two parts of the bursa meet.This feature plus the morphological apomorphies (particularly the thick basal margin of elytron and protruding humeral tooth) and its geographic distribution (NW of the Iberian Penin sula) indicate its monophyly.
These four subgenera have a simple distal tubular region of the bursa (only Z. tenebrioides has some ves tigial infoldings), a character state that should be consid ered primitive compared to the complex pattern of infoldings found in other subgenera (Pelor, Iberozabrus).This ancestral character of this simple bursa does not sup port a close relationship between these four subgenera, which agrees with the conclusion of Andujar & Serrano (2001) based on a cladistic analysis of the external mor phology.
From this study it is concluded that the female repro ductive tract of Zabrus is a valuable source of characters for the phylogenetic analysis of the genus.These include (at least) the length, shape and setation of the gonocoxites, the number of furrow pegs, the microtrichia on the articulating membrane between the gonocoxites, the development (infoldings) of the distal tubular bursa copulatrix, and the presence of extra lobules at the origin of the distal bursa.Further studies now in progress on all the subgenera and species of Zabrus will assess the phy logenetic value of these characters.It is expected that they, together with others based on external morphology, the sequence of cytochrome oxidase I (Sánchez-Gea et al., in prep.) and the karyotype data (Galián et al., 1991, and unpubl. data), will contribute to a better under standing of the phylogeny and evolution of this complex genus.