PHYLOGENETIC RELATIONSHIPS OF A NEW SPECIES OF APOSTOLEPIS FROM BRAZILIAN CERRADO WITH NOTES ON THE ASSIMILIS GROUP (SERPENTES: COLUBRIDAE: XENODONTINAE: ELAPOMORPHINI)

The assimilis species group is one of the several assemblages that can be distinguished within the genus Apostolepis. The group is characterized by a uniform red dorsal pattern, broad nucho-cervical collars, enlarged light snout blotch, dark ventral head, and entirely black terminal shield. A new species is here recognized as a member of the assimilis group and its phylogenetic relationships to A. assimilis and A. cearensis is inferred by means of cladistic analysis. Apostolepis ammodites sp. nov. can be easily diagnosed from all congeners by many autapomorphies and an exclusive combination of features. Comparative data are presented on scalation, coloration, dentition, osteology, and geographic distribution, with some notes on feeding and reproductive biology. The new species occurs through central Brazilian Cerrado, filling a gap between the areas occupied by A. assimilis and A. cearensis. Although superficially resembling A. assimilis, parsimony analysis based on morphological data indicates its phylogenetic relationships closest to A. cearensis. Systematic analysis shows that two recently described species, A. freitasi and A. tertulianobeui, must be relegated as junior synonymies of A. cearensis and A. assimilis, respectively.


INTRODUCTION
The South American fossorial snakes of the genus Apostolepis Cope 1862 consist of a highly speciose clade of about 30 species, and a number of yet undescribed ones.The taxonomy of such an already difficult group (Vanzolini, 1986;Ferrarezzi, 1993;Harvey, 1999) has become increasingly complex, not only due to the lack of a comprehensive systematic review but, also because of a recent proliferation of poorly described new taxa (e.g., Lema, 2002Lema, , 2003Lema, , 2004a,b),b).This is the case of two new species, lately described by Lema (2004a,b) and based on a single specimen each: Apostolepis freitasi from central Bahia, and Apostolepis tertulianobeui from Minas Gerais, both being undistinguishable from Apostolepis cearensis and Apostolepis assimilis, respectively, but clearly referred to the assimilis group as defined by Ferrarezzi (1993).
The Instituto Butantan received, in the last few years, several specimens of a clearly distinctive but unnamed elapomorphine species of the genus Apostolepis, that seems to be quite common within the Cerrado domain of central Brazil.It appears to be intermediate between Apostolepis assimilis and A. cearensis, in superficial morphology and through it geographical distribution.The present paper refers to the formal definition and diagnoses of the assimilis species group along with an attempt to elucidate the taxonomic status and systematic relationships of its component species.

MATERIAL AND METHODS
Specimens: A total of 25 museum specimens known to represent the new species were examined.In addition to published figures and descriptions, representative individuals or samples of most species of Apostolepis were analyzed (Ferrarezzi, 1993), in order to fundament a brief character analyses of the group.Relevant material observed for external features (scalation, color pattern and proportions), cranial osteology, and hemipenial anatomy is listed in Appendix 1. Specimens from the following institutions with there respective acronyms were analyzed (used in the text, figures, and appendix): IB, Laboratório de Herpetologia, Instituto Butantan, São Paulo; MNRJ, Museu Nacional, Universidade Federal do Rio de Janeiro; CHUNB, Coleção Herpetológica da Universidade de Brasília; MZUSP, Museu de Zoologia da Universidade de São Paulo; FUNED, Fundação Ezequiel Dias, Belo Horizonte, MG.
Along with the description of the new species we report the state observed for nearly all external characters currently thought to be diagnostic for the new species relative to its congeners (Ferrarezzi, 1993;Harvey, 1999;Harvey et al., 2001).Morphological information about members of the dorbigny species group were obtained from Harvey, 1999 and Embert (pers. comm.) on Ventral shields were counted using Dowling's (1951) method.
In addition to the observation of external morphology, we prepared osteological and hemipenial material of the new species and its closest relatives Apostolepis assimilis and A. cearensis, as well as of A. pymi and A. flavotorquata, used as outgroups.Skulls of preserved specimens were prepared either dried (with the aid of dermestid beetles and by chemical maceration).Hemipenes were mounted with agar jelly, following the procedures described by Pesantes (1994) for inverted organs from preserved specimens, or by Manzani & Abe (1988) for freshly killed ones.The nomenclature used for hemipenial structures is based on Dowling & Savage (1960) and Zaher (1999).
Species habitat correlations were made by plotting distribution localities in the South American vegetation map of Hueck & Siebert (1972).
Comparative analyses: Monophyly of an ingroup comprising the assimilis species group (Apostolepis assimilis, A. cearensis and A. ammodites sp. nov. -sensu Ferrarezzi, 1993) has been assumed as a working hypothesis.Representatives of the dorbignyi group (A.dorbignyi, A. multicincta) were included as the closest outgroup, whereas those belonging to the flavotorquata group (A.flavotorquata, A. pymi) were taken as a more distant outgroup to root the tree resultant from cladistic analysis.The empirical justification for this assumption about ingroup-outgroup demarcation can be found in Ferrarezzi (1993), but it is also implicit in our character matrix.Character states observed were numerically coded in discrete binary and multistate transformation series, in order to construct the data matrix ready to be subjected to a parsimony analysis using Hennig86 applicative (Farris, 1982).Statements regarding evolutionary direction (polarity) of morphological transformation series (character states) were only assessed a posteriori to parsimony analyses, following the principles recommended by Farris (1982) and Nixon & Carpenter (1993).
The criterion used to evaluate phylogenetic relationships between the new species and other ingroup taxa was apomorphy based (Hennig, 1966).Systematic relationships and biogeographic history of the new species are briefly discussed in a cladistic and vicariance context.

The assimilis species group
Definition and diagnosis: Apostolepis of medium size; nasal separated from preocular; fifth supralabial scale only in contact with the parietal; temporal 0+1; terminal shield pointed in a sharply edge; large internasal white blotch covering the snout and reaching the frontal shield; moderate to extremely broad and sharply evident white and black nucho-cervical collars; dorsal pattern uniformly red (without any trace of dark stripes); white ventral surface, except the gullar region which is dark pigmented (black spoted) to mostly black; terminal shield black, undifferentiated from the tail tip coloration.Duvernoy's gland moderately developed; harderian gland well developed, reaching the temporal region, adductor jaw muscles moderately developed, reaching the dorsal surface of the brain case.
Distribution: Northeastern, central, and southeastern Brazil to neighboring northeastern Argentina; through the Caatinga and Cerrado domains to mesophytic forest of upper Paraná.

Remarks:
The monophyly of the assimilis group, as here defined, is still uncertain due to the complete lack of osteological information for several species not analyzed in the present study.Elapomorphus assimilis Reinhardt, 1861:235.Type-locality: Minas Gerais, Brazil.Apostolepis tertulianobeui Lema, 2004b:155-160 (New synonym).
Natural history: Apostolepis assimilis is an inhabitant of open vegetation areas from Atlantic forest and Cerrado, also considered an urban species (Pereira et al., 2004).It preys on small amphisbaenians and scolecophidian snakes (Ferrarezzi, 1993;Marques et al., pers. comm.).A female had six vitellogenic follicles and the largest measuring was 30,91 mm.
Remarks: A. tertulianobeui Lema, 2004 is clearly a member of the assimilis species group (sensu Ferrarezzi, 1993), judging from color pattern and scalation features.In most relevant diagnostic features presented by Lema (2004b), the holotype of A. tertulianobeui does not differ from the range of variation we have observed in a large sample of A. assimilis (Ferrarezzi, 1993).Eventually, a narrower cervical collar can be found in a few individuals as an intrapopulational polymorphism, but a reduced dark pigmentation, as referred by Lema (2004b), may be better explained by fading in the long time preserved holotype specimen.Although there is no specific type-locality, besides the state of Minas Gerais, the distrbution of A. tertulianobeui is cincident with the distributional area of Apostolepis assimilis (Fig. 6).Therefore, even though we did not examine the holotype of A. tertulianobeui, we have no doubt that this name must be relegated as a junior synonym of A. assimilis.
Remarks: The recently described species A. freitasi Lema, 2004 is clearly a member of the assimilis species group (sensu Ferrarezzi, 1993), judging from the features of color pattern and scalation.In all diagnostic features presented by Lema (2004a), the holotype of A. freitasi is identical to A. cearensis.It is not surprising that Lema (2004a) traces no comparison of his new species with the last one, since if this have been done, the author would have failed to find any diagnostic feature at all.The same can be said about the typelocality which is within of the geographic distribution area of A. cearensis (Fig. 6).Therefore, even without having examined the holotype of A. freitasi, we have no doubt that this name must be included as a junior synonym of A. cearensis.
Apostolepis ammodites sp.nov.Etymology: Ambivalent connotation, from the Greek, αµµοσ (ammos) meaning sand plus δψτεσ (dytes, the internal 'y' may be rendered by 'i' in Latin) meaning anyone that likes immersions or diving, but furthermore from the Latin ammos meaning around or having two plus dites meaning richly adorned or sumptuous.
In allusion both to the habitat and habits of the new species and to its unique extravagant nucho-cervical color pattern having two white collars around a black collar.
Scalation: (Fig. 4) Rostral somewhat prominent but roundish, the portion visible from above about 1/2 to 1/3 of its distance to frontal.Nasal always separated from preocular by a broad prefrontal contact with second supralabial.Six supralabials (second and third entering eye).Temporals 0+1, usually only the fifth supralabial in contact with parietal (occasionally also the forth making a slightly contact).Occipitals 3-5, the lateral very large.Synfisial separated from chinshields by contact between first infralabials; usually seven infralabials, the first four in contact with anterior (mean = 28.5) in females (n = 13) (Table 1; Fig. 8); terminal caudal shield short, high and blunt pointed, in contact with 5-6 scales around.
Dentition and cranial osteology: (Table 2; Figs. 9 and 10) Tooth series short and with stout teeth.Maxillary very short, with four solid teeth increasing in size posteriorly followed, after a distinct interspace, by a pair of large deeply grooved fangs, situated bellow the eye.Five palatine followed by three or four pterygoid teeth; the anterior end of pterygoid barely extending beyond its articulation with ectopterygoid, and just touching the palatine.Dentary short, bearing 9-10 subequal teeth, increasing in lenght to the forth and then gradually decreasing to the last one.Reduced posterior process of dentary extending just a behind splenial-angular hinge; meckelian channel closed along most of the dentary length.Ectopterygoid flattened and deeply forked anteriorly, forming a fairly large maxiloectopterygoid fenestra.Premaxillae very thick and robust, the lateral processes bounded to the maxillae, and with a stout dorsal process making very broad contact with the dorsal laminae of nasals, which is very wide and nearly rectangular, broader than and making an extensive transversal suture with the frontal and with the antero-dorsal edge of prefrontal.Supratemporals small, distantly separated from parietal and not reaching the posterior level of occipitals.Postorbital bone absent.
Hemipenis: The organ is subcilindrical and stout with a narrower base.Apex is just slightly bilobate, calyculate, and semicapitate.Spines are nearly uniformly distributed around the body.The sulcus spermaticus is forked slightly above the mid-length of the organ, with the branches running centrolineally to the extreme apex.
Coloration in life: (Fig. 3) Dorsum uniform vivid orangered (without any vestige of dark stripes); head black postero-laterally (to firsts occipital rows), with a large reddish blotch on snout (covering entirely the prefrontals to reach the anterior end of frontal and supraoculars), a small to indistinct cream supralabial spot posterior to the eye is mostly restricted, if not confined, to the forth supralabial; a broad white nuchal collar (3-6 scales long) followed by a similar extensive black cervical collar (4-7 scales long); and an additional white postcervical collar (1-3 scales long); ventral surface uniformly light (unpigmented), except under the head and neck, where the infralabial, chin and gullar regions are mostly black.The dorso-ventral tail coloration is similar to that of body, and with a distinct black band around the tail tip (7-11.5 scale long); terminal caudal shield uniformly black (not differentiated from tail tip coloration).
Distribution: All the known distribution records for the new species fall within the central and northeastern portion of the Cerrado morphoclimatic domain (Fig. 6).
Natural History: Apostolepis ammodites sp.nov. is a inhabitant of open vegetation areas of Cerrado (Fig. 5), where it appears to be restricted to sandy soils (F.Curcio, C. Nogueira and M.T. Rodrigues, pers. comm.).
A specimen (IB 64613) contained digested remains of skin of a small unidentified Amphisbaena and the head of an ant of the subfamily Attinae.The ant's head is most probably the secondary remain from the stomach content of the amphisbaenid prey.
The new species is oviparous (A.Silveira, pers.comm.).A female (IB 65261, 502 mm SVL) had three vitellogenic follicles and the largest measuring was 15,99 mm.Five males (IB 62593, IB 65262 IB 65263, IB 65268, and IB 65664) had large testes and convoluted deferent ducts.The largest male (IB 62593, 450 mm SVL) had the largest test, with 13,86 mm and was collected in August/2000.The smallest male (IB 65268) with large testes and opaque deferent ducts had 282 mm SVL.It was collected between December/2001 and January/2002.

Characters and character states coded for the cladistic analysis
Characters and respective character states numerically coded and listed below correspond to those in the data matrix of Table 3, which are optimized in the cladogram presented in Figure 11.Linearly ordered multistate transformation series (denoted by NA) were managed under the assumption of additivity during parsimony analysis.

Results of Cladistic analysis
Parsimony analysis of the data matrix in Table 3 resulted in a single most parsimonious tree, which was rooted in the flavotorquata species group (Fig. 11).This cladogram presents a highly consistent pattern of character congruence, as denoted by its consistency and retention indexes: length = 43; CI = 0.97, RI = 0.97 (the values are the same under both Wagner and Fitch parsimony options applied to ordered multistate characters).Even if the total amount of data is partitioned into different character classes (vis: coloration, scalation, osteology, or ecology), the same single tree topology is obtained from each independent analyses of the four subsets.
The results of the cladistic analysis demonstrate that A. ammodites sp.nov. is closer to A. cearensis than A. assimilis (Fig. 11).This last species appears as the most conservative lineage within the clade.At least with regard to the character sample analyzed here, it remains nearly identical to the common ancestor of the tree species.On the other hand, the lineage leading to A. ammodites sp.nov.and A. cearensis is the most divergent lineage morphologically, judging from an amount of synapomorphic and autapomorphic traits of coloration and, especially, cranial osteology (Figs. 9,10 and 11).
Regarding the evolutionary rate of the different classes of morphological data, the scalation features represent the most conservative ones, while the cranial osteology is the most divergent.

DISCUSSION AND CONCLUSIONS
At least three other species exhibiting entirely black terminal shield may be further proven to belong to the assimilis group Ferrarezzi, unpublished data).These are Apostolepis arenaria Rodrigues, 1993, andA. gaboi Rodrigues, 1993, a pair of closely related species that shares apomorphies with both A. ammodites sp.nov.and A. cearensis, plus a few additional features only with the latter, (and thus, may be sister to A. cearensis).On the other hand A. quirogai Giraudo & Scrocchi, 1998 lacks most derived features of the other species, and may represent the sister taxa of the whole group.The phylogenetic relationships of all of them are left to be discussed in a further study.By now, it is sufficient to explain that keeping them out of here will not disqualify our analysis about the pattern of cladistic relationships of the other species, since they have also been excluded from outgroup comparison.
The present discovery of new diagnostic characters arranged in a simple congruent pattern of relationships, although just for a few species, stands as a first step towards a comprehensive resolution of the whole group.The original synapomorphy squeme proposed here represents a testable phylogenetic hypothesis to be corroborated, modified or (mostly important) even refuted, after an advanced survey on these character states for other putative members of a broad-FIGURE 11.Cladogram depicting the phylogenetic relationships among the components of the assimilis species group of Apostolepis.This is the single most parsimonious tree derived from the cladistic analysis of the data matrix of Table 3 (length = 43; CI = 0.97, RI = 0.97 under both Wagner and Fitch parsimony options applied to ordered multistate characters).The head and neck external morphology was reconstructed for the hypothetical common ancestors, as a corollary of our theory about cladistic relationships and character state polarities.Differently colored numbers represents four sets of characters.The four small cladograms below represents the tree topology derived from independent analyses of each character set.
based assimilis group, as well as other Apostolepis in general.
Finally, given the allopatric pattern of distribution (Fig. 6), a hypothesis of the biogeographic history under the assumption of a vicariance model is developed (Fig. 12).The biological area cladogram resulting from the recovered cladistic relationships for the assimilis group of Apostolepis indicates that the northeastern portion of the Cerrado is more closely connected to the Caatinga than to the southwestern Cerrado region.Further area cladograms derived from other groups of organisms exhibiting the same areas of endemism are needed in order to test the generality of this conjecture.

FIGURE 5 .
FIGURE 5. Physiography of an area inhabited by Apostolepis ammodites sp.nov., in the locality of Correntina (Bahia state), where specimen CHUNB s/nº were collected.Photo by C. Nogueira.

FIGURE 6 .
FIGURE 6. Geographic distribution of Apostolepis ammodites sp.nov.(plotted stars represent localities for the type-series -an encircled star indicates the type-locality), with regards to the distribution area of its closest relatives A. cearensis (vertical lines) and A. assimilis (horizontal lines).Note the tight distributional limits of this allopatric pattern exhibited by the components of the assimilis species group.

FIGURE 8 .
FIGURE 8. Numbers of ventrals and subcaudals plotted for the type series of Apostolepis ammodites sp.nov.(circles), in comparison with a sample of A. assimilis (triangles) and A. cearensis (squares).Empty symbols represent females and full symbols represent males.

FIGURE 12 .
FIGURE 12. History and pattern of biogeographic relationships among the distribution areas of the species belonging to the assimilisgroup, as hypothesized following a vicariance model of speciation.

TABLE 2 .
Tooth counts for the species of the assimilis group of Apostolepis.

TABLE 1 .
Selected external features diagnostic of Apostolepis ammodites sp.nov.relative to A. cearensis and A. assimilis.