Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 97, Num. 1, 2002, pp. 65-72

Mem Inst Oswaldo Cruz, Rio de 
  Janeiro, Vol. 97(1) 2002, pp.  65-72
A Cladistic Analysis of Inaequalium 
  (Coscarón & Wygodzinsky, 1984), with Information on Geographical Distribution 
  (Diptera: Simuliidae) 
MN Strieder/+, V Py-Daniel* 
Laboratório de Entomologia, Centro de Ciências 
  da Saúde, Universidade do Vale do Rio dos Sinos, Av. Unisinos 950, 93001-970 
  São Leopoldo, RS, Brasil *Instituto Nacional de Pesquisas da Amazônia, 
  Manaus, AM, Brasil

  + Corresponding author. Fax: +55-51-2590.8122. E-mail:strieder@cirrus.unisinos.br
This research received financial support from 
  the Universidade do Vale do Rio dos Sinos, Fapergs, and was partially supported 
  with a grant from MEC/Capes, Brazil, through the Programa Institucional de Aperfeiçoamento 
  de Pessoal de Nível Superior - PICDT, and is part of the first author's 
  PhD thesis. 

  
Received 19 January 2001 

  Accepted 18 September 2001 
Code Number: oc02011
The black flies of the genus Inaequalium 
  present a Neotropical distribution, with Panama at the northern limit, and the 
  Argentinian pampas at the southern, but do not occur in the Central Amazon. 
  This study offers a cladistic analysis establishing a hypothesis of relationships 
  between the species of Inaequalium. A total of 37 characters have been 
  considered in order to establish the hypothetic phylogenetic relationships. 
  Cerqueirellum (Py-Daniel, 1983) was considered as outgroup. Data were 
  analyzed using Henning 86 version 1.5. Wich the ie* command and implicit enumeration 
  a unique possible cladogram was obtained in Inaequalium with 52 steps, 
  and a CI of 0.76 and RI of 0.81. Two well-defined clades was obtained in the 
  resulting cladogram, the "botulibranchium" species-group, includes 
  I. travassosi, I. souzalopesi, I. botulibranchium and I. petropoliense, 
  and the "inaequale" species-group, includes I. rappae, 
  I. nahimi, I. inaequale, I. leopoldense, I. subnigrum, I. diversibranchium, 
  I. mariavulcanoae, I. nogueirai, I. beaupertuyi, I. clavibranchium and I. 
  subclavibranchium. 
Key words: Neotropical - aquatic insects - black 
  flies - Simuliidae - Inaequalium 
The geographic distribution of the genus Inaequalium 
  (Coscarón & Wygodzinsky, 1984) comprises South and Central America; 
  its northern limit is Panama and to the south it reaches the Pampas region in 
  Argentina, but is not found in Central Amazon. Most species are recorded from 
  the coastal mountains of Brazil (Coscarón & Wygodzinsky 1984, Coscarón 
  1987, 1991, Py-Daniel & Moreira 1989, Strieder et al. 1992, Coscarón 
  & Coscarón-Arias 1995, 1997, Strieder & Py-Daniel 1999, 2000). 
  
Coscarón and Wygodzinsky established the 
  taxon Inaequalium in 1984 as a subgenus of Simulium Latreille, 
  1802, having Simulium (Inaequalium) inaequale (Paterson 
  & Shannon, 1927) as the type species. Adults of the eight species initially 
  included in Inaequalium, are black of medium size with wings between 
  2.3-2.8 mm long; thorax laterally and posteriorly with a gray stripe; cibarium 
  of females with an elevated base in the median area, with conspicuous, well 
  sclerotized spines. Females also present short paraprocts, much wider than long. 
  Males have basistyle with length and width almost equal and longer than the 
  dististyle; dististyle presents a sub-triangular, sub-quadrate or sub-cylindrical 
  shape and has strong apical or pre-apical spines. Pupae have cocoons with a 
  dorsal length between 2.1-3.8 mm and ventral length between 2.8-4.5 mm; filamentous 
  gills with six terminal filaments ("inaequale" species-group) 
  or thick gills with two to six branches ("botulibranchium" 
  species-group). Larvae present 1 + 1 sub-triangular posterior ventral tubercles; 
  generally deep gular cleft, with a sub-triangular shape or reduced cleft, with 
  a semicircular format (I. souzalopesi); the third article of the antenna in 
  general darker and longer than the first and second (Coscarón & Wygodzinsky 
  1984). 
According to PyDaniel (1990) and PyDaniel and 
  Moreira Sampaio (1994, 1995), the recognition and utilization of the levels 
  of subfamily, tribe, genus and subgenus varies in the Simuliidae and that depends 
  on the point of view of the taxonomist. These authors, based on a revision of 
  Nearctic and Neotropical genera originally considered as Prosimuliini and the 
  genus Simulium from Simuliini, proposed to eliminate the tribal level 
  and to elevate all the subgenus of Simulium s.l. to generic rank. Under 
  these circumstances, Simulium Latreille, 1802 would be restricted to 
  Simulium s. str., in the Holarctic Zoogeographical region. Although many 
  taxonomists follows Crosskey and Howard (1997) which maintain the higher classification 
  of Crosskey (1987) and critique Py-Daniel and Moreira Sampaio (1994, 1995) hypothesis. 
  Coscarón et al. (1999) studied the species of Simulium from the 
  Neotropical region, after meticulous phylogenetic studies, these authors concluded 
  that the revised Neotropical species form an independent supraspecific group 
  of Simulium s. str., for which the name Asphatia was suggested. 
  
In this work, the elevation of the subgenus to 
  genus in Simuliidae is followed, according to the nomenclature used by PyDaniel 
  and Moreira Sampaio (1994, 1995), Py-Daniel (1997), Strieder and Py-Daniel (1999, 
  2000) and Andrade et al. (2000). Since species of the genus Inaequalium 
  show a homogeneous adult morphology it is necessary to intensify the search 
  for new characters to facilitate recognition and in order to correlate the characters 
  of different stages. The larvae and pupae present features that allow the differentiation 
  of species (Coscarón & Wygodzinsky 1984, Strieder 1998, Strieder & 
  Py-Daniel 1999, 2000). The present study provides new characters for recognition 
  of species of Inaequalium and by a comparative analysis of these characters, 
  presents a phylogenetic hypothesis for the genus. 
MATERIALS AND METHODS 
  
Following Coscarón and Wygodzinsky (1984), 
  Crosskey (1987), Crosskey and Howard (1997), Coscarón (1987, 1991), Py-Daniel 
  and Moreira (1989), Py-Daniel and Moreira Sampaio (1995), Strieder and Py-Daniel 
  (1999, 2000) a preliminary list of the species of simuliids included in the 
  genus Inaequalium (Coscarón & Wygodzinsky, 1984) was prepared. 
  From the revision of types, the examination of material available in important 
  collections and bibliographic data a matrix of characters was elaborated. New 
  characters that allow a better differentiation of the species were verified. 
  Elaboration of the relationship between the species of Inaequalium was 
  based on the phylogenetic systematics developed by Henning (1966). On the table 
  that originated the data matrix of characters a code for each one was designed, 
  in which the number "0" (zero) means hypothetically the plesiomorphic 
  state, the numbers "1" (one) and "2" (two) indicate the 
  apomorphic state and "?" indicate that the character is not comparable 
  or unknown for the taxon. In the polarization of the characters considered for 
  the elaboration of a phylogenetic hypothesis among the species of Inaequalium, 
  the taxon Cerqueirellum (Py-Daniel, 1983), which correspond to a monophyletic 
  group nearly related, was considered as outgroup (Coscarón 1987). Data 
  were analyzed using the Tree Gardener 1.0 program (Ramos 1996), which was developed 
  based on Henning 86 version 1.5 (Farris 1989). The ie* command and implicit 
  enumeration were used for calculating the cladogram. For the majority of the 
  characters there was no distinction or emphasis. However, the character 12 was 
  considered particularly additive. The convergent characters (c) and the reversions 
  (r) are indicated on the cladogram obtained. 
Only 15 species of the genus Inaequalium: 
  I. beaupertuyi (RamírezPérez, Rassi & Ramírez, 1977), 
  I. botulibranchium (Lutz, 1910), I. clavibranchium (Lutz, 1910), 
  I. diversibranchium (Lutz, 1910), I. inaequale (Paterson & 
  Shannon, 1927), I. leopoldense Strieder & Py-Daniel, 2000, I. 
  mariavulcanoae (Coscarón & Wygodzinsky, 1994), I. nahimi 
  (PyDaniel, 1984), I. nogueirai (D'Andretta & González, 1962), 
  I. petropoliense (Coscarón, 1980), I. rappae (PyDaniel & 
  Coscarón, 1982), I. subclavibran-chium (Lutz, 1910), I. subnigrum 
  (Lutz, 1910), I. souzalopesi (Coscarón, 1980) and I. travassosi 
  (D'Andretta & D'Andretta, 1947) were considered as final taxa in the phylogenetic 
  analysis. The others related species: I. clarki (Fairchild, 1940), I. 
  lurybayae (Smart, 1944), I. parimaensis (Ramírez Pérez, 
  Yarzábal, Takaoka, Tada & Ramírez, 1986), I. pseudoexiguum 
  (Nunes & Almeida, 1974), were not included due to their uncertain taxonomic 
  situation and/or insufficient data. 
RESULTS AND DISCUSSION 
  
In the present phylogenetic study 37 characters 
  (2 for females, 2 for males, 22 for pupae and 11 for larvae) were used (Table 
  I a b). 
  Table II shows the basic matrix related 
  to the results of the analysis of the 37 characters considered. Using the program 
  Hennig 86, ie* command, a unique possible cladogram presenting data of the characters 
  considered was obtained. It presents a length of 52 steps, and a CI of 0.76 
  and RI of 0.81 (Fig. 1). 
Cerqueirellum represents a monophyletic 
  taxon sustained by three synapomorphies derived from the morphology of the larva, 
  as follows: setae of cephalic apotome with internal branching; second external 
  teeth of the mandible absent or extremely reduced; and mandible with a row of 
  setae reaching the supramarginal inferior area. In the resulting cladogram of 
  Inaequalium, the species of the whole internal group are grouped independently 
  of Cerqueirellum, and this shows that these taxa are monophyletic. 
The monophyly of the genus Inaequalium is 
  sustained by the following characters: basal portion of female cibarium with 
  small teeth arranged over median prominency (1:1); and third anterior part of 
  distal brush of larval mandible presenting setae with bifurcation near insertion 
  base (36:1). This synapomorphies observed to Inaequalium justify the 
  treatment of this group as genus, instead of subgenus. This is in agreement 
  with Coscarón and Wygodzinsky (1984), Coscarón (1987) and Coscarón 
  and Coscarón-Arias (1997), who concluded that Inaequalium (as a 
  subgenus) form a monophyletic taxon. The cladogram obtained in Inaequalium 
  has two well-defined clades, the "botulibranchium" species-group 
  and the "inaequale" species-group, similar to those of Coscarón 
  (1987) and Coscarón and Coscarón-Arias (1997). 
The "botulibranchium" species-group 
  includes I. travassosi, I. souzalopesi, I. botulibranchium and 
  I. petropoliense. This clade is defined by three synapo-morphies and 
  two convergences: (5:1) texture of pupal gill filaments without reticulate; 
  (6:1) pupae with thick gill branches (c); (17:1) pupal frontoclypeus with multinodulous 
  tubercles; (25:1) pupal abdomen tergites I and II with spines; and (31:0) larvae 
  without setae on dorsal-posterior region of abdominal cuticle (c). This group 
  has a distribution restricted to the mountains of southeastern Brazil (Fig. 
  2). 
The "inaequale" species-group 
  includes I. rappae, I. nahimi, I. inaequale, I. leopoldense, I. subnigrum, 
  I. diversibranchium, I. mariavulcanoae, I. nogueirai, I. beaupertuyi, I. clavibranchium 
  and I. subclavibranchium. This group is defined by the following 
  synapomorphies: (23:1) and (23:2) pupa with thorax tubercles wider or as wide 
  as the diameter of the thoracic trichomes at point of insertion; (30:1) larval 
  anal gills with 13 to 20 lobes in each external branch. 
This second species-group is also predominantly 
  from the mountains of southeastern Brazil with a larger diversity in the Serra 
  do Mar region, where only I. beaupertuyi and I. nahimi do not 
  occur (Fig. 3). The species I. inaequale 
  and I. subnigrum present a large range, covering most of the tropical 
  area of South America, except the lowland region of the Central Amazon. The 
  only two species that are absent from the mountains of southeastern Brazil occur 
  respectively on the Escudo Venezolano and in the southern region of Amazon, 
  restricted to the Mato Grosso in the old western Brazilian shield. 
In relation to the two groups obtained in Inaequalium, 
  the results are similar to those presented by Coscarón (1987, 1991) and 
  Coscarón and Coscarón-Arias (1997). The results only disagree with 
  these authors in relation to the position of I. travassosi, which they 
  include in the "inaequale" group. 
The first branching of the "botulibranchium" 
  species-group separates I. travassosi from I. souzalopesi subgroup. 
  I. travassosi present a homoplasy: (27:1) antenna of larva smaller than 
  the stem of labral fan (c). 
I. souzalopesi subgroup present seven 
  synapo-morphies: (2:1) female paraproct basally longer than wide and acuminate 
  on distal half; (3:1) male distimere longer than twice its width at base; (4:2) 
  distimere with subcylindrical shape; (10:1) larval gill with ventral branch 
  curved; (14:1) pupal frontoclypeus and thorax with few tubercles; (29:1) larval 
  cervical sclerites big and fixed on cephalic apotome; and (33:1) larval hypostomial 
  bridge equal to or bigger than hypostomium. 
The characters that separate I. souzalopesi 
  from I. botulibranchium and I. petropoliense are the following: 
  (15:1) and (16:1) pupal frontoclypeus with subdivided tubercles and without 
  nodulosity; (18:1) and (24:1) larva with simple cephalic and thoracic trichomes; 
  (28:1) antennal articles of larva with transversal striations. 
I. botulibranchium subgroup is defined 
  by a synapo-morphy and a homoplasy: (8:1) pupal gill branches with appendages 
  (microfilaments or spines); (22:0) pupae with rounded and subtriangular tubercles 
  (r). 
One character of the pupa, (25:0) abdominal tergites 
  I and II without spines (r), characterize I. botulibranchium. 
I. petropoliense is characterized by one 
  homoplasy and two autoapomorphies of the pupa, as follows: (14:0) frontoclypeus 
  with abundant tubercles (r); (19:1) antennal sheaths with tubercles; and (21:1) 
  thoracic tubercles subtriangular shaped and with uniform distribution. 
The first branching of the "inaequale" 
  group separates I. rappae from the other species. 
I. rappae presents a homoplasy and an 
  autopomorphy: (6:1) pupal gill with thick branches (c); (20:1) pupal cephalic 
  genae with trichomes. 
One synapomorphy and a homoplasic state: ventral 
  cocoon length almost equal (12:1) or smaller (12:2) than length of gill branches 
  supported the monophyly of the species with filiform gills. 
The second branching of the "inaequale" 
  group results in two clades, respectivaly supported with a reversion and two 
  synapomorphies. These two monophyletic groups are composed by four and six species 
  respectively. The homoplasy: (35:0) lateral mandibular process straight or with 
  a slight depression in the middle area (r), is the character which congregates 
  the following species: I. nahimi, I. inaequale, I. leopoldense and 
  I. subnigrum. The synapomorphies: (4:1) distimere subquadrate shaped; and 
  (23:2) pupa with wide thoracic tubercles, relating to the diameter of thoracic 
  trichomes at point of insertion, sustain the monophyly of the following species: 
  I. diversibranchium, I. mariavulcanoae, I. nogueirai, I. beaupertuyi, 
  I. clavibranchium and I. subclavibranchium. 
The first branching of the first clade of species 
  with filiform gills separates I. nahimi from I. inaequale subgroup. 
  
I. inaequale subgroup is defined for: 
  (26:1) presenting tubercles in the abdomen tergite I of the pupa. The next branching 
  separates I. inaequale from I. subnigrum and I. leopoldense. 
  
I. inaequale presents a synapomorphy and 
  two homoplasies: (9:1) filiform gills with the primary ventral branch and the 
  two secondary dorsal branches generally of the same length; (12:2) cocoon length 
  smaller than the maximum length of the gill filaments (c); and (23:0) pupal 
  thoracic tubercles small, relative to the diameter of the thoracic trichomes 
  at the point of insertion (r). 
I. subnigrum and I. leopoldense 
  present a homoplasy: (31:1) setae from abdominal cuticle on the dorsal-posterior 
  region of larva absent or in a reduced number (c). 
The first branching of the second clade of species 
  with filiform gills separates I. diversibranchium and I. mariavulcanoae 
  from I. nogueirai subgroup. 
I. diversibranchium and I. mariavulcanoae 
  present a synapomorphy: (13:1) filiform gills with the primary dorsal branch 
  presenting bigger or equal length to ventral primary branch. 
The next dichotomy separates I. nogueirai 
  from I. beaupertuyi subgroup. 
I. beaupertuyi subgroup presents a homoplasy: 
  (12:2) ventral length of cocoon smaller than the maximum length of the gill 
  filament (c). 
I. clavibranchium and I. subclavibranchium 
  establish a monophyletic group, apart from I. beaupertuyi by a synapomorphy 
  and a homoplasy: (7:1) gill filaments of pupa with distal portion inflated; 
  (35:0) lateral mandibular process almost straight and not crossing inferior 
  margin of mandible (r). 
This work presents a cladistic analysis of the 
  genus Inaequalium (Coscarón & Wygodzinsky, 1984), which allows 
  the following conclusions to be made: (1) the analysis of 37 characters confirms 
  the existence of two clades morphologically well defined in this taxon. The 
  "botulibranchium" group, which brings together the species: 
  I. travassosi, I. souzalopesi, I. botulibranchium and I. petropoliense; 
  and the "inaequale" group, which brings together the following 
  species: I. rappae, I. nahimi, I. inaequale, I. subnigrum, I. leopoldense, 
  I. diversibran-chium, I. mariavulcanoae, I. nogueirai, I. beaupertuyi, I. 
  clavibranchium and I. subclavibranchium; (2) Inaequalium is 
  a monophyletic group, which presents a wide geographic distribution in the Neotropical 
  region occurring predominantly in the coastal region of Brazil, with a larger 
  diversity on the slopes of the Serra do Mar mountain range. 
ACKNOWLEDGEMENTS 
  
To Dr Sixto Coscarón, Universidad Nacional 
  de La Plata, Museo de La Plata, Argentina, for the incentive and for lending 
  specimens; to Dr Cecília Volkmer-Ribeiro, Fundação Zoobotânica 
  do Rio Grande do Sul; Dr Gervásio Silva Carvalho, Pontifícia Universidade 
  Católica do Rio Grande do Sul; Dr Gilson Moreira, Universidade Federal 
  do Rio Grande do Sul for helpful discussions. To Dr Paulo Günter Windisch, 
  Fernando Joner and José Eloy dos Santos Jr, Universidade do Vale do Rio 
  dos Sinos, for reviewing and improving the manuscript. 
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