approximately 150,000 species of flies are described in the world and more than 24,000 species are described in the neotropics .
the genus zaprionus ( diptera , drosophilidae ) consists of approximately 60 species of which about 10 are grouped in the subgenus anaprionus and 50 in the subgenus zaprionus .
phylogenetic relationships within groups and subgroups of drosophilids as well as the phylogenetic position of zaprionus within the drosophilidae family are still uncertain .
although most phylogenetic analyses associate the genus zaprionus to the subgenus drosophila , new comparative analyses are needed to test the robustness of this association .
ultrastructural sperm analyses are important tools for study of the taxonomy and phylogeny of insects .
characterized the primary evolutionary radiation that occurred in the drosophila tripunctata group based on the ultrastructure of the mitochondrial derivatives and the number of sperm per cysts .
the authors highlighted the need for new ultrastructural studies of the gametes of these insects to provide additional clarification of their evolutionary relationships .
ultrastructural analyses of sperm in zaprionus are restricted to the species zaprionus indianus and zaprionus sepsoides .
the authors described important characteristics of these drosophilid gametes , such as the presence of granules in the peritoneal sheath , the presence of 2 mitochondrial derivatives of different sizes , the presence or absence of paracrystalline material in the derivatives , the arrangement of the axoneme , and the number of sperm per cyst .
this study aimed to characterize the ultrastructure of sperm of 6 other species of zaprionus ( z. africanus , z. camerounensis , z. davidi , z. gabonicus , z. megalorchis and z. tuberculatus ) and the species drosophila willistoni ( subgenus sophophora ) .
scaptodrosophila latifasciaeformis ( subgenus scaptodrosophila ) was used as an outgroup ( table  1 ) .
in addition , data from the literature for other species of zaprionus and drosophila were used to help understand the relationships between zaprionus and drosophila .
all species showed globular granules in the cytoplasm of the coating layer of the testicular envelope , called the peritoneal sheath ( fig .  1a , supplementary figs . 
16 ) , except for z. davidi ( fig .  1b , supplementary fig . 
these pigmented granules are responsible for the color of the peritoneal coating sheath of the testes and for the formation of a physical barrier that can protect the testes and store nutrients .
note peritoneal sheath ( ps ) filled with granules ( gr ) of different sizes and electron densities in z. africanus and their absence in z. davidi .
scale : figure  a : 11000 x ; figure  b : 10000 x. 
  tem micrographs of zaprionus testes .
note peritoneal sheath ( ps ) filled with granules ( gr ) of different sizes and electron densities in z. africanus and their absence in z. davidi .
scale : figure  a : 11000 x ; figure  b : 10000 x. the color of the peritoneal sheath of the testes is critically important for taxonomy . in the genus drosophila ,
the peritoneal sheaths of the zaprionus species analyzed in this study and those of d. willistoni and s. latifasciaeformis showed yellowing .
yellowing has also been observed in the sheaths of z. indianus , z. sepsoides and z. spinipilus that were analyzed previously .
however , z. vittiger was polymorphic for sheath color , which may be yellow or brownish purple .
the sperm of pterygota ( a primitive group of insecta ) has 2 mitochondrial derivatives that flank the axial filament . in drosophilids ,
used the size of these mitochondrial derivatives as an evolutionary tool to understand the radiation of the genus drosophila .
they observed that the 2 derivatives differed in size and that this size difference was greater in sophophora than in drosophila . in the present study , although measurements were not taken , the simple observation of prints showed that our results are consistent with those of mojica et  al .
: the difference in the size of these mitochondrial derivatives was greater in d. willistoni ( which belongs to the sophophora subgenus ) ( fig . 
suggested that the relative size of the mitochondrial derivatives may have changed as drosophila species have evolved . 

figure 2.ultrastructure of transverse sections of the spermatozoal tail of z. gabonicus ( a ) showing the paracrystalline material ( p ) on both mitochondrial derivatives ; the axonemes in d. willistoni ( b ) and z. davidi ( c ) have the arrangement of 9 + 9 + 2 microtubules ; the cysts containing 64 spermatozoa in z. camerounensis ( d ) and z. tuberculatus ( e ) .
scale : figures  a , b , c : 84000 x ; figures  d , e : 10000 x. 
 figure 3.ultrastructure of transverse section of the spermatozoal tail of s. latifasciaeformis ( a ) , z. tuberculatus ( b ) and z. megalorchis ( c ) note the presence of the axoneme ( ax ) and 2 mitochondrial derivatives of different sizes : larger mitocondrial derivative ( lm ) , smaller mitochondrial derivative ( sm ) ; the accumulation of paracrystalline material ( p ) is visible in s. latifasciaeformis and z megalorchis .
scale : figures  a , b , c : 84000 x. 
 table 1.studied species and their geographical origin.subgenusgroupcomplexspeciesgeographic location of the collectionzaprionusvittigerindianuszaprionus africanuskibale ( uganda)zaprionusvittigerindianuszaprionus gabonicusmakokou ( gabo)zaprionusvittigerindianuszaprionus megalorchiscongozaprionusvittigervittigerzaprionus camerounensisamani ( tanznea)zaprionusvittigerdavidizaprionus davidiso tom ( so tom)zaprionustuberculatustuberculatuszaprionus tuberculatusithala ( south africa)sophophora  willistonidrosophila willistonimato / sp ( brazil)scaptodrosophilascaptodrosophila latifasciaeformisso jos do rio preto / sp ( brazil ) 
 table 2.ultrastructural parameters of sperm used for comparisons of species . 
mitochondrial derivativesparacrystalline materialaxoneme 64 spermspeciesgranulessame sizedifferent sizelargest derivativesmallest derivative9 + 9 + 2per cystzaprionus africanus++++++zaprionus camerounensis++++++zaprionus davidi+++zaprionus gabonicus++++++zaprionus megalorchis++++++zaprionus tuberculatus++++zaprionus indianus*++++++zaprionus sepsoides*++++++drosophila willistoni+++++drosophila melanogaster*na++++drosophila simulans*na++++drosophila cardini*na+++drosophila dunni*na+++drosophila hydei*na++++drosophila subobscura*na++scaptodrosophila latifasciaeformis++++++note.*rego et  al .
2000 ) , * noguchi and miller ( 2003 ) , * pasini et  al .
( 1996 ) , * henning ( 1992 ) , * ramamurthy et  al . ( 1980 ) , * hauschteck - junguen and maurer ( 1976 ) , na : not analyzed . 
 
ultrastructure of transverse sections of the spermatozoal tail of z. gabonicus ( a ) showing the paracrystalline material ( p ) on both mitochondrial derivatives ; the axonemes in d. willistoni ( b ) and z. davidi ( c ) have the arrangement of 9 + 9 + 2 microtubules ; the cysts containing 64 spermatozoa in z. camerounensis ( d ) and z. tuberculatus ( e ) .
scale : figures  a , b , c : 84000 x ; figures  d , e : 10000 x. ultrastructure of transverse section of the spermatozoal tail of s. latifasciaeformis ( a ) , z. tuberculatus ( b ) and z. megalorchis ( c ) note the presence of the axoneme ( ax ) and 2 mitochondrial derivatives of different sizes : larger mitocondrial derivative ( lm ) , smaller mitochondrial derivative ( sm ) ; the accumulation of paracrystalline material ( p ) is visible in s. latifasciaeformis and z megalorchis .
scale : figures  a , b , c : 84000 x. studied species and their geographical origin .
( 2000 ) , * noguchi and miller ( 2003 ) , * pasini et  al .
( 1996 ) , * henning ( 1992 ) , * ramamurthy et  al . ( 1980 ) , * hauschteck - junguen and maurer ( 1976 ) , na : not analyzed . except for z.
davidi and z. tuberculatus ( figs .  2c and 3b ) , the analyzed species have paracrystalline material on both mitochondrial derivatives ( figs . 
this same characteristic has been observed in z. indianus , z. sepsoides and d. hydei . however , in most species of the genus drosophila ( table  2 ) , the paracrystalline material is present only in the larger mitochondrial derivative .
the structure of the sperm axoneme is of great importance for phylogenetic studies in insects .
most species of insects present the  9 + 9 + 2 arrangement , consisting of one pair of central microtubules and 9 double peripheral microtubules , surrounded by 9 additional accessory microtubules , although some species have a peculiar number , such as 9 + 9 + 3 in neuroptera , 9 + 9 + 1 in culicidae ( diptera ) , 9 + 7 in tricoptera , and 9 + 0 in ephemeroptera .
moreover , as the majority of species of the suborder brachyocera , all drosophilids species analyzed had an axoneme structure of the 9 + 9 + 2 configuration , which is the typical arrangement of 9 + 2 internal microtubules surrounded by 9 additional accessories microtubules ( figs . 
spermiogenesis in all analyzed species occurs within cysts where the sperm are organized and exist at the same developmental stage ( fig . 
this phenomenon is referred to as cystic spermatogenesis and is characterized by synchronized cell division within a given cyst .
so far , studies have indicated that all insects have cystic spermatogenesis . in triatominae ,
the cysts develop independently ; that is , a cyst does not influence the developmental stage of neighboring cysts . in drosophilids , as described for plalycentropus ( trichoptera : limnephilidae ) , we suggest that neighboring cysts are also synchronized for cystic spermatogenesis ( fig . 
for all of the species analyzed , we observed the presence of 64 cells inside a cyst ( fig . 
, this number varies from 44 to 56 sperm per cyst ; in d. cardini , it varies from 36 to 40 sperm per cyst ; in d. melanogaster , this fixed number is 64 ; and in d. subobscura , this number can reach up to 128 sperm per cyst . in the cluster analysis , on the basis of the analyzed characteristics ,
5 species of zaprionus as well as 6 species of drosophila were grouped , suggesting their relatedness within each genus ( fig . 
although an increase in the number of characteristics and species is necessary to validate these results , they are already indicative that the ultrastructure of sperm is a promising tool for phylogenetic and taxonomic studies of insect groups . 

figure 4.cladogram obtained from cluster analysis using a presence - absence matrix for the characteristics analyzed in the present study and taken from literature . 
 
cladogram obtained from cluster analysis using a presence - absence matrix for the characteristics analyzed in the present study and taken from literature .
the species and strains of zaprionus and other species used in this study and their geographic location are shown in table  1 .
the testes of 24 3-day - old adults of each species were processed according to the methods of cotta - pereira et  al . with modifications .
ultrathin sections of 70  nm , contrasted with uranyl acetate and lead citrate , were examined with a transmission electron microscope .
a cluster analysis was conducted using the euclidean distance and joining method ( statistica ; statsoft inc . ) with the data from table  2 from which a presence - absence matrix of the characteristics was generated .
jean david ( center national de la recherche scientifique ( cnrs ) / gif - sur - yvete / france ) for providing some of the lines of zaprionus , rosana silistino souza ( academic support assistant ii ) for technical assistance in sample preparation and luis fernando segala ( assistant professor ) for help with statistical analyses .
this paper was supported by fundacao de amparo a pesquisa do estado de sao paulo ( fapesp ) ( process number 2010/011939 ) and coordenadoria de aperfei , coamento do pessoal do ensino superior ( capes ) .
 abstractthe genus zaprionus consists of approximately 60 species of drosophilids that are native to the afrotropical region . 
 the phylogenetic position of zaprionus within the drosophilidae family is still unresolved . in the present study , ultrastructural features of spermatozoa of 6 species of zaprionus as well as the species drosophila willistoni and scaptodrosophila 
 latifasciaeformis were analyzed . 
 the ultrastructure revealed that the species have the same flagellar ultrastructure . 
 two mitochondrial derivatives , one larger than the other , close to the axoneme were present , primarily in d. willistoni ( subgenus sophophora ) . except for z. 
 davidi and z. tuberculatus , the analyzed species had paracrystalline material in both mitochondrial derivatives . 
 moreover , the testes showed 64 spermatozoa per bundle in all of the species . in the cluster analysis , 
 6 zaprionus species were grouped closely , but there were some incongruent positions in the cladogram . 
 the results indicated that sperm ultrastructure is an important tool for elucidating the phylogeny and taxonomy of insects . 
Introduction
Results and discussion
Materials and methods
Supplementary Material
Disclosure of potential conflicts of interest
Acknowledgments
Funding