Reappraisal of Rhamdia branneri Haseman , 1911 and R . voulezi Haseman , 1911 ( Siluriformes : Heptapteridae ) from the rio Iguaçu with notes on their morphometry and karyotype

The species Rhamdia branneri Haseman, 1911 and the subspecies Rhamdia branneri voulezi Haseman, 1911 from rio Iguaçu are currently recognized as synonyms of Rhamdia quelen (Quoy & Gaimard, 1824). However, recent karyotype and ecomorphology studies distinguish R. branneri and R. voulezi as different species. Examination of Rhamdia populations from rio Iguaçu, including type specimens, together with other congeners from rio Tibagi, allowed to properly reexamine the situation of these Haseman’s taxa and references given to Rhamdia in the Iguaçu. The species R. branneri and R. voulezi have strong serrae with large basis on both margins of the pectoral-fin spine, uncommon in the remaining species of Rhamdia and different from the fine serrate margins of the pectoral spine of R. quelen; a regular dorsal profile, slightly curved between supraoccipital and dorsal fin; with dorsal darkbrown or light-gray coloration along body, abdomen pale, without profuse small black spots, common in populations of the upper Paraná species and the type-material of R. quelen. Also the following morphometric characters discriminate these species: Rhamdia voulezi, adipose fin elongate; smaller distance between dorsal and adipose fin; smaller length between adipose fin to base of caudal fin and smaller distance from posterior margin of eye to opercular border. Rhamdia branneri, larger distance from dorsal and adipose fins; deeper caudal peduncle; higher trunk depth in the vertical distance through adipose, between pelvic and anal fins; larger scapular bridge; shorter maxillary barbel; shorter external mental barbel; shorter interorbital distance; shorter length of dorsal fin basis and shorter adipose-fin base length. A PCA between the populations of Rhamdia from Iguaçu and a population from rio Tibagi, upper Paraná basin, discriminates the population from Tibagi on basis of dorsal to adipose fin distance, dorsal-fin spine length, maxillary barbel length, eye diameter, and pectoral-fin spine length. This morphometric study allied to the karyotype known differences suggest R. branneri and R. voulezi as valid species. The complex state of R. quelen with the neotype recently designated from rio Samiria recommend new studies on basis of molecular genetics and provision of the names R. branneri and R. voulezi in the Iguaçu basin.


Introduction
Rhamdia Bleeker, 1858 is recognized by the combination of the following characters proposed by Silfvergrip (1996): three pair of barbels; vomer bone edentulous; double lip fold; posterior process of the fourth parapophysis expanded distally with one large and several smaller indentations; supraoccipital process free from supraneural plate; orbital rim free; adipose fin with free posterior margin, posterior fontanel closed and posterior process of cleithrum (humeral process) well developed.One additional feature that diagnoses Rhamdia is the forked caudal fin with rounded lobes.None of these characters, however, are exclusive to Rhamdia and there is no attempt to test the monophyly of Rhamdia or to determine the synapomorphies that support the genus.
On the other hand, during the last century the genus Rhamdia appeared with 66 described species, as listed by Gosline (1945) and accepted by Mees (1974).Silfvergrip (1996) revised the genus and recognized only 11 valid species, distributed from Mexico to Argentina and confined to the Cis-Andean region of South America as accepted by Bockmann & Guazelli (2003) and Ferraris (2007).With respect to the Iguaçu Rhamdia, Shibatta & Garavello (1995) studied populations of this genus in accord that this area perhaps retain exclusive species of Rhamdia.Also Garavello & Shibatta (2007), Garavello et al. (2012) and Baumgartner et al. (2012), inform the Iguaçu basin as an area of medium endemism with native species inclusive those of Rhamdia.
From this area, Haseman (1911) described Rhamdia branneri, Rhamdia branneri voulezi and identified other sympatric species as Rhamdia quelen (Quoy & Gaimard, 1824), combining the following characters: supraoccipital longer in R. branneri voulezi compared to R. branneri; head proportionally larger than trunk; and adipose fin and barbels relatively shorter than in R. quelen.As originally identified by Haseman, the subspecies R. branneri voulezi was distinguished from R. branneri on basis in the very elongate post-mentonian barbel that surpass the pectoral-fin insertion, and the long maxillary barbel reaching to adiposefin origin.Silfvergrip (1996) refute the characters utilized by Haseman arguing they are labile and put Haseman´s species in the synonymy of R. quelen.
Further, Abucarma & Martins-Santos (2001) studying on Rhamdia karyotypes from Iguaçu, and Garcia et al. (2010) on karyotype evolution trends, origin and differentiation of supernumerary chromosomes in R. quelen, favored to reappraise the species of Rhamdia from the rio Iguaçu.Recently, Mise et al. (2013), also in a collection from the Iguaçu, revealed that morphological characters split the species R. branneri and R. voulezi.
In the present study, collections of Rhamdia undertaken by LISDEBE in the 1980s and NUP in 1990s at rio Iguaçu, allow us to reexamine the populations of Rhamdia from this area on basis of morphometry and karyotype available data.The result herein provided is the reappraisal and redescription of R. branneri and R. voulezi and the recommendation for the maintenance of these names for the Rhamdia species from Iguaçu basin.

Material and Methods
The morphometric data were obtained with help of a vernier caliper with 0.05 mm of precision.The following set of measurements were taken point to point (Fig. 1): standard length (SL); pre-dorsal length (DPD); dorsalfin base length (CBD); dorsal-fin height (AD); dorsal-fin to adipose-fin distance (DDAd); adipose-fin base length (CBAd); adipose-fin to caudal-fin base distance (DAdBC); pre-pelvic length (DPV); pelvic-fin to anal-fin distance (DVA); anal-fin base length (CBA); anal-fin to caudal-fin base distance (DAC); caudal-peduncle depth (APC); trunk depth at vertical through adipose fin between pelvic and anal fins (ATAA); first dorsal-fin ray to first pelvic-fin ray distance (D1RD1RV); first pelvic-fin ray to last dorsal-fin ray distance (D1RVUD); last dorsal-fin ray to first analfin ray distance (DURD1A); pectoral-girdle width (LCE); pectoral-fin spine length (CEP); dorsal-fin spine length (CED); maxillary-barbel length (CBM); external mentalbarbel length (CBME); head length (CC); snout length (CF); eye diameter (DO); eye-operculum distance (DOOp); interorbital distance (DIO); and mouth width (LB).Counts taken included: 1) dorsal-fin rays; 2) pectoral-fin rays; 3) pelvic-fin rays; 4) anal-fin rays; 5) caudal-fin rays; 6) gillrakers on first left branchial arch.Morphometric analysis.Body proportions were obtained for each character in relation to standard length (SL) except subunits of head that were taken in relation to head length (HL).The principal component analysis (PCA), that included the holotype measurements of R. branneri and R. voulezi, was applied to discriminate the body forms of these two Rhamdia species from rio Iguaçu.Also these two species were compared with a population of Rhamdia from rio Tibagi of the upper Paraná basin adjacent to the Iguaçu.Patterns of size and shape variation among the morphometric variables listed above were used from the studied samples that were evaluated through PCA, a multivariate statistical procedure designed to summarize major patterns of variation among metric characters.The values for all characters were transformed to logarithms to equalize variances of allometric relationships (Bookstein et al., 1985).The principal component scores for the first axis were plotted to assess patterns of size and shape variation within and among R. branneri and R. voulezi populations.We investigate the species delimitation of these two species based on distinctive morphology, morphometrics, meristics and coloration to redescribe them.In all these cases, reliable morphometric differentiation requires attention to allometry.Statistical analysis were performed using the statistical program PAST (Hammer et al., 2001).
Institutional abbreviations: LISDEBE, Laboratório de Ictiologia Sistemática do Departamento de Ecologia e Biologia Evolutiva da Universidade Federal de São Carlos, São Carlos; MZUEL, Museu de Zoologia da Universidade Estadual de Londrina, Londrina; NUP, Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura-Coleção Ictiológica, Maringá; CAS-IC, California Academy of Sciences-Ichthyological Collection, San Francisco; FMNH, Field Museum of Natural History, Chicago; NRM, Swedish Museum of Natural History, Stockholm.A large collection of Rhamdia from rio Iguaçu housed in these institutions and listed below was studied and 46 specimens were chosen for morphometric analysis, diagnosis, descriptions and photographs.Main karyotype data were taken from Abucarma & Martins-Santos (2001), Garcia et al. (2010) and Martinez et al. (2011).The examined type material of R. branneri and R. branneri voulezi come from FMNH and that of Rhamdia quelen from NRM. Haseman, 1911 (Figs.2-4) Haseman, 1911: 377   Diagnosis.Rhamdia branneri differs from the remaining Rhamdia species, with exception of R. voulezi, and Rhamdia cf.quelen from rio Tibagi by having both sides anterior and posterior of the pectoral-fin spine serrate in large dents (vs.slightly serrate in fine dents in Rhamdia cf.quelen).Rhamdia branneri is also distinct by its dorsal-fin length not reaching the origin of the adipose fin when adpressed (vs.adpressed dorsal fin reaching adipose-fin origin in R. voulezi and R. cf.quelen); maxillary barbel short not reaching the origin of adipose fin (vs.maxillary barbel long, surpassing adipose fin origin in R. voulezi and longer than the SL in R. cf.quelen); R. branneri dorsal profile straight between the snout and supraoccipital process and convex from this point to the dorsal fin insertion (vs.dorsal profile almost straight from tip of snout to dorsal-fin insertion in R. voulezi and R. cf.quelen).

Rhamdia branneri
Ground color of preserved R. branneri specimens dark or light gray with few large spots and without small black dots over trunk (vs.ground color grayish on dorsum to light orange on abdomen in R. voulezi and dark brown spotted in R. cf.quelen); dorsal-fin without a hyaline horizontal stripe immediately dorsal to base (vs.a hyaline horizontal stripe higher to base of dorsal fin in R. voulezi and R. cf.quelen).
Differs in corporal proportions from R. voulezi in the higher distance between dorsal and adipose fins; higher distance between adipose ending and caudal fin base; higher distance between orbit and opercular bone and larger scapular bridge width; R. branneri also differs in chromosome number and morphology: 2n=58-62 chromosomes, plus one submetacentric B chromosome and multiple NOR system (vs.Description.Morphometric characters of R. branneri are summarized in Table 1 and the meristic characters in Table 2. Dorsal profile almost straight from tip of snout to supraoccipital and slight convex from this point to dorsal-fin insertion; body depth at anterior vertical through adipose fin and between pelvic and anal fin shorter than the distance between the ending of dorsal fin insertion and pelvic fin.Distance from dorsal to adipose fin generally shorter than the length of dorsal-fin base.Caudal peduncle depth small nearly7.8-12.0,mean 10.7% SL.Head almost conic in dorsal view, covered by thick skin, except for eyes with free margin, occipital process covered by thick skin (not visible).
Orbit relatively large nearly 12.9-17.Color pattern of preserved specimens.Ground color generally grayish and with small dispersed black dots; abdomen generally yellowish-orange.Dorsal-fin with a light horizontal line upper to its base; over to this light line the interradial membranes with the same ground color and the branched rays may appear blackish highlighted.
Remaining fins with nearly same ground color of body and trunk; interradial membranes hyaline.

Discussion
The Silfvergrip (1996) decision to include the species Rhamdia branneri and R. branneri voulezi under the synonymy of Rhamdia quelen (Quoy & Gaimard, 1854) contrast with the present study.The examination of specimens from the type series of the neotype of Rhamdia quelen from rio Samiria (Fig. 8) gives opportunity to get in touch with differences between this species and R. branneri and R. voulezi.They differ concerning to morphology of dorsal and adipose fins, structure and length of mental and maxillary barbels, pigmentation and morphometric traits from the Iguaçu species and the population of Rhamdia cf.quelen from rio Tibagi basin.Also, the examination of the Iguaçu specimens studied by Silfvergrip (1996) revealed that the specimens from São Miguel do Iguaçu (NUP-MU 2664 and NUP-MU 3893) in fact belong to R. voulezi.In addition, the examination of the specimens from rio Iguaçu at Porto União, CAS-SU 62523 and CAS-SU 62686 revealed to be R. branneri.The pectoral spine double serrate, the karyotype differences and ecomorphological data, as soon as color pattern and morphometric trends herein studied, suggest that R. branneri and R. voulezi are significantly distinct justifying the present adjustment.
They also recognize the presence of two types of B chromosomes: a medium sized B metacentric chromosome and a sub-metacentric chromosome, more frequent in R. branneri; 0 to 2 medium sized metacentric and a B submetacentric chromosome in R. voulezi.These differences in karyotype of these Iguaçu Rhamdia, including one submetacentric B chromosome in R. branneri, vs. one metacentric B chromosome in R. voulezi, plus a simple nucleolus organizer region (NOR) in R. voulezi, vs. multiple NOR system in R. branneri may suggest that differentiation occurred on the karyotype structure of these species.These described chromosomes are considered by the authors as safeguard for inter-specific variation between R. branneri and R. voulezi.
It is also important to recognize the karyotype description of a Rhamdia quelen population by Moraes et al. (2007) from Bodoquena Plateau, Mato Grosso, Brazil, composed by a diploid number of chromosomes 2n=58: 36 metacentric; 16 sub-metacentric and 6 sub-telocentric chromosomes.One to three B chromosomes of medium size and the nucleolus organizer region (NOR) situated in terminal region of the shorter arm of the sub-metacentric chromosome pair 20.Also, Martinez et al. (2011) offered a study on three populations of a species defined as Rhamdia quelen from two large Brazilian rivers: Paraná and Araguaia, showing that the diploid number found for the three analyzed populations is 58 stick chromosomes.Following Garcia et al. (2010), "Rhamdia quelen invariably presents 58 (2n) chromosomes in all populations that have been analyzed so far.Since R. quelen is the most widely species cytogenetically studied among the Heptapteridae, the stability of this diploid number can be regard as characteristic of this species".They observed various karyotype formulae in populations identified as R. quelen from the upper and lower Paraná, and from Paraíba do Sul rivers.In both cases the karyotype formula: 36m, 10sm and 12st (see Garcia et al., 2010) was the most frequent in identifying R. quelen populations from Paraná, while 40m, 10sm and 8st was the commonest formula in the Paraiba do Sul populations.Perdices et al. (2002) based on sequencing data of mitochondrial genes show that the genus Rhamdia has diverged from other heptapterids very recently, suggesting that Rhamdia is undergoing an initial process of karyotype differentiation, reinforcing their reproductive isolation leading to speciation.For those authors R. quelen, sensu Silfvergrip, is polyphyletic under mitochondrial DNA perspective.
As second consideration, concerning to morphometry, Mise et al. (2013) advanced a PCA on populations of R. branneri and R. voulezi that indicates an obvious segregation between these two species along the first axis of their figure 2. Those authors also revealed that most of the morphologic characters that separate the Rhamdia species from Iguaçu are related to body shape.They clearly discussed that R. voulezi can be characterized by having more compressed and less depressed bodies, longer caudal peduncle, wider mouth and larger aspect ratio of the anal fin.On the other hand, R. branneri can be characterized by having a higher mouth opening and higher anal fin.
Our morphometric analysis corroborate Mise et al. (2013), concluding that there are two distinct species of Rhamdia in the rio Iguaçu basin.The sheared principal component analysis, herein applied to populations of Rhamdia from Iguaçu, mainly suggest that R. branneri and R. voulezi may be considered independent specific taxa (Tables 3-4 and Fig. 9).Additionally, a comparison through a principal component analysis between populations of R. branneri, R. voulezi from Iguaçu versus a population of Rhamdia cf.quelen from rio Tibagi highlighted their differences as showed in Table 5 and Figs.9-10.On basis of the accurate examination of the holotype specimens of R. branneri and R. branneri voulezi, the Iguaçu material studied by Silfvergrip (1996) from CAS and NUP plus a large number of fresh specimens, we conclude that the serrae on both sides of the pectoral spine, the length of the internal and external mental barbels and the length of adipose fin are the main characters to distinguish these species and are in agreement with the pattern established by Haseman for R. branneri and R. voulezi.
Also R. branneri have the ground color of trunk and abdomen pale brown with rare large dark dots dispersed through body and dorsal-fin without a hyaline horizontal strip immediately dorsal to base.Rhamdia voulezi has the ground color generally gray and without large black dots; abdomen generally orangish-yellow and the dorsal fin with a light horizontal line dorsal to its base, distinct from R. branneri and other species.As a result, the main differential morphometric characters between R. branneri and R. voulezi that could be seen in Table 4 are: R. branneri have smaller interorbital distance, smaller dorsalfin base length, smaller adipose-fin base length, larger mouth and larger snout length; while R. voulezi have: larger interorbital distance; larger dorsal-fin base length; larger adipose -fin base length, smaller distance between adpressed dorsal fin and adipose-fin base.
Finally, the taxonomical simplification proposed by Silfvergrip (1996) when strongly reduced the number of species of Rhamdia recognizing many species as synonym of R. quelen (Quoy & Gaimard, 1824) may be re-evaluated.Perdices et al. (2002) already advanced in this way considering the identification by Silfvergrip of R. quelen from Central America a mistake, because there are morphological and molecular evidence to consider the Central America species different from Rhamdia species of South America.So, we agree with Perdices et al. (2002) that genus Rhamdia needs revision under molecular genetic analysis, which may lead to the evaluation of the species actually in synonymy of Rhamdia quelen.

Fig. 1 .
Fig. 1.Diagram of measurements taken from Rhamdia specimens for the principal component analysis.See Materials and Methods for explanation of abbreviations.

Fig. 5 .
Fig. 5. Geographical distribution of Rhamdia branneri and R. voulezi in rio Iguaçu.Each symbol may represent more than one sample.
R. xetequepeque, R. jequitinhonha, R. muelleri and R. poeyi by having shorter orbital diameter: R. voulezi 12. 20.4% in R. jequitinhonha,.Rhamdia voulezi is further distinguished from R. jequitinhonha by their longer caudal peduncle (18.4-21.8% vs. 16.3%SL) and from R. muelleri by the longer interorbital distance 29.8-39.9%HL in R. voulezi (vs.24.2% in R. muelleri) and shorter maxillary barbel length: 178.5-374.2%HL in R. voulezi (vs.73.9-129.0% in R. muelleri); R poeyi with small serrae of spines on posterior margin of pectoral spine differ from R. voulezi with strong and well developed serrae on both sides; R. voulezi also differ from R. poeyi in caudal fin upper lobe shorter than lower and margin of lower lobe rounded (vs.longer and pointed lower caudal fin lobe in R. poeyi).small teeth, almost with same size on premaxillae and dentary.Maxillary barbel flattened, longer and surpassing beginning of adipose-fin insertion.In young specimens, this barbel surpasses anal-fin base; external mental barbel surpassing beginning of pectoral-fin; inner mental barbel surpassing gill opening in juveniles, but not in adults.Posterior cleithral process short.Dorsal-fin spine smooth on both sides.