Re-description and distribution of Rivulus punctatus BOULENGER , 1895 ( Teleostei : Rivulidae ) and its habitats in Paraguay

The neotropical killifi sh genus Rivulus POEY, 1860 of the aplocheiloid family Rivulidae (subfamily Rivulinae) comprises more than 100 species (COSTA, 2003a). Members are distributed from Middle-America and the Caribean Islands to northern Argentina and occur in a variety of habitats ranging from savannah and forest creeks, small ponds, swamps and temporary ditches. Based on morphological data the genus is recognized as a monophyletic assemblage (HUBER 1999; COSTA Re-description and distribution of Rivulus punctatus BOULENGER, 1895 (Teleostei: Rivulidae) and its habitats in Paraguay

Members are distributed from Middle-America and the Caribean Islands to northern Argentina and occur in a variety of habitats ranging from savannah and forest creeks, small ponds, swamps and temporary ditches.Based on morphological data the genus is recognized as a monophyletic assemblage (HUBER 1999;COSTA 2004COSTA , 2006)).However, in a phylogenetic context it is poorly defi ned among the basal lineages of the Rivulidae (COSTA, 2004(COSTA, , 2006a(COSTA, , 2006b)).In analyses of molecular data it turned out as paraphyletic (HRBEK & LARSON, 1999;HRBEK et al., 2004).Nevertheless, Rivulus contains well-supported monophyletic subunits, which are currently recognized as subgenera (HU-BER, 1999;COSTA, 2006).One of these subgenera is the recently described Melanorivulus COSTA (2006).Melanorivulus includes the species of the R. punctatus-group (COSTA, 1995).This monophyletic unit comprises about 20 species (COSTA, 2005(COSTA, , 2006a(COSTA, , 2006b(COSTA, , 2007a(COSTA, , 2007b)).The type species R. punctatus is the most southern species in Melanorivulus.It is distributed in the río Paraguay, lower río Paraná and middle río Uruguay drainage in Brazil, Bolivia, Paraguay and Argentina (COSTA, 1995).
Rivulus punctatus was described by BOULENGER (1895) on the basis of specimens collected at Colonia Risso in Paraguay.Thus it is the oldest taxon within the subgenus.The species was already the subject of taxonomic treatments (HUBER, 1992;STAECK & SCHIN-DLER, 1994;COSTA, 1995).However, none of these focused on specimens from Paraguay.During fi ve fi eld studies of the second author between 1984 and 2006 it was possible to catch several samples of R. punctatus in Paraguay, even in the vicinity of the type locality.Although there are several collecting sites of R. punctatus in Paraguay listed (e.g.HUBER, 1992HUBER, , 1995;;ET-ZEL, 1985, 1991), none of them contains a description of its ecology and habitats.Therefore the objectives of the present article are to describe R. punctatus from Paraguay, to analyse its variation and to document the habitats of this species.

Methods
Measurements and counts follow HUBER (1992).He and the majority of authors (e. g.THOMERSON & TAPHORN, 1992;LASSO-ALCALÁ et al., 2006) use the snout tip as the most anterior point of reference.COSTA (1988), however, used the middle of the posterior limit of the depression between upper jaw and the neurocranium, and his morphometric data are compatible only to a certain extent with those published here.Measurements were made with an electronic calliper reading to the nearest 0.1 mm.Fin-ray counts were made with light transmitted through the fi ns and included all discernible elements.Using this method the counting of the number of fi n rays in alcohol specimens is not without diffi culties, and it may happen that sometimes the very minute anterior rays of anal-dorsal-or caudalfi n are overlooked and not counted (see HUBER, 1992).
The material of R. punctatus from Paraguay used is listed under species description (see also Table 1).The majority of the specimens are very small, weak or otherwise not well enough preserved.Therefore 20 representative specimens (10 males and 10 females) are selected to obtain the morphometric and meristic data.Additional counts from four cleared & stained specimens are included.Terminology of colour pattern, shapes and frontal squamation follow HUBER (1992).In order to make comparison of the species easier, the description follows the general format used by COSTA (2005).
Principal components analysis was applied to investigate patterns of sexual dimorphism in multidimensional space and in order to visualize the pattern of variation in morphometric data along geographic locations.The geographic locality is represented by scores on the fi rst PCA axis obtained from the untransformed decimal equivalents of latitude and longitude of the collecting sites of each included specimens (see WOODMAN, 2000 for detailed description of this procedure).Two separate PCA were calculated for the geographic position: fi rst one with the localities of 20 specimens (10 males, 10 females) used for morphometric analysis (factor loadings on PC1: latitude = -0.989and longitude = 0.148) and a second with localities of 27 males used for the coding of the caudalfi n pattern (factor loadings on PC1: latitude = 0.994 and longitude = -0.103).The PC1 axes accounts for 95.4% (fi rst analysis) and 88.2% (second analysis) of variance respectively.Thus the scores reliably describe the relative geographic position of the localities (cf.Woodman, 2000).To correct the effect of size all measurements were standardized by expressing them as proportions of standard length as recommended by VAN VELZEN et al., (1992).For this purpose the equation log(x') = log(SL) -log(x) is used (BLACKITH & REYMANT, 1971).To make the caudal-fi n pattern in males available for statistic analyses, it is coded into fi ve stages (see description below).Since this character (stages are treated as ordered) is correlated with SL (Fig. 14), the codes are size adjusted for further statistic evaluation (Fig. 16) by subtraction of a linear regression line from the code values (although the correlation between un-adjusted codes of caudal-fi n pattern and geographic locality is similar).
PCA (using the variance-covariance matrix), correlation coeffi cients and regressions lines were calculated with the programme PAST version 1.71 (HAM-MER et al., 2001, 2007).For assumptions, necessary data and the descriptions of the statistic applications see HAMMER & HARPER (2006).Results are visualized using the programme KyPlot (YOSHIOKA, 1997(YOSHIOKA, -2000)).The signifi cance of pairwise differences between males and females in the one-dimensional standardized data (expressed as percent of SL) is tested with the t-test Vertebrate Zoology ■ 58 (1) 2008 as described in LOZAN (1990).The p-values were corrected for multiple tests by the sequential Bonferronicorrection (see SCHINDLER, 2003).

Description
The description is based on specimens from Paraguay (Table 1).Morphometric data are summarized in Maximum recorded adult size in males 35.2 mm SL, in females 30.9 mm SL.Dorsal profi le slightly convex from snout to end of dorsal-fi n base, straight on caudal peduncle.Ventral profi le of head convex, almost straight or slightly convex between head and posterior end of anal-fi n base; on caudal peduncle slightly concave.Body slender, subcylindrical anteriorly, slightly deeper than wide, posterior of dorsal-fi n origin laterally compressed.Greatest body depth at pelvic-fi n base.Dorsal and anal fi n slightly pointed in males, round in females.Caudal fi n horizontally elongated, posterior margin round.Pectoral fi ns rounded, 19.6% of SL in males, 18.8% of SL in females.Pelvic fi ns slightly pointed, reaching between urogenital papilla and anterior anal-fi n base in males, and between anus and urogenital opening in females.Dorsal-fi n origin on vertical through base of 10 th or 11 th anal-fi n ray.Dorsal-fi n rays 7, 8(4) or 9; anal-fi n rays 13(2) or 14(2); caudal-fi n rays 28(1), 29(2) or 30(1); pectoral-fi n rays 12-13; pelvic-fi n rays 6-7.Scales large, cycloid; basal radii 18-22.Body scales extending over caudal-fi n base (about 30% of caudal-fi n length), posterior margin of the scaled area convex.No scales on dorsal-and anal-fi n bases.Frontal squamation F-patterned; rarely E-patterned.Scales in longitudinal series 30 to 32; transverse series 8 to 9; 22 to 24 predorsal scales.Gill Vertebrate Zoology ■ 58 (1) 2008 rakers on fi rst arch 8 or 9. Vertebrae 29(1) or 30(1).Dorsal portion of preopercle comparatively short and pointed.Vestigal ventral process of angulo-articular not particularly developed.First epibranchial moderate (in younger specimens < 15 mm SL) to strongly bent (in adult specimens > 25 mm SL).Hypural forms two plates separated by a median gap.Epural and parhypural thin in smaller specimens and broad in larger specimens.

Colouration
Males.Sides of body metallic bluish-green (yellowish brown or greyish in alcohol) with dark reddish dots arranged into oblique chevron-like bars (number of red dots and arrangement is variable).Laterodorsal brown spots on anterior portion of body; dark grey longitudinal stripe between eye and caudal-fi n base.Dorsum dark grey, venter white.Postorbital region metallic bluish-green.Chin and ventral part of head whitish.Lower jaw dark grey.Dorsal fi n light bluish with red dots or thin reddish lines in the posterior parts.Anal fi n light bluish with red dots and/or reddish oblique bars in posterior parts; margin with black stripe.Dark pattern on caudal fi n very variable.Five different patterns on caudal fi n of males are recognized.The following descriptions can give only a rough impression of the typical patterns, because there are variations within and continuities between several classes of caudal-fi n patterns: Caudal fi n with (1) vertical bars (mostly distally less prominent), (2) with bars interrupted in dorsal and ventral parts, or with scattered vertical lines, (3) dotted, (4) reticulated or (5) without conspicuous dark pattern.There is a signifi cant correlation between SL and the coded caudal-fi n patterns (r = 0.49, p < 0.01), for larger specimens tend to posses the caudal-fi n pat-terns 3, 4 or 5 (the latter two states are observed in adult ones only).Females.Sides of body grey to slightly purple with faint brown to dark longitudinal stripe and reddish dots.Dorsal, anal and pelvic fi ns with dark stripe on distal margin.Entire caudal fi n with dark border; centre of caudal fi n with blotchy spots, reticulated or marbled in larger specimens.Caudal spot present, slightly apart from dorsal margin (sometimes masked by marbled pattern or dark blotches).
In Paraguay R. punctatus is reported from Colonia Risso (terra typica) in the north (río Paraguay drainage) to Ayolas in the south (río Paraná drainage) and from San Juan Neomucero in the east to Pozo Colorado in the west, that means a maximal distance of 200 to 300 km from the río Paraguay channel.Central and western Paraguay is mainly characterized by the wide, hot and semi-arid lowlands (only up to 200 m asl) of the Chaco region.The Paraneña region (east of the río Paraguay), a mixture of plateaus and valleys, raises up to about 400 to 850 m asl (Cordillera de Amambay).Until now R. punctatus is only found in the lowlands (see Table 1 for the collecting data obtained by the second author).

Habitat
Rivulus punctatus occurs in the lowlands of Paraguay and prefers both fl ooded regions near the río Paraguay  channel and creeks in (at least formerly) forest areas.It is diffi cult to determine now the original structure of some regions in Central-Paraguay, because at present most of them are deforested and used for extensive agriculture (mostly cattle-breeding, but also soybean, maize and sunfl ower plantations).There is the impending danger that even the rests of the primary forest will disappear in the next decade if the speed of denaturalization is not reduced.
Rivulus punctatus occurs in three typical types of habitats: fi rstly the more or less fl ooded margins of creeks and rivers with submerged vegetation (grass, bush and frequently Nymphea) at the embankments (Fig. 8 and 9, locations "La Rosada" and "Loreto").In this kind of habitat R. punctatus was observed only 2 to 5 cm under the surface of the water between fl ooded grass and bushes.Sometimes it was also found in small ponds or puddles which remained after heavy rainfalls.
The second types of habitats (Fig. 10, location "Ayolas") are swamps as they are found in southern Paraguay between San Ignacio and Pilar.In this region the Tab. 1. Locations, habitat characteristics and examined specimens of Rivulus punctatus in Paraguay.n1 = number of specimens in the jar, n2 = number of specimens used in the morphometric data analyses, n3 = number of males used in the caudal-fi n pattern analyses, μS = electric conductivity (μS/cm expansion of the swampy areas is extensive.Usually their bottom is muddy and the water clear and shallow (deepness approximately 20 to 50 cm).A lot of different aquatic or semi-aquatic plants like Azolla, Ludwigia, Nymphea and Pistia inhabit the swamps (see Fig. 11).There Rivulus punctatus was found -mostly sympatric with Apistogamma borelli -2 to 3 cm below the surface of the water between the plants.
The third type of habitat of R. punctatus is found in the Chaco Paraguayo (Fig. 12, Chaco near Concepcion).In the dry season (i.e. the winter from June to September) small rivers and creeks often dry up, and only small pools with murky grey to brownish-red water are left over.In these habitats R. punctatus is accompanied by Trigonectes balzanii.Data of important water parameters are summarized in Table 1.

Discussion
Rivulus punctatus is the type species of Melanorivulus COSTA, 2006 and therefore its subgeneric arrangement is without question.This subgenus was formerly known as the punctatus-superspecies (HUBER, 1992) or as the punctatus-complex (COSTA, 1995(COSTA, , 2005)).
The diagnoses published for the taxon R. punctatus neither acknowledge the variation in colour pattern (cf.COSTA, 1995) nor take into account the most closely related taxa (e.g.HUBER, 1992;STAECK & SCHINDLER, 1994).All earlier publications suffer from the lack of specimens collected at or near to the type-locality.This is why we provide here an evaluated diagnosis based on specimens of Rivulus punctatus from Paraguay.As  it is the oldest taxon (described by BOLENGER, 1895) within Melanorivulus, it is not in the danger of being regarded as a junior synonym of another taxon.
There are four further species of the subgenus Melanorivulus (viz.R. cyanopterus COSTA, 2006, R. dapazi COSTA, 2006, R. bororo COSTA, 2007and R. paresi COSTA, 2007) distributed in the upper río Paraguay drainage (COSTA, 2006).Rivulus cyanopterus is differentiated from R. punctatus by possessing comparatively broad oblique red bars on the anal fi n in both sexes and by the lack of distinctive colours or dark patterns in the caudal fi n.R. punctatus is distinguished from R. dapazi by less scales in the longitudinal series (30-32 in R. punctatus vs. 34-35 in R. dapazi), by oblique bars of red dots on the sides of the body vs. chevron-like red lines in R. dapazi and by the lack of dark orange stripes on the caudal fi n.Rivulus bororo and R. paresi differs from R. punctatus by a pattern of red dots on fl ank, arranged in chevronlike series.Further R. bororo is distinguished from R. punctatus by a fewer number of mandibular neuromasts and R. paresi by possessing a reticulate colour pattern on the dorsal fi n and the basal portion of the anal fi n in males (COS-TA, 2007a).With regard to the pattern of reddish dots on body fl anks and anal fi n R. punctatus is similar to R. rossoi COSTA, 2006 from rio Paraná drainage and to R. zygonectes MYERS, 1927 distributed in río Araguaia, río Tocantins and río Xingu basins (see COSTA, 1995).However, R. punctatus clearly differs from R. rossoi by a higher number of gill rakers on fi rst gill arch (1+8    versus 1+6 in R. rossoi) and the lack of orange stripes on the caudal fi n of males.From R. zygonectes it is distinguished by a lower number of dorsal-fi n rays (8-9 in R. punctatus versus 9-11 in R. zygonectes), less scales in longitudinal series (30-32 mode 31 in R. punctatus versus 32-38 in R. zygonectes) and elongated caudal fi n (versus caudal fi n more or less rounded and not particularly elongated in R. zygonectes).
Because of the variation in caudal-fi n and body fl ank patterns in R. punctatus (see description above, COLLIER & STEINBERG, 1977;HUBER, 1992;STAECK & SCHINDLER, 1994;SEEGERS, 2000) the possibility appears not unlikely that more than one species is included in the current concept of this taxon.However, the facts that there is no determined correlation neither between morphometrics and geographic position (r=-0.388,p>0.05, fi g 15 for scatter plot) nor between caudal-fi n pattern and locality (r=0.032,p>0.1, see fi g. 16), make us regard all the examined populations of R. punctatus as members of a single evolutionary entity in the sense of the ESC (WILEY, 1978).This view is additionally supported both by the observation of a change of caudal fi n pattern during ontogenesis in at least some specimens (STAECK & SCHINDLER, 1994) and the high variability of colour patterns and morphological data reported for closely related species (HUBER, 1992(HUBER, , 2007;;COSTA, 1995).
Further the lack of any clear physiogeographical barriers in the lowlands apparently prevent the isolation of populations and make it more likely that only one species is involved.Consequently it can be assumed that R. punctatus inhabits the fl oodplains of the río Paraguay and the río Paraná lowlands in Brazil, Bolivia, Paraguay and Argentina (ETZEL, 1985(ETZEL, , 1991;;HUBER, 1992;STAECK & SCHINDLER, 1994;COSTA, 1995COSTA, , 2003a)).
The habitats preferred by R. punctatus are characterised by a low depth (usually less than 30 cm) of water, slow or no currents and muddy or sandy (<3 mm diameter) substrates.Rivulus punctatus usually inhabits the zone close to the surface of the water between marsh and water plants or submerged bushes and grass.The analysis of the food composition of R. punctatus shows that major components are allochtone insects (STAECK & SCHINDLER, 1994) which probably correlates with the preferred habitats (shallow water close to the surface).The biotopes of R. punctatus documented here are generally similar to those described for other species of Melanorivulus (e. g.BASTOS & LOURENÇO, 1983 for R. pictus [misidentifi ed as R. punctatus] and COSTA, 2003bCOSTA, , 2003cCOSTA, , 2005COSTA, , 2007b for further species) and seem to be characteristic of all the members of this group.
The documented variability for R. punctatus raises the question about the species limits within the subgenus.Acknowledging the variability in chromatic character states COSTA (1995) synonymized the taxa R. apiamici COSTA, 1989, R. pinima COSTA, 1989, and R. vittatus COSTA, 1989, with R. pictus COSTA, 1989. But in 2005 COSTA revalidated all three species and described additional similar taxa without a comprehensive discussion of species limits and taxonomy.Therefore, the diagnoses of all these species may be considered as provisional hypotheses until a comprehensive phylogeographic analysis of Melanorivulus is available.At the moment it is diffi cult to obtain such one, because every year new populations of endemic evolutionary units are discovered in the upper drainages of the river systems (see COSTA, 2005COSTA, , 2006bCOSTA, , 2007aCOSTA, , 2007b)).

Rivulus (Melanorivulus) punctatus BOULENGER, 1895
Type species of the subgenus Melanorivulus COSTA, 2006.It is distinguished from the remaining members of the subgenus by the combination of the following character states: (1) caudal fi n in both sexes spatula shaped, elongated longitudinally (versus caudal fi n round, not particularly elongated), (2) red dots on body fl anks not arranged in complete bars nor in longitudinal stripes (versus red dots arranged in chevron-like, oblique bars, longitudinal stripes or fl anks of body plain), (3) scales in longitudinal series 30-32 (versus >31 scales), (4) no red bars, no orange stripe or yellow pattern on caudal fi n (versus red bars, orange stripe or yellow pattern present), and (5) gill rakers on fi rst arch 1+8 (versus 1+6).

Table 2
. For general appearance see Figs. 1-7.Osteological data based on cleared & stained specimens only.Meristic data include counts of 26 specimens from Paraguay (number in parentheses after counts indicate the data of four cleared & stained specimens).
Morphometric data in percents of SL of Rivulus punctatus from Paraguay (males = 10, females = 10), results of pair-wise t-tests (males vs. females) and coeffi cients of loading of a PCA of morphometrics.Mean = arithmetric mean, dev = standard deviration, min = lowest value, max = highest value, p = results of t-test, p' = corrected p-value, PC1 = fi rst principal component axis, PC2 = second principal component axis.