A new species of the characid genus Hollandichthys Eigenmann from coastal rivers of southern Brazil ( Teleostei : Characiformes ) with a discussion on the diagnosis of the genus

Hollandichthys taramandahy, new species, is described from the rio Tramandaí, rio Mampituba, and rio Araranguá basins, coastal drainages of Rio Grande do Sul and Santa Catarina States, Brazil. The new species is distinguished from H. multifasciatus, the single recognized species of the genus, by the presence of a small black spot covering the base of median caudal-fin rays, by the lower number of dorsal and ventral procurrent caudal-fin rays, by the absence of a black spot located along anterior half of adipose-fin, and by the absence of humeral spot in specimens larger than 60 mm of standard length. Comments about the geographic distribution and diagnosis are provided for the genus.


Introduction
The genus Hollandichthys was proposed by Eigenmann (1909: 257), and diagnosed in his identification key based on the "maxillary with teeth along its entire edge, lateral line very short, and with dark lines along the sides" [sic].In that study, Eigenmann listed Hollandichthys multifasciatus (Eigenmann & Norris, 1900) in the new genus and considered Pseudochalceus affinis Steindachner, 1908 andP. perstriatus Miranda-Ribeiro, 1908 as junior synonyms.After that, Eigenmann (1910: 432) designated Tetragonopterus multifasciatus Eigenmann & Norris, 1900 as the type species of the genus, and maintained the synonyms previously proposed.Tetragonopterus multifasciatus was briefly described based on three specimens collected in Cubatão [São Paulo State], Pseudochalceus affinis was described from the rio Jaraguá in Joinvile, Santa Catarina State, and P. perstriatus from "Corregos de Iporanga" [São Paulo State].Although proposed in 1909, only more than a decade later Eigenmann (1921) presented a brief description for the monotypic genus Hollandichthys, and redescribed the type species.
Based on the absence of any important anatomical differences among the species of Hollandichthys and Pseudochalceus Kner, Shultz (1966) considered the two genera as synonyms.Later, Géry (1972) proposed Hollandichthys as a subgenus of Pseudochalceus.Weitzman et al. (1986) revalidated Hollandichthys considering the differences between those genera presented by Géry (1972) and their distributions (coastal Atlantic drainages of southern and southeastern Brazil for Hollandichthys and coastal Pacific drainages of Colombia and Ecuador for Pseudochalceus).Further phylogenetic studies with different inference tools positioned Hollandichthys in distinct characid clades.Based mostly on osteology and external morphology, Mirande (2009) proposed Hollandichthys as a member of the group named "Bramocharax clade" including Pseudochalceus, Bramocharax Gill plus Oligosarcus Günther.However, posteriorly Mirande (2010) assigned Hollandichthys to a group named "Pseudochalceus clade" formed by Hollandichthys and Pseudochalceus and considered closely related to the Rhoadsiinae.Based on molecular data, both Javonillo et al. (2010) and Thomaz et al. (2010) hypothesized Hollandichthys as the sister-group of Rachoviscus Myers, instead of Pseudochalceus.
The unique valid species of the genus, Hollandichthys multifasciatus, was hypothesized to be a third lineage of inseminating characids with modified spermatozoa by Azevedo (2004), along with the inseminating species of the Stevardiinae and the inseminating species of the tribe Compsurini of the Cheirodontinae.Quagio-Grassiotto et al. (2012) deeply explored sperm morphology in these fishes and further supported the hypothesis of close relationships among the species of Hollandichthys and Rachoviscus based on unique characters observed in the spermiogenesis and sperm ultrastructure.
Hollandichthys was previously known from coastal river basins between Rio de Janeiro and Santa Catarina States and from the upper rio Tietê, São Paulo State.Bertaco (2003) extended the southern limit of the genus to coastal rivers from northern of Rio Grande do Sul State, distribution reiterated by subsequent authors (Lima et al., 2003(Lima et al., , 2007;;Reis et al., 2003;Abilhoa et al., 2009).
Based on a comparison of several geographically isolated populations of Hollandichthys along all the geographic distribution of the genus conducted by the senior author, a new species was revealed and is described herein from the small tributaries of rio Tramandaí, rio Mampituba and rio Araranguá basins, southern Brazil.Further evidences of close relationships of Hollandichthys and Rachoviscus are discussed and a new diagnosis is provided for the genus.

Material and Methods
Counts and measurements taken following Fink & Weitzman (1974), except for the number of scale rows below the lateral line counted from the scale row ventral to lateral line to the scale row nearest to the origin of first pelvic-fin ray.Counts of vertebrae, supraneurals, gill rakers on the first arch, teeth, and procurrent caudal-fin rays were taken from cleared and stained specimens (c&s) prepared according to Taylor & Van Dyke (1985).Vertebral counts included the four vertebrae of the Weberian apparatus, and the terminal centrum was counted as a single element.Procurrent caudal-fin-ray counts were also taken in some alcohol preserved specimens after the removal of the musculature with a scalpel.Lower and upper jaws of c&s specimen were prepared for SEM (scanning electronic microscopy) by drying in acetone followed by gold metallization.
Measurements were taken point to point with a caliper on the left side of specimens.Measurements are expressed as percents of standard length (SL) except for subunits of the head, which are recorded as percents of head length (HL).Sex of adult specimens of Hollandichthys was recognized by visual examination of their gonads, anal-fin profile and/or by the presence or absence of bony hooks in fin rays.Values for the holotype are indicated by an asterisk.
Specimens examined belong to the following institutions: We designated six paragenetypes (sensu Chakrabarty, 2010), including one of the vouchers previously listed as Hollandichthys multifasciatus by Thomaz et al. (2010;UFRGS 11793) that is also included in our paratypes list for H. taramandahy.COI sequences for the remaining five paragenetypes (MCP 26969) were taken by A. T. Thomaz (unpubl. data).Methods for DNA extraction and sequencing of three mitochondrial genes, 16S, cytochrome c oxidase subunit I (COI) and the NADH dehydrogenase 2 (ND2), and two nuclear genes, the nuclear gene seven in absentia (Sia) and the intron 5 of the a-tropomyosin gene (Trop) given for the paragenetypes are explained by Thomaz et al. (2010).

Hollandichthys taramandahy, new species Figs. 1-6
Hollandichthys multifasciatus (non Eigenmann & Norris, 1900).-Reis et al., 2003: 125 (endangered    Diagnosis.Hollandichthys taramandahy can be diagnosed from H. multifasciatus by the presence of a small, vertically expanded black spot at base of median caudal-fin rays (Figs.1-4), clearly visible after fixation in formalin or alcohol (vs.absence of a black spot on caudal fin, Fig. 7); adipose fin without distinctive marks (vs. anterior portion of adipose fin black, Fig. 7); absence of humeral spot on specimens larger than 60.0 mm SL (vs.humeral spot always present, Fig. 7; two black humeral spots present on small specimens of both species).Furthermore, H. taramandahy can be distinguished from H. multifasciatus by the number Description.Morphometric data summarized in Table 1.Body compressed and elongate; greatest body depth anterior to dorsal-fin origin.Dorsal head profile gently concave.Snout slightly pointed.Dorsal body profile slightly convex from supraoccipital bone to base of last dorsal-fin ray and straight from this point to adipose-fin origin.Ventral body profile convex from anterior tip of lower jaw to pelvic-fin of dorsal procurrent caudal-fin rays (8-11, mode = 10, n = 20, vs. 10-15, mode = 12, n = 72, respectively) and ventral procurrent caudal-fin rays (7-8, mode = 8, n = 20, vs. 9-12, mode = 10, n = 72, respectively), and by the shorter length of the penultimate branched anal-fin ray in adult males (9.0-12.3 in SL, mean = 10.5 vs. 11.1-20.8 in SL, mean = 14.9, respectively).Premaxilla with two tooth rows; outer row with two or three*, rarely four, tricuspid teeth with central cusp longest; inner row with five teeth, with three to five cusps (fourth or fifth cusp diminute); central cusp three or four times longer and broader than other cusps; symphyseal tooth much longer than remaining teeth.Maxilla with 13-17 (usually 13-14, 15*) tricuspid teeth, with central cusp longer.Four or five anteriormost dentary teeth large, with three to four cusps (fourth cusp diminute), followed by one medium sized tricuspid tooth, and 7-12 teeth with two to three cusps, rarely conical; central cusp in all teeth three or four times longer and broader than other cusps.All cusp tips slightly curved towards oral cavity (Fig. 5).Color in alcohol.Ground color dark yellow in dorsal region of body and head, gradually lighter on sides.Dark brown longitudinal lines usually forming a zigzag pattern between longitudinal rows of scales, except immediately above anal-fin base; more conspicuous along the longitudinal axis of the body, more visible in specimens larger than 50.0 mm SL (Fig. 1).Small black chromatophores scattered over rays of all fins.Adipose fin without distinctive marks, except for the anterodorsal border smoothly light gray pigmented.Small black spot vertically expanded at base of median caudal-fin rays (Figs.1-2).Specimens measuring 42.0-50.0mm SL with one or two faint and vertically elongate humeral spots.Color pattern of specimens smaller than 41.0 mm SL similar to that of large specimens, but always with two black vertically elongate humeral spots; anterior diffuse, located over first to fourth perforated lateral-line scales and sometimes extending over horizontal series of scales just above lateral line.Second humeral spot darker, located over seventh to eighth perforated lateral-line scales and sometimes extending over horizontal series of scales just above lateral line.Specimens larger than 60.0 mm SL without humeral spots (Figs.1-2).Specimens up to 45 mm SL with chevron-shaped striae posteriorly diverging from longitudinal line, following miosepta.The remaining fins with dispersed dark Anal-fin rays iii-v,27-30 (iii,28*; rarely 26, mode = 29, n = 51).First unbranched ray usually only apparent in c&s specimens.Anal-fin origin at vertical through base of third to fourth branched dorsal-fin rays.Unbranched and anterior two to three branched anal-fin rays bearing a large posterior flap along their posterolateral borders, more developed in larger specimens of both sexes, extending along entire length of anterior unbranched rays and proximal midlength of last unbranched and anterior branched rays.

Color in life.
Overall coloration light golden to greenish, except for the lateral and ventral portions of head and belly with ground coloration white.Anterior portion of dentary and dorsal portion of snout yellow.Iris dark brown above and below pupil and bright yellow anterodorsally.Tip of pelvic fin and adipose fin yellow; tip of the anterior lobe of the anal fin reddish, caudal fin light orange to reddish and dorsal and pelvic fins hyaline in a young male (Fig. 3).All fins yellow in an unsexed young specimen with 41 mm SL (Fig. 4).Specimens smaller than 41.0 mm SL with two black vertically elongate humeral spots, first spot diffuse and second spot more conspicuous (Fig. 4).Caudal fin spot hardly discernible in live specimens (Figs. 3 and 4).Dark brown longitudinal lines more conspicuous along the longitudinal axis of the Conservation note.Hollandichthys taramandahy was listed as endangered species in the state of Rio Grande do Sul, Brazil (Reis et al., 2003), but named as H. multifasciatus (popular name Lambari-listrado).The species is threatened due to its very limited distribution range, in areas subject to high level of anthropogenic disturbances, e.g., deforestation of stream margins.et al. (2010), Thomaz et al. (2010), andQuagio-Grassiotto et al. (2012) presented strong support to the hypothesis that Hollandichthys is more closely related to corresponding approximately to the position of the 20 th to 25 th rays; three or four posteriormost branched anal-fin rays longer than other rays, forming a conspicuous anal-fin lobe; penultimate ray longest (Figs. 1 and 3).Anal-fin profile nearly straight in all its extension in females (Fig. 2).

Javonillo
Males and females also differ in proportions of the pectoral-and pelvic-fin lengths (Table 1).Caudal peduncle depth slightly wider in males than females, and procurrent caudal-fin rays more developed in males.Gill glands (Burns & Weitzman, 1996) were not observed macroscopically on first gill arch in either males or females.
Distribution.Hollandichthys taramandahy in known from smaller tributaries of the rio Maquiné and rio Três Forquilhas, rio Tramandaí system, Rio Grande do Sul and from rio Mampituba and Araranguá drainages, Santa Catarina, Brazil.Rio Maquiné represents the meridional limit of distribution of the genus (Fig. 8).
Etymology.The species name, taramandahy, is in reference to the drainage of the type locality.In Tupi Guarani language, Taramandahy (=Tramandaí) means winding river, or where is about to harvest (related to fish harvesting).A noun in apposition.
Ecological notes.All specimens were caught in lateral puddles of rivers and very small tributaries with shallow and lentic black waters, with mud and leaf on the bottom, and very dense riparian vegetation, similar to what was observed by Sabino & Castro (1990) for H. multifasciatus in an Atlantic Forest stream in Ubatuba, São Paulo State, southeastern Brazil.The larger specimens (70.0-80.0mm SL) were found isolated and small specimens (30.0-40.0mm SL) were captured in groups of 3 to 6 fishes, as also observed by Sabino & Castro (1990) for H. multifasciatus.Although extensive collection efforts have been applied in sampling fishes along  at base of the lower caudal-fin lobe, extending posteriorly to vertical through half-length of middle caudal-fin rays, no glandular tissue of granular appearance are observed in mature males, as described for the Glandulocaudinae and Stevardiinae sensu Menezes & Weitzman (2009).
Monophyly of Hollandichthys was demonstrated by Thomaz et al. (2010: fig. 1), who found two well supported sister clades among fourteen samples of Hollandichthys in a Bayesian analysis with Sia, Trop, 16S, COI and ND2 genes.Hollandichthys taramandahy is part of the southern Hollandichthys clade of their cladogram, represented by two lots that cluster together from the rio Maquiné and rio Mampituba (UFRGS 11793 and UFRGS 11792, respectively).The remaining lots from the southern clade (MCP 38333, Florianópolis Island;UFRGS 10579, São Francisco do Sul;MHNCI uncat, Paranaguá) do not belong to Hollandichthys taramandahy.In all these specimens the caudal peduncle spot is absent.
Rachoviscus than to Pseudochalceus or any other characid genera included in those analyses.This hypothesis was advanced in an unpublished thesis by Quevedo (2006), based mostly on osteology and general morphology.It is hard to argue in favor of the old hypothesis (Schultz, 1966;Géry, 1972) of a close relationship of Hollandichthys to Pseudochalceus, reinforced by Mirande (2009Mirande ( , 2010)), due to the absence of Rachoviscus species in Mirande's analyses and therefore the lack of a testable hypothesis of relationships among the three genera in those two papers.Hollandichthys is very similar to Pseudochalceus in the color pattern and in the almost fully toothed maxilla, but as pointed out by Thomaz et al. (2010) this similarity seems to represent convergence or shared plesiomorphic traits.
The clade formed by Hollandichthys and Rachoviscus can be diagnosed from other characiforms by a unique type of spermiogenesis and by the presence of unique long and spiraling mitochondria in the midpiece of the spermatozoa of the species of both genera (Quagio-Grassiotto et al., 2012: fig.4).The last character is shared with H. taramandahy, as observed in transmission electronic microscopy (TEM) preparation of the testis of one of the paratypes of MCP 29244 (M. A. Azevedo, unpubl.).A third character that supports a close relationship between the species of the two genera is the presence of a body cavity ventrally between the pelvic and anal fins in males and females that houses internally the anus and urogenital opening (Fig. 9).This arrangement may play some role in the sperm transference from males to females (Quevedo, 2006).This is not observed in Pseudochalceus or any other Characidae, but is shared by Hollandichthys multifasciatus, H. taramandahy, and Rachoviscus species and thus is considered herein a putative synapomorphy of the two genera.
Hollandichthys multifasciatus and H. taramandahy share an unique sexually dimorphic character among characids represented by the posterior region of the anal fin of males with a concavity in the distal margin, formed by reduction in length of branched anal-fin rays 20-25 and elongation of the posterior ones (Figs. 1, 3 and 7).This is not observed among other species of characid fishes and is hypothesized herein as a putative synapomorphy for Hollandichthys species that diagnoses the genus from Rachoviscus.Another character shared by both species of Hollandichthys is the branched anteriormost ray of the pelvic fin (Fig. 6).The first pelvic-fin ray is unbranched in almost all characids (except in the genera Glandulocauda and Mimagoniates of the subfamily Stevardiinae) and further diagnoses Hollandichthys from Rachoviscus.The co-occurrence of the first pelvic-fin ray branched in Hollandichthys and some stevardiines seems to be homoplastic, since Glandulocauda and Mimagoniates are deeply nested with Clade A characids (Malabarba & Weitzman, 2003;Javonillo et al., 2010;Mirande, 2010;Oliveira et al., 2011) that excludes Hollandichthys and Rachoviscus.Although Hollandichthys possesses small scales Currently, Hollandichthys multifasciatus has two junior synonyms, Pseudochalceus affinis (rio Jaraguá, tributary of the rio Itapocu, coastal drainage of Santa Catarina State) and P. perstriatus (córregos de Iporanga, rio Ribeira de Iguape basin, São Paulo State).Morphological (Bertaco, 2003), molecular (Thomaz et al., 2010) and karyotypic variability (Balen et al., 2013) among the populations currently identified as Hollandichthys multifasciatus, however, suggests the existence of a species complex under this taxon name, and the validity of these nominal species is under review by the authors.None of these names, however, are applicable for H. taramandahy.The type series of Pseudochalceus affinis and examined specimens from the type locality of P. perstriatus (rio Ribeira de Iguape basin) lack the small black spot covering the base of the median caudal-fin rays that is diagnostic for H. taramandahy and have more dorsal and ventral procurrent caudal-fin rays (10-13, mode = 12, and 8-11, mode = 10, n = 5, in the type series of P. affinis, and 11-15, mode = 12, and 9-12, mode = 11, n = 46, in specimens from the rio Ribeira de Iguape basin) than H. taramandahy (8-11, mode = 10, and 7-8, mode = 8, n = 20).Furthermore, specimens from the rio Ribeira de Iguape basin possess a small black spot located on the proximal portion of the adipose-fin and a black humeral spot in specimens larger than 60.0 mm SL (both absent in H. taramandahy).
The species of the genus Hollandichthys inhabit coastal rivers and marine islands from northern Rio Grande do Sul State to southern Rio de Janeiro State, and the upper rio Tietê in the upper rio Paraná drainage, included in the area of the Atlantic Rainforest biome.Such a distribution pattern strongly corroborates the recognition of a large area of endemism comprising the coastal drainages of south and southeastern Brazil, including the headwaters of rio Tietê.This is similar or largely congruent with the distribution of species of the genera Deuterodon Eigenmann (Lucena & Lucena, 1992, 2002), Kronichthys Miranda-Ribeiro (Ferraris, 2007), Mimagoniates Regan (Menezes et al., 2008;Menezes & Weitzman, 2009), Spintherobolus Eigenmann (Weitzman & Malabarba, 1999), Scleromystax Günther (Britto & Reis, 2005), and Pareiorhaphis Miranda-Ribeiro (Pereira & Reis, 2002;Pereira, 2005).
Dorsal-fin rays ii,9* (n = 51); first unbranched ray approximately one-half the length of second ray.Dorsal-fin origin posterior to middle of SL and posterior to vertical through pelvic-fin origin.Dorsal-fin profile slightly rounded.Unbranched and anterior 3 to 4 dorsal-fin rays bearing large posterior flap along posterolateral borders, more developed in specimens larger than 60 mm SL, extending along entire length of first unbranched ray and proximal midlength of second unbranched and anterior branched rays.Adipose fin located approximately at vertical through insertion of 20 th -22 th anal-fin rays.