A new glanapterygine catfish of the genus Listrura ( Siluriformes : Trichomycteridae ) from southeastern Brazil , corroborated by morphological and molecular data

Listrura costai, new species, is described from small streams in a swampy coastal plain in the rio Jurumirim basin, Angra dos Reis Municipality, Rio de Janeiro State, southeastern Brazil. The new species is morphologically very similar to L. nematopteryx and L. picinguabae, all possessing only one long pectoral-fin ray. It differs from its congeners by possessing an autapomorphic character: first hypobranchial with an anterior process (vs. process absent). Other features such as coloration, numbers of opercular and interopercular odontodes, number of anal-fin rays, head length, and shape of some bone structures help to distinguish the new species from L. nematopteryx and L. picinguabae. Molecular analyses using partial sequences of the mitochondrial DNA genes cytochrome oxidase c subunit 1 and cytochrome b from the new species and morphologically similar species are provided. The results about both molecular markers corroborate the validity of the new species by significant genetic distance values between it and congeneric species, and by its phylogenetic position in the hypotheses performed by maximum-parsimony method.

The recent discoveries of new populations of Listrura are possibly a consequence of more frequent sampling directed to the specialized habitat occupied by their species, characterized by leaf litter and soft mud bottom of small, shallow water bodies commonly associated with small mountain streams.Other fish species are uncommon in this kind of habitat (see Habitat notes in Landim & Costa, 2002;Villa-Verde & Costa, 2006), making difficult the discovery of new populations of Listrura in ordinary ichthyological surveys.Some populations of Listrura were discovered by herpetologists who frequently collect frogs and tadpoles in biotopes with similar environmental features (e.g., L. picinguabae, firstly collected by the herpetologists Richard Schasse and Sérgio Potsch Carvalho e Silva; see paratypes in Villa-Verde & Costa, 2006).
Recent taxonomic studies have employed molecular analysis methods, commonly using the mitochondrial DNA gene cytochrome oxidase c subunit 1, as a supplement to the morphological data in the recognition of new fish species (e.g., Victor, 2007;Benine et al., 2009;Melo et al., 2011).This practice is very useful for morphologically similar species, such as in the case of the species of Listrura.
In August 2005, five specimens of a new population of Listrura were collected in rio Jurumirim basin, Angra dos Reis Municipality, Rio de Janeiro State, in an expedition coordinated by the second author.Later, new samples were collected in the same locality, totalizing 22 specimens examined herein.Morphological and molecular data revealed that the specimens from rio Jurumirim basin constitute a distinct taxon, which is herein formally described as a new species.Costa (1992), with modifications according to Villa-Verde & Costa (2006).Measurements are presented as percentages of standard length, except for subunits of head, which are presented as percentages of head length.Counts of number of dorsal and anal-fin rays include all rays (principal and predorsal/preanal, respectively).Osteological observations and counts were made only on cleared and stained ("CS") specimens prepared according to Taylor & van Dyke (1985).Osteological terminology follows Bockmann et al. (2004).Laterosensory system terminology follows Arratia & Huaquín (1995).

Measurements and counts follow
Fragments of the mitochondrial DNA cytochrome b (Cytb) and cytochrome oxidase c subunit I (COI) genes from L. nematopteryx (one specimen), L. picinguabae (three), the new species (four), L. tetraradiata (five) and Listrura sp. from rio São João basin, Rio de Janeiro State (four) were sequenced.Total DNA was extracted from muscle tissue using the phenol/chloroform method described by Sambrook et al. (1989).The extracted DNA was precipitated with sodium acetate and ethanol, re-suspended in 50 µL of ultrapure water, and stored at -20°C.
Partial sequences of Cytb gene were amplified using primers Cytb Siluri F (5'-CCA CCG TTG TAA TTC AAC TA -3') and Cytb Siluri R (5'-GAT TAC AAG ACC GGC GCT TT -3'), both designed by the last author.For COI gene primers L5698-ASN (5'-AGG CCT CGA TCC TAC AAA GKT TTA GTT AAC -3'; Inoue et al., 2001) and H7271-COI (5'-GTG GTG GGC TCA TAC AAT AAA -3'; C. Oliveira, pers.comm.) were used.The polymerase chain reaction (PCR) amplification and sequencing for Cytb were as follows: one initial cycle of 4 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C, and a final extension at 72°C for 5 min.The same conditions were used for COI, except for the use of 46°C as annealing temperature.The PCR products were purified and both strands were sequenced in an ABI 3730XL automatic sequencer.
Sequences were edited using the Seqman II (DNAStar, Inc., http://www.dnastar.com)and aligned in ClustalW software (Thompson et al., 1994).Levels of gene divergence between the sequences (Kimura 2-parameter model; Kimura, 1980) and phylogenetic analyses based on maximum-parsimony (MP) method (Swofford & Berlocher, 1987) were performed in the MEGA5 software (Tamura et al., 2011), and made for Cytb and COI genes separately.The most parsimonious trees were obtained by max-mini branch-&-bound algorithm (Purdom et al., 2000).The resultant topologies were statistically tested by the bootstrap method (Felsenstein, 1985) with 1,000 pseudoreplicates.The rooting was always taken on segment of L. tetraradiata, based on current hypothesis that it is a basal taxon of Listrura (Landim & Costa, 2002).
The Diagnosis.Listrura costai is autapomorphically distinct from its congeners by possessing a small anterior process on the first hypobranchial (vs.process absent; Fig. 3).The new species is also distinguished from its congeners, except L. nematopteryx and L. picinguabae, by possessing only one pectoral-fin ray (vs.two to four rays).It differs from L. nematopteryx and L. picinguabae by having an inferior row of dots extending only on posterior half of body (vs.over almost the entire body), and posterior tip of ceratobranchial 5 strongly concave (vs.slightly concave or straight; Fig. 3).It further differs from L. nematopteryx by possessing a discontinuous lateral midline row of spots (vs.continuous row), lateral processes of urohyal reaching the anterior region of posterior ceratohyal (Fig. 4) [vs.reaching the posterior region of posterior ceratohial; see fig.
Description.Morphometric data given in Table 1.Body elongate, subcylindrical at anterior portion of trunk, and strongly compressed at caudal peduncle.Dorsal and ventral profiles straight.Skin papillae minute.Head depressed and trapezoidal.Snout blunt and slightly elongated.Mouth subterminal and narrow.Teeth conical, tips pointed and curved.Two rows of teeth in jaw.Premaxilla teeth 15-26; dentary teeth 13-21.Eyes anteriorly located on head, nearer to snout tip than to opercular patch of odontodes.Nasal, maxillary and rictal barbels well developed.Tip of nasal barbel reaching between posterior margin of interopercular patch of odontodes and posterior margin of opercular patch of odontodes.Tip of maxillary barbel reaching just posterior margin of opercular patch of odontodes.Tip of rictal barbel reaching between middle of interopercular patch of odontodes and posterior margin of opercular patch of odontodes.Anterior nostril opening anteriorly at base of nasal barbel.Posterior nostril located on anterior half of distance between anterior nostril and eye.Interopercular odontodes 8-12, opercular odontodes 6-8; odontodes conical, tips pointed and slightly curved.
Dorsal and anal fins approximately triangular.Dorsalfin origin on posterior half of trunk, at vertical between centra of 30 th and 35 th vertebrae.Anal-fin origin anterior to dorsal-fin origin (dorsal-fin origin on vertical through base of 3 rd anal-fin rays) to posterior to it, on vertical through base of 4 th dorsal-fin ray (Fig. 5), and between centra of 31 st Laterosensory system extremely reduced, without supraorbital, infraorbital, mandibular, preopercular, and otic canals (Fig. 6a).Postotic canal with two pores: anteriormost at vertical through anterior margin of opercular patch of odontodes; posteriormost at vertical just posterior to opercular patch of odontodes.Lateral line on trunk very short, with two pores: anteriormost largest, at vertical just posterior to pectoral-fin base; posteriormost below former in lateral view and just posterior to it.
Osteology.Anterior cornua of mesethmoid straight in dorsal view (Fig. 6a).Lateral ethmoid with lateral projection.Frontal and parieto-supraoccipital fully joined by sutures; cranial fontanel absent.Sphenotic, prootic and pterosphenoid entirely fused to each other.Vomer without posterior process.Parasphenoid without posterior process.Pterotic with lateral laminar expansion (Fig. 6a).Basioccipital and exoccipital fully fused to each other and posteriorly fused to Weberian capsule.Co-ossified basioccipital and exoccipital approximately triangular.Weberian capsule with small lateral opening on each side.
Autopalatine nearly square with curved lateral process (Fig. Hyomandibula with elongated and narrow anterior process (Fig. 6b).Anterodorsal portion of quadrate laminar.Metapterygoid extremely reduced and articulating only with anterodorsal portion of quadrate through cartilaginous block.Preopercle straight.Odontodes disposed vertically on opercle and obliquely on interopercle.
Urohyal with narrow pointed and elongated lateral processes reaching to anterior region of posterior ceratohyal (Fig. 4).Two small condyles on anterior region of urohyal.
Cleithrum flattened, slightly triangular and with large fenestra on central region (Fig. 6d).Scapulocoracoid narrow, fully cartilaginous and with narrow process on distal portion.Single proximal radial rounded and fully cartilaginous.Single pectoral-fin ray narrow and segmented.myomeres.Venter yellowish white.Head pale brown on dorsal region, light yellowish gray on lateral and ventral regions; dark brown to black chromatophores concentrated over dorsal portion of head and snout, and interopercular and opercular regions, but absent or rare on infraorbital region, making distinctively unpigmented zone between snout, orbit and interopercle; small unpigmented zone on center of dorsal portion of head; dark chromatophores extending to ventral portion of head to produce dark transverse zone on chin and short dark zone on branchiostegal region.Barbels hyaline with brown base.Distal portion of dorsal and anal fins hyaline, proximal portion yellowish hyaline with dark brown chromatophores on fin rays.Narrow marginal black line on fin membrane adjacent to procurrent caudal-fin rays.Caudal fin yellowish hyaline with sparse dark brown chromatophores on fin rays.Pectoral fin hyaline.
Distribution.Listrura costai is known from two neighboring small unnamed coastal streams adjacent to the road RJ-155, Angra dos Reis Municipality, Rio de Janeiro State, Brazil, both draining into a swampy portion of the rio Jurumirim, close to its mouth (Fig. 7).
Coloration.Body light yellowish gray with minute dark brown to black dots on dorsal portion of flank and dorsum, more concentrated on lateral midline, and laterodorsal portion and dorsal midline of trunk to form three longitudinal rows of small, closely placed dark brown to black spots.Lateral midline spots to form discontinuous row; above lateral midline row of spots, dots often coalesced to form short, dark brown oblique lines near central portion of flank and oblique rows of small dark brown spots on side of caudal peduncle, all following myomeres; below lateral midline row of spots, minute brown dots to form narrow longitudinal row extending to posterior half of trunk.Few minute brown dots below lateral midline of caudal peduncle, usually arranged in oblique rows following through well-preserved areas of Atlantic forest in the mountain range of the Serra da Bocaina.The rio Jurumirim forms a grassy lowland that becomes partially, and sometimes fully, flooded during periods of high rainfall.In this region, the grass vegetation has replaced the original Atlantic forest probably due to human action.
Most specimens of Listrura costai were collected in a narrow (about 1 m wide) and shallow (about 50 cm deep) clear water stream located in a swampy plain area densely vegetated with grass (Fig. 8).This site located in rio Jurumirim lowland, just upstream from a paved road (RJ-155), is right next to the pipe that runs underneath the road, draining the stream into a larger tributary of the main river.Most specimens were collected in a shallower portion of this stream (about 10 cm deep) either in the mud bottom or the thin layer of litter covering it (Fig. 8a).Some specimens were also collected at shallowest pools (less than 5 cm deep) adjacent to the stream (Fig. 8b).The locality where the holotype was found has similar characteristics.During the collection of the holotype, the water was clear with pH 6.1 and temperature 25.5ºC at 10:25 am.
Conservation remarks.The extremely restrict distribution, together with a high specificity of its natural habitat (swampy streams in lowland plain areas that rarely occur in southern Rio de Janeiro State watersheds) and relative proximity of human settlements make L. costai prone of becoming critically endangered or even extinct in a very short time period due to effects of human activities or stochastic events.However, no studies about population or geographical range reductions are available.Considering the IUCN categories and criteria to evaluate threatened species (IUCN, 2001(IUCN, , 2010)), the actual information about L. costai allows only apply the criterion D2 (area of occupation less than 20 km²) for the category Vulnerable (VU).Conservation status of L. costai may be better circumscribed with a detailed analysis of its population density.Approval of conservation measures for this area is important not only to preserve this species but also for the conservation of other endemic species, such as Rivulus lazzarotoi.
Etymology.The species epithet is given for Dr. Wilson J. E. M. Costa, in recognition of his significant contributions to Neotropical Ichthyology, including the study with trichomycterid catfishes.Molecular analyses.Partial sequences of the Cytb gene were taken from 17 specimens which resulted in a matrix with 1085 base pairs (bp) from which 949 sites were conserved, 136 were variable, and 123 parsimony informative.The nucleotide frequencies were 29.8% thymine, 29.6% cytosine, 26.2% adenine, and 14.4% guanine.In COI gene, partial sequences of only 12 specimens were amplified (amplification did not work in one specimen of L. costai, one of L. picinguabae, and three of Listrura sp.), resulting in a matrix with 789 bp from which 698 sites were conserved, 91 were variable, and 73 parsimony informative.The nucleotide frequencies were 30.0%thymine, 28.3% cytosine, 23.3% adenine, and 18.4% guanine.A single haplotype was found in both mitochondrial markers for each species with more than one sample, except for L. tetraradiata which presented two haplotypes.Values of molecular distances between L. costai, L. nematopteryx, L. picinguabae, Listrura sp., and L. tetraradiata are given in Table 2.The intraspecific distance values for all species were zero in both mitochondrial genes, except in COI gene of L. tetraradiata that was 0.001 ± 0.001, due to a single nucleotide different on one specimen.Interspecific distance values for Cytb gene range from 0.036 ± 0.006 between Listrura sp. and L. nematopteryx to 0.091 ± 0.009 between L. tetraradiata and L. nematopteryx.For COI gene, the range was from 0.038 ± 0.007 between Listrura sp. and L. nematopteryx to 0.076 ± 0.011 between Listrura sp. and L. tetraradiata.Phylogenetic analyses for Cytb gene obtained 70875 most parsimonious trees, with length of 165 steps, consistency index 0.855263 and retention index 0.957115.For COI gene, 315 most parsimonious trees were obtained, with length of 108 steps, consistency index 0.844444 and retention index 0.936364.Figure 9 presents two consensus trees obtained from most parsimonious Cytb-and COI-gene trees, respectively, with the bootstrap values for 1,000 pseudoreplicates.Both consensus topologies present a same monophyletic clade (L.nematopteryx plus Listrura sp.) well-supported by bootstrap values equal than 100%.Listrura costai also shows wellsupported by 100% bootstrap in both topologies.

Discussion
de Pinna (1988) suggested the absence of the posterior process of vomer as a putative synapomorphy for the genus, corroborated by Landim & Costa (2002) and Villa-Verde & Costa (2006).This feature was herein found in Listrura costai (Fig. 6a), as well as in all specimens of Listrura examined, reinforcing the synapomorphic condition of this character.
Listrura costai, L. nematopteryx, and L. picinguabae are morphologically very similar to each other when compared with the remaining congeners.They are the only valid species of the genus that present a single pectoralfin ray.The glanapterygines Pygidianops cuao Schaefer, Provenzano, de Pinna & Baskin and P. amphioxus de Pinna & Kirovsky also present this feature, but in the former it is unsegmented and shorter (0.3 times head length), with the fin expanded at the base (Schaefer et al., 2005).de Pinna & Kirovsky (2011) did not inform the length of pectoral-fin ray for P. amphioxus, but the figures presented indicate  that it is less than 0.4 times head length.In opposition, those species of Listrura present a segmented and longer (0.7-1.1 times head length) pectoral-fin ray, with the fin narrow at the base.The presence of a single pectoral-fin ray in both taxa is clearly a convergence.The molecular analyses present interesting results about the relationships among the species used in this study.The population of Listrura from rio São João basin (herein treated as Listrura sp.) is clearly a distinct taxon by absence of the dorsal fin.It also displays only one pectoral-fin ray.However, the overall results of the molecular analyses indicate a close relationship between Listrura sp. and L. nematopteryx.The lowest values observed in genetic distance were between these two species for both Cytb and COI genes, 0.036 ± 0.006 and 0.038 ± 0.007 respectively (Table 2).On other hand, the observed values between L. costai and the remaining species were higher, range from 0.045 ± 0.007 to 0.078 ± 0.009 for Cytb gene and from 0.039 ± 0.007 to 0.070 ± 0.010 for COI gene.It means that the degree of genetic divergence of L. costai is consistent with those observed in its congeners (see the interspecific values for remaining species analyzed; Table 2).The phylogenetic analyses of both genes indicate that L. costai comprises a well-defined distinct monophyletic taxon with 100% bootstrap value (Fig. 9).In fact, it has been observed in a more inclusive phylogenetic analysis for Listrura using morphological data (Villa-Verde, 2006).Phylogenetic analyses of the genus using both morphological and molecular data are being made (by senior author) and the conclusive results also corroborate the species status of Listrura costai.
An anterior process on the first hypobranchial was observed in all CS specimens examined (5) of L. costai, although there is a variation in the degree of development (Figs. 3a,6c).The presence of this process contrasts with its absence in all other species of the genus (see Comparative Material Examined for a relation of total number of CS specimens examined), therefore this character can be considered as an autapomorphy for the new species.A similar process was also reported and considered as a putative synapomorphy for basal trichomycterid subfamilies Trichogeninae and Copionodontinae (de Pinna, 1998).On the basis of the current hypotheses of phylogenetic relationship among the subfamilies of Trichomycteridae (de Pinna, 1998: fig.10), the process is herein considered as homoplastic character for basal trichomycterids and L. costai.
The occurrence area of L. costai is geographically situated between the distributional range of L. nematopteryx and L. picinguabae, about 120 km and 70 km of distance, respectively (Fig. 7).The new species occurs in the lowland of a small coastal river basin, draining a restricted area between the Serra da Bocaina slopes and the sea, and isolated from adjacent coastal basins by smaller mountains.Thus, L. costai is not likely to be found elsewhere out of this area.

Fig. 5 .
Fig. 5. Scheme of anal-fin origin in relation to dorsal-fin origin of Listrura costai, new species, showing the anteriormost (left) and posteriormost (right) positions of anal fin.

Fig. 9 .
Fig. 9. Two Maximum Parsimony consensus trees for the mitochondrial DNA genes Cytochrome b (left) and Cytochrome Oxidase C subunit I (right), respectively.Numbers indicate the bootstrap values with 1,000 pseudoreplicates.Segments with values < 50% were condensed and values not shown.

Table 1 .
Morphometric data of holotype and 14 paratypes of Listrura costai, new species.