Profundulus kreiseri, a new species of Profundulidae (Teleostei, Cyprinodontiformes) from northwestern Honduras

Abstract A new species of Profundulus, Profundulus kreiseri (Cyprinodontiformes: Profundulidae), is described from the Chamelecón and Ulúa Rivers in the northwestern Honduran highlands. Based on a phylogenetic analysis using cytochrome b and the presence of synapomorphic characters (dark humeral spot, a scaled preorbital region and between 32-34 vertebrae), this new species is placed in the subgenus Profundulus, which also includes Profundulus (Profundulus) oaxacae, Profundulus (Profundulus) punctatus and Profundulus (Profundulus) guatemalensis. Profundulus kreiseri can be distinguished from other members of the subgenus Profundulus by having less than half of its caudal fin densely scaled. Profundulus kreiseri can further be differentiated from Profundulus (Profundulus) oaxacae and Profundulus (Profundulus) punctatus by the absence of rows of dark spots on its flanks. The new species can further be differentiated from Profundulus (Profundulus) guatemalensis by the presence of fewer caudal- and pectoral-fin rays. The new species is distinguished from congeners of the profundulid subgenus Tlaloc (viz., Profundulus (Tlaloc) hildebrandi, Profundulus (Tlaloc) labialis, Profundulus (Tlaloc) candalarius and Profundulus (Tlaloc) portillorum) by having a scaled preorbital region and a dark humeral spot. Profundulus kreiseri and Profundulus portillorum are the only two species of Profundulus that are endemic to the region south of the Motagua River drainage in southern Guatemala and northwestern Honduras.

portillorum) by having a scaled preorbital region and a dark humeral spot. Profundulus kreiseri and P. portillorum are the only two species of Profundulus that are endemic to the region south of the Motagua River drainage in southern Guatemala and northwestern Honduras.

Keywords
Central America, nuclear Middle America, Cyprinodontiformes, Kreiser's Killifish, phylogeny, systematics introduction The genus Profundulus Hubbs, 1924, is a depauperate lineage of northern Central American and southern Mexican (Fig. 1) cyprinodontiforms that belong to Profundulidae. The family comprises a single genus, Profundulus, which has seven valid species (Matamoros and Schaefer 2010). The northernmost distributed species, Profundulus oaxacae (Meek, 1902), is found in the Río Verde basin, in the Mexican Pacific slope in the state of Oaxaca (Miller et al. 2005). The southernmost boundary of the genus is delimited by the distributional range of P. portillorum Matamoros and Schaefer, 2010, which is found in the headwaters of the Ulúa and Nacaome River basins in the Atlantic and Pacific slopes of Honduras (Matamoros et al. 2009;Matamoros and Schaefer 2010;Matamoros et al. 2012).
Among neotropical members of Cyprinodontiformes, profundulids are the least studied. Although some progress has been made over the past two decades in advancing our understanding of the genetics (Doadrio et al. 1999;Valencia-Diaz andEspinoza-Perez 2011), morphometrics (Gonzales-Diaz et al. 2005) and conservation (Velazquez-Velazquez and Schmitter-Soto 2004;Velazquez-Velazquez et al. 2009) of the family, many aspects of the ecology, life history and general biology of Profundulus species remain unknown. Furthermore, most papers that address Profundulus are focused on Mexico, the northernmost range of the family, and little is known about the species that occur south of Mexico (e.g., P. guatemalensis and P. portillorum). Recent ichthyological work in Honduras (Matamoros et al. 2009) and El Salvador (McMahan pers. comm.) has produced new localities and records of undescribed species for the family (e.g. P. portillorum). The aim of this paper is to describe a new species of Profundulus from the Chamelecón and Ulúa River basins in the Atlantic slope of Honduras.

Morphology
All specimens examined are housed at the University of Southern Mississippi Ichthyology Collection (USM), Louisiana State University Museum of Natural Science (LSUMZ), and University of Michigan Museum of Zoology (UMMZ). Measurements and counts were taken following Miller (1948), including standard length, snout length, head length, predorsal length, anal-fin origin to caudal-peduncle base, anal-fin length, eye diameter, head depth, caudal-peduncle depth, head width and maximum body width. All measurements were made using digital calipers. Counts include midline scales, scales around the caudal peduncle, anal-fin rays, dorsal-fin rays, pectoral-fin rays, and caudal-fin rays.

Molecular phylogeny
Genomic DNA was extracted from ethanol-preserved fin tissue using a DNeasy Tissue Kit (QIAGEN Inc.). We amplified the mitochondrial cytochrome b gene using the GluF and ThrR primers described by Machordom and Doadrio (2001). Initial sequences were generated with these two primers and used to design the following internal primers: ProcytbintF (5'-ACTCGATTCTTYGCCTTCCA-3') and ProcytbintR (5'-GGGTGAAATGARATTTTGTCG-3'). Subsequent amplifications and sequencing were conducted using the primer pairs GluF-ProcytbintR and ThrR-ProcytbintF. Amplifications were conducted in a total volume of either 25 ml or 50 ml using 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.01% gelatin, 200 mM dNTPs, 2 mM MgCl2, 0.5 units of Taq polymerase (New England Biolabs), 0.3 mM of each primer, 20-150 ng of template DNA, and water to the final volume. Cycling conditions consisted of an initial denaturing step of 1 min at 95º C followed by 30 cycles of 1 min at 95º C, 1 min at 50º C and 1 min at 72º C. A final elongation step of 72º C for 3 min completed the reaction. PCR products were cleaned using the ExoSAP-IT system (USB Co.), and then used as the template in a cycle sequencing reaction with an ABI BigDye Terminator cycle sequencing kit (Foster City, CA, USA) using the primers described above. Sequencing reactions were cleaned using sephadex (Princeton Separations, Adelphia, NJ, USA) and then sent to the Iowa State University DNA Sequencing and Synthesis Facility. The sequences were edited and aligned using Sequencher v. 4.10.1 (Gene-Codes Co.). Sample information and GenBank accession numbers for ingroup and outgroup species used in the analysis are listed in Table 1.
Sequence divergence (uncorrected p-distance) was estimated in PAUP* (Swofford 2002). The AIC (Akaike's information criterion) model selection procedure was implemented in jModelTest (Posada 2008) and used to obtain an appropriate substitution model and parameter values for phylogenetic analysis. Phylogenetic relationships were inferred by maximum parsimony (MP) using PAUP* (Swofford 2002) and Bayesian analysis (BA) using MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). All valid species of Profundulus were included in the analysis. Two species from the family Goodeidae, Allodontichthys hubbsi Miller & Uyeno, 1980, and Alloophorus robustus  (Bean, 1982), were used as outgroups in the two phylogenetic analyses: these taxa were selected because Costa (1998) recovered goodeids as the sister group of Profundulidae. For the MP analysis, a heuristic search was performed to find the most parsimonious tree(s). Nonparametric bootstrapping (Felsenstein 1985) was used to measure clade support, with 1000 total pseudoreplicates and TBR branch-swapping with 10 random sequence addition replicates per pseudoreplicate. For BA analysis we performed four simultaneous analyses, each with ten Markov chain Monte Carlo simulations run for 1,000,000 generations, sampling trees every 1000 generations. At the end of the analysis, the average standard deviation of the split frequencies was < 0.01, indicating that the runs had converged. The first 100 trees from each run before reaching equilibrium were discarded as burn-in. The remaining trees were used for reconstruction of a 50% majority-rule consensus tree with posterior probabilities values. One of the paratypes (USM 39024; tissue 08-2921) was sequenced (GenBank accession number JQ254935) and therefore constitutes a "paragenetype cytochrome b" following the nomenclature of Chakrabarty (2010).

Usage of "genetypes" nomenclature
The "genetypes" nomenclature was proposed by Chakrabarty (2010) to flag sequences from type specimens. Many genetypes can be created from a single specimen and these may be a single gene region or an entire genome; for instance "paragenetype COI" and "paragenetype ND2" could be added from USM 39024 at a later date, as could genetypes from other type specimens of this species (e.g., paratypes, holotype) from which DNA can be extracted. This nomenclature is simply a flag to alert molecular biologists and taxonomists that sequences are available from type specimens. The genetype terminology is not used here in a strict nomenclatural sense, as it is not formally accepted by the International Code for Zoological Nomenclature. However, we consider this terminology useful and expect increased application in the future. The present paper is one of the first uses of the genetype terminology.
Diagnosis Description. Morphometric and meristic data for type material are summarized in Table 2. The largest specimen is 81.2 mm SL. The body is elongate with the dorsal and ventral profiles nearly symmetrical. The narrowest point on the body is the tip of the snout, with the body expanding gradually dorsally and ventrally to the deepest point slightly anterior to the verticals through the dorsal-and anal-fin origins. The vertical through the origin of the dorsal fin is slightly anterior to the origin of the anal fin. The body depth narrows in the region of these two unpaired fins, and the dorsal and ventral body margins are straight and parallel on the caudal peduncle before diverging out slightly at the origin of the caudal fin.
The head (including cheek, infraorbital and preorbital regions) is covered with scales that are deeply embedded in the skin. The mouth is terminal, the lower jaw protruding slightly beyond the upper. The posterior portion of the maxilla extends ventrally to a vertical through the anterior region of the orbit.
The number of dorsal-fin rays ranges from 10-12 (holotype=11). The number of pectoral-fin rays ranges from 13-16 (holotype=14). The posterior edge of the pectoral fin does not reach the pelvic-fin origin. The number of anal-fin rays ranges from 9-14 (holotype=12; mode=12). The caudal fin is rounded, the number of fin rays ranging from 13-18 (holotype=17). All specimens examined have six pelvic-fin rays. The number of scales along the midline of the body ranges from 33-34 (holotype=33). The number of scales around the caudal peduncle ranges from 9-10 (holotype=10). The number of vertebrae ranges from 31-33 (holotype=32).
Live coloration. In life this species is brown, with a golden-yellow blotch that covers most of the operculum and reaches the base of the pectoral fin. An inconspicuous dark stripe is present along the midline of the body starting at a vertical between the dorsal-and anal-fin origins and terminating at the origin of the caudal fin.
Preserved coloration. The body is a uniform dusky brown with a prominent dark humeral spot posterior to the upper insertion of the pectoral fin. A dark stripe is present along the midbody; this stripe is more conspicuous in preservation than in life. The distal margins of the unpaired fins are opaque, but the basal ¾ of the fins are covered with scattered melanophores.
Distribution. Profundulus kreiseri is only known from the middle reaches of the Chamelecón and Ulúa rivers in Honduras (Figs. 1, 3).
Ecological notes. The only known localities of P. kreiseri are both characterized as small tributaries ranging in width from 0.8 to 4 meters with stones (from pebbles to boulders) as the dominant substrate. The canopy cover of both localities is estimated to be 70-80%. Both creeks feature a variety of run, pool, riffle, rapid and small waterfall habitats (Fig. 3).

Conservation.
The limited range of this species makes it vulnerable to extinction via habitat loss. The creation of a new hydroelectric dam on the Chamelecón River will likely drastically impact populations of this new species.
Etymology. The specific epithet is in honor of Dr. Brian R. Kreiser, the doctoral advisor and friend of the first author.
Suggested English name: Kreiser's Killifish Suggested Spanish name: El Escamudo de Kreiser

Molecular analysis
The phylogenetic analysis included sequences from 12 taxa (10 in the ingroup and 2 outgroups). Novel sequences were deposited in GenBank under accession numbers JQ254929-JQ254935 (Table 1). A total of 990 bp of cytochrome b were analyzed, of which 280 sites (28.3%) were parsimony informative. The GTR + I + G model was selected as the best fit for the dataset by jModelTest using the AIC. The optimized parameters were: A=0.2692A=0. , C=0.2640A=0. , G=0.2334.0000), the gamma distribution was 1.4610, and the proportion of invariable sites was 0.4970. The same tree topology was recovered by MP (a single tree, length: 585, CI: 0.735; RI: 0.796) and BA (Fig. 4). Two monophyletic groups corresponding to the two subgenera were recovered. Node support was generally high throughout the tree (Fig. 4). The new species was recovered as the sister group of P. guatemalensis with high bootstrap and posterior probability values. P-distances between the members of the two subgenera ranged from 17.8 to 19.3%. Within the subgenus Tlaloc, sequence divergence ranged from 1 to 9.2%. The shortest genetic distance was found between P. labialis and P. candalarius (1%). Sequence divergence between the new species (two specimens) and each of the three other species in the subgenus Profundulus ranged from 4.1 to 6.5% (Table 3). Table 3. Uncorrected pairwise genetic divergence based on cytochrome b sequences. C= chamelecón river, u= ulúa river, n= nacaome river.

Discussion
Profundulus portillorum has the southernmost range of any species in the family; it is found in the Ulúa River in the Atlantic slope of Honduras and the Nacaome River in the Pacific slope of Honduras (Matamoros and Schaefer 2010). The Chamelecón River locality reported for Profundulus kreiseri represents a new drainage for the family. Most species of Profundulus have restricted distributional ranges (found only among a few adjacent river systems) and only one species has a widespread distributional range, P. guatemalensis. This species is the only Profundulus to cross the Motagua River Fault into nuclear Middle America, a region that extends from southern Guatemala to northern Nicaragua (Matamoros et al. 2012). The Motagua River Fault may be a biogeographic boundary for Profundulus. Profundulus punctatus and P. labialis also reach the Motagua River, but are not found south of it (Miller 1955;Miller et al. 2005;Matamoros and Schaefer 2010) and P. portillorum and P. kreiseri are restricted to nuclear Middle America but are not found north of the Motagua River. The combination of the tree topologies, node support, uncorrected pairwise divergences values and morphological comparisons between the new species and its congeners fully support the distinctiveness of Profundulus kreiseri.
Profundulus kreiseri has higher intraspecific genetic variation (0.016) than is found between some species (e.g., 0.010 in P. labialis versus P. candalarius). The low sequence divergence found between P. labialis and P. candalarius may be a reflection of a recent divergence. These two taxa are currently allopatric and can be differentiated based on morphological characters (e.g., fin ray counts, body depth). However, additional work may be needed to verify the taxonomic status of these taxa given the results from our molecular analysis. Intraspecific variation in P. kreiseri is also twice as high as what was recovered for the only other species for which multiple individuals were sampled in our study, P. portillorum (0.008). This variation in P. kreiseri may reflect a relatively long period of isolation between the populations in the Chamelecón and Ulùa rivers.
Our molecular phylogenetic analysis recovered two distinct clades corresponding to the nominal subgenera and placed P. kreiseri within the subgenus Profundulus as the sister taxon of P. guatemalensis. The caudal-fin scales of P. kreiseri do not extend beyond the anterior half of the fin (Fig. 2D), whereas they cover half or more of the caudal fin in other members of the subgenus Profundulus (e.g., P. punctatus and P. guatemalensis- Fig.  2E, F). Because the presence of squamation on at least half of the caudal fin is a defining character of the subgenus (Miller 1955), our recognition of P. kreiseri as a member of the subgenus Profundulus is based on the molecular data and the presence in P. kreiseri and other members of the subgenus of a dark humeral spot and scaled preorbital region. The new species exhibits none of the diagnostic features of the subgenus Tlaloc. In addition to the caudal squamation, species status of Profundulus (P.) kreiseri is warranted based on its pigmentation pattern and numbers of pectoral-and caudal-fin rays. A dichotomous key to all of the Central American species of Profundulus is presented below.