Description of Priapella lacandonae sp . n . – a new poeciliid fish from the río Tulijá basin , Grijalva system , Chiapas , Mexico ( Teleostei : Cyprinodontiformes : Poeciliidae )

Priapella lacandonae, new species, is described from río Mizola, río Tulijá basin, río Grijalva system, Chiapas, Mexico. It is clearly distinguished by metrics, gonopodial and other morphological characteristics from all other species of the genus. A mitochondrial DNA-sequence based molecular phylogenetic analysis revealed similar results on the status of the new species as a separate taxon and its relation to the other closest related species.


Introduction
Fish of the poeciliid genus Priapella, which includes P. chamulae Schartl, Meyer & Wilde, 2006, P. compressa Alvarez, 1948, P. intermedia Alvarez, 1952, P. olmecae Meyer & Perez, 1990 and the type species P. bonita (Meek, 1904) inhabit clear riverine and lacustrine freshwater biotopes in southeastern Mexico.P. compressa, P. chamulae and the new species are found in the upper reaches of the río Grijalva system, Chiapas, Mexico.In this area P. compressa and the new species are not syntopic as far as known.The present paper describes the new species of Priapella from the headwaters of the río Tulijá basin, Chiapas, Mexico.Parenti (1981), Parenti & Rauchenberger (1989) established the supertribus Poeciliini and in-cluded Priapella and all remaining genera of Poeciliinae except Tomeurus.The tribe Poeciliini sensu Rosen & Bailey (1963) was adopted by the authors.According to Rosen (1979) and Schartl, Meyer & Wilde (2006) Xiphophorus is the most closely related genus to Priapella.Meyer et al. (1994) generally confi rmed by DNA-sequence based phylogenetic methods earlier studies that used morphological and metric analyses.On the contrary, Rodriguez (1997) strongly advocated for an exclusion of Priapella from Poeciliinae.Gheodotti (2000) fi nally established for the genus Priapella an own tribe, Priapellini, and points out the close relationship to Alfarini and Gambusiini.Finally Hrbek et al. (2007) ordered Priapella together with Scolichthys, Carlhubbsia, Xiphophorus, Heterandria (however, see Hrbek et al. 2007 for the status of H. formosa), Gambusia and Belonesox in a group, which then represents a supertribus or tribus Gambusiini.Within this group Priapella must be recognized as the main primitive Gambusiini member that occurred already approximately 44 million years ago in Middle Central America (Hrbek et al. 2007).
Measurements were made by vernier calipers, reading to 0.1 mm.Measurements and counts follow standard practice (Miller, 1948).The length of the distal tip of gonopodium ray 4a and 3 was measured on a horizontal line from the distal tip of ray 4p to the distal tip of the gonopodial hook.The depth of gonopodium was measured on a vertical line from ray 3 to ray 5p exactly where the serrae 4p starts to nestle against ray 4a.The number of specimens for all counts is greater or equal to 5. The total gill-raker count of the fi rst gill arch includes all gill rakers in the angle of the gill arch.The last two rays in the dorsal fi n were counted as a single ray.Vertebral counts include the hypural plate as one vertebra.The nomenclature of the sensory canal system of the head follows the standard of Gosline (1949) and parts of the gonopodial system follow Rosen & Gordon (1953), Rosen & Kallman (1959) and Rosen & Bailey (1963).
Genomic DNA was isolated from pooled organs of individual fi sh as described by Schartl et al. (1995) or from dorsal fi n clips according to Altschmied et al. (1997).Sequences from the mitochon drial cytochrome b gene were amplifi ed by PCR.For cytochrome b the primer pair L14725 (5'GA YTTGAARAACCAYCGTTG3' and H15982 (5'CCT AGCTTTGGGAGYTAGG3') described by Hrbek et al. (2007) was used.The PCR was done under the following conditions: denaturation 95 °C for 30 sec, annealing 48 °C for 40 sec, exten sion 72 °C for 45 sec.31 cycles were run from less than 100 ng genomic DNA.In each case a single PCR product was obtained and sequenced directly by GATC Biotech (Konstanz, Germany).Nucleotide sequences were analyzed using the program BioEdit Sequence Alignment Editor (copyright http://www.mbio.ncsu.edu/BioEdit/bioedit. html).Multiple sequence a lign ments were generated using the same program.Phylogenetic analyses were done with MEGA 3.1 (Ta mura et al., 2007).For calculating the distance matrix the transition/transversion rate was set to 2.0.Minimum evolution and Neighborjoining (NJ) trees were constructed.Robustness of the trees was tested by bootstrap analyses using 2000 and 10 000 replicas.All DNA sequences generated in this study are deposited in GenBank under accession numbers JF 892547 -49.Diagnosis.Priapella lacandonae is a large sized species of Priapella (max.SL 48 mm), which is distinguished from all other species of the genus by the following characters: membranous hook of the gonopodium ray 3 weakly developed and distally faintly bent, vs. slightly bent in P. bonita, P. compressa, P. inter-media, P. olmecae and strongly bent in P. chamulae; subdistal plate-like membranous process on gonopodium ray 3 faintly developed, vs. well developed in P. chamulae and not very prominent in P. compressa and P. intermedia; distal spines of gonopodium ray 3 long and densely crowded, vs. not densely crowded in P. compressa and P. intermedia, and short and not densely crowed in P. olmecae; tip of gonopodium ray 4p reaching the tip of the membranous hook, vs. not reaching the tip of the hook in P. bonita, P. compressa, P. intermedia, P. chamulae and P. olmecae.P. lacandonae is also distinguished by the following unique combination of characters: length of gonopodium short; frequency distribution of SL/GL radius 3.30 -3.60, vs. 2.60 -2.75 in P. olmecae, 3.00 -3.20 in P. compressa, 2.60 in P. bonita and 3.50 -3.60 in P. intermedia; 11 to 12 gill rakers, vs. 15 -16 in P. bonita, 12 to 13 in P. compressa, 12 to 14 in P. compressa; 8 to 9 dorsal fi n rays, vs. 8 in P. bonita, 10 in P. compressa.ray 4p straight and distally slightly bent, proximal serrae with 8 to 9 thorns; ray 5 shorter than rays 3 and 4. Rays 6 and 7 thickened distally, tips broadly expanded and curved dorsally.

Priapella lacandonae, new species
Gonopodial suspensorium with three well developed gonapophyses, gonapophyses I, II and III long and slender, each with an uncinus (interpreted by Rosen & Bailey (1963) as parapophyses); parapophyses present on gonapophysis I, II and III; shaft tips of gonapophysis I and II angular and curved ventrally, gonapophysis III curved anteriorly.Ligastyle very long and slender.Gonactinost 1 without inferior winglike appendage, gonactinostal complex 2 to 4 in front with a superior lateral wing, gonactinosts 5 to 9 without bony plates or outgrowths.
Dorsal fi n with 8 -9 rays (fi rst 3 rays simple, all others branched), origin of dorsal fi n posterior to the insertion of anal fi n; caudal fi n with 27 to 28 rays (15 branched); anal fi n 10 rays (fi rst 2 to 3 rays simple, all others branched); pectoral fi n with 13 to 14 rays (fi rst 2 and last 2 rays simple, all others branched); ventral fi n with 6 rays (fi rst and last ray simple, all others branched), in females not reaching to the anal fi n base and in males reaching to the base of the gonopodium, fi rst ray thin and bent distally.Adult males with a weakly developed keel, starting on the edge of caudal peduncle and ending near base of gonopodium; keel with small scales.
Males and females without sex specifi c coloration (Fig. 3).Body color of adult females and males greyish; iris iridescent blue.Border of dorsal fi n and upper and lower ramus of caudal fi n with light whitish color, all other fi ns hyaline.
Etymology.The new taxon is named in appreciation of the native population of Lacandons, who live Description.Body deep, head long and sharply pointed, 25.5 -27.5 % of SL.Longitudinal scale series 26 -(rarely) 28; predorsal scale series 13 -14; scale series around caudal peduncle 16.Number of vertebrae 29 to 30.Gill rakers on fi rst arch 11 to 12 + 1.
Teeth of upper and lower jaws unicuspid and recurved; those of outer row enlarged and spear-like shaped, not numerous and widely spaced; spear-like shaped inner teeth numerous.Upper pharyngeal bones kidney-shaped.Teeth of the medial region somewhat enlarged, each side with a series of 5 -6 rows, teeth small and conical.Lower pharyngeal bone (ceratobranchial 5) with a total of 120 -140 large unicuspid teeth, 6 -8 on posterior rows, 6 -7 on middle rows.Teeth of medial region very large.Both halves of lower pharyngeal antler-shaped, closely together on a small part along midline.Arms of pharyngeal long and split at the ends.Ceratobranchial 4 without teeth, hypobranchial 4 absent.
Gonopodium short and compact, 3.3 to 3.6 times in SL; ray 3 broadly expanded, terminating in a small and faintly bent membranous hook, distal spine-like bony segment of ray 3 long, angular and directed towards subdistal spine-like series, 8 to 9 slightly angulate spine-like series long and slender, plate-like process of membranous tissue ventral to spine-like series not well developed, base of ray 3 somewhat thickened and subdivided in 4 to 5 segments; ray 4a longest and generally totally bent over the gonopodium hook of ray 3, subdistal segments of ray 4a broader than long; is recognized as a separate species, because it does not share the following synapomorphy between P. compressa and P. intermedia: distal tip of gonopodium long (LDG/DDG radius 0,85 -0,95).In addition there is one autapomorphy of P. lacandonae: ray 4a totally bent over the hook of gonopodium ray 3. P. lacandonae is further distinguished from P. intermedia and P. compressa by fewer subdistal spines of gonopodium ray 3 (3 -5 vs. 6 -7, 5 -7), from P. intermedia by the much longer fi rst isolated spine of gonopodium ray 3, and a much longer distal spine-like bony segment of ray 3, from P. olmecae by longer spines of gonopodium ray 3; and from P. compressa by fewer dorsal fi n rays (8 -9 vs. 10).
A molecular phylogenetic analysis was performed using mitochondrial cytochrome b sequences.The resulting data set consists of approx.1130 bases for each species.The genetic distances calculated (Table 2) revealed that the genetic distance between the species of the genus Priapella are in the same range as for instance over the whole genus Xiphophorus (Meyer & Schartl, 2003).In particular the genetic mainly in northeastern Chiapas, Mexico and Petén, Guatemala.
Comparison and relationships.Rodriguez (1997) gives the following derived characters for the genus Priapella: gonopodium ray 3 with the most distal segment modifi ed into a forward-curved bony hook, a long membranous segment posterior to the ray joining with the last segments of ray 4a, the membranous segment becoming fl at at the junction and gonopodium ray 4a with 10 -13 distal segments extending far behind the tip of ray 4p.All these characters are present in P. lacandonae.On the basis of these synapomorphies, P. lacandonae is unequivocally attached to the genus Priapella.
By morphological criteria P. lacandonae is most closely related to P. chamulae and P. compressa.There are several synapomorphies that unite P. lacandonae with P. compressa and P. chamulae, namely: numerous, long and pointed subdistal spines of gonopodium ray 3; subdistal segments of gonopodium ray 4a opposite serrae shorter than high.However, P. lacandonae  distance values between P. lacandonae and the most closely related species, P. compressa and P. chamulae are even higher than corresponding values for the well recognized species of Southern swordtails.The genetic distance between one individual of P. lacandonae collected above the waterfall and one below the waterfall was zero.The molecular data were analyzed with minimum evolution and neighbour-joining methods which yielded almost identical phylogenetic results.The topology of the resulting trees was always the same.Two major branches were resolved, which unite P. intermedia and P. olmecae in one group and P. compressa, P. chamulae and P. lacandonae in the other.The new species is clearly separated from P. chamulae; similar to the split between P. intermedia and P. olmecae.It is also separated from P. compressa on a branch, the length of which is absolutely in the range of those for the other previously recognized species.Of note, all nodes within the genus Priapella are supported by high bootstrap values, which underlines the robustness of the molecular phylogenetic tree.
Distribution.Priapella lacandonae is only known from the type locality.
Habitat notes.The collection site is a small stream (río Mizola), approx.7 to 8 m wide, above Cascada Misol-Ha, that fl ows into to the río Tulijá basin, río Grijalva system.At the type locality (280 m eleva-

Table 1 .
Measurements (in mm) of holotype and paratypes of Priapella lacandonae sp.n.
Phylogram of Priapella lacandonae and related Priapella species based on mitochondrial DNA sequences.50% majority rule consensus tree rooted on Xiphophorus maculatus as outgroup.Minimal and maximal bootstrap values obtained using different types of analysis (see Material and Methods) are indicated above the branches.tion,17° 23′ 15.9′′ N, 91° 59′ 17.4′′ W) on March, 9 th , 2004 at noon the water had a temperature of 25 °C, a conductivity of 520 μS, pH 8.3, and a total hardness of 16.The water was clear and fast fl owing with no aquatic vegetation.The substratum consisted mainly of rocks, gravel, sand and leaves.The stream is shadowed by the vegetation.Accompanying poeciliid fi shes were Pseudoxiphophorus bimaculatus, Poecilia mexicana, cichlids and a Characid, Astyanax mexicanus.The habitat below the waterfall is similar, except that there are large rocks in the river that structure the environment and create basins of different fl ow velocity.On May 8 th , 2007, at noon the water had 27 °C (air 32 °C), a conductivity of 72 μS, pH 8 and a total hardness of 8. Accompaning poeciliid fi shes were Xiphophorus hellerii, Poecilia mexicana and a Characid, Astyanax mexicanus.

Table 2 .
Pairwise distance matrix between Priapella species and X. maculatus using the Kimura 2-parameter as correction method.