Description of Gambusia zarskei sp. n. – a new poeciliid fish from the upper Rio Conchos system, Chihuahua, Mexico (Teleostei: Cyprinodontiformes: Poeciliidae)

Since the systematic listing of the representatives of the Gambusiini by ROSEN & BAILEY (1963), numerous new descriptions and changes in nomenclature have occurred within the tribus, particularly in the case of the Gambusia. RAUCHENBERGER (1989) designates a total of three subgenera for Gambusia, namely Arthrophallus, Heterophallina and Gambusia. The subgenera Arthrophallus, according to RAUCHENBERGER (1989), includes three groups, namely the affi nis species group, nobilis species group and senilis species group, whereby the here described new species is clearly a member of the later species group, together with alvarezi HUBBS & SPRINGER, 1957, amistadensis PEDEN, 1973, atrora ROSEN & BAILEY, 1963, gaigei HUBBS, 1929, geiseri HUBBS & HUBBS, 1957, hurtadoi HUBBS & SPRINGER, 1957, longispinis MINCKLEY, 1962 and senilis GIRARD, 1859. It should be mentioned that due to cytochrome b sequence variations, LYDEARD et al. (1995) developed differently weighted molecularphylogenetic pedigrees from some Gambusiini groups. In contrast to RAUCHENBERGER (1989), LYDEARD et al. (1995) classify hurtadoi as an independent complex together with vittata, marshi and panuco, whereas the later three taxa are included in the subgenus Heterophallina and hurtadoi in the subgenus Arthrophallus by RAUCHENBERGER (1989). Furthermore geiseri is not associated by LYDEARD et al. (1995) with any member of the senilis species group, but with the affi nis species group. In the light of this confl icting classifi cation independent studies with additional material on the systematics of the genus Gambusia appear to be timely and necessary. The genus Gambusia is distributed widely over continental and coastal habitats, from eastern North Description of Gambusia zarskei sp. n. – a new poeciliid fish from the upper Rio Conchos system, Chihuahua, Mexico (Teleostei: Cyprinodontiformes: Poeciliidae)


Introduction
Since the systematic listing of the representatives of the Gambusiini by ROSEN & BAILEY (1963), numerous new descriptions and changes in nomenclature have occurred within the tribus, particularly in the case of the Gambusia.RAUCHENBERGER (1989) designates a total of three subgenera for Gambusia, namely Arthrophallus, Heterophallina and Gambusia.The subgenera Arthrophallus, according to RAUCHENBERGER (1989), includes three groups, namely the affi nis species group, nobilis species group and senilis species group, whereby the here described new species is clearly a member of the later species group, together with alvarezi HUBBS & SPRINGER, 1957, amistadensis PEDEN, 1973, atrora ROSEN & BAILEY, 1963, gaigei HUBBS, 1929, geiseri HUBBS & HUBBS, 1957, hurtadoi HUBBS & SPRINGER, 1957, longispinis MINCKLEY, 1962 and senilis GIRARD, 1859.It should be mentioned that due to cytochrome b sequence variations, LYDEARD et al. (1995) developed differently weighted molecularphylogenetic pedigrees from some Gambusiini groups.In contrast to RAUCHENBERGER (1989), LYDEARD et al. (1995) classify hurtadoi as an independent complex together with vittata, marshi and panuco, whereas the later three taxa are included in the subgenus Heterophallina and hurtadoi in the subgenus Arthrophallus by RAUCHENBERGER (1989).Furthermore geiseri is not associated by LYDEARD et al. (1995) with any member of the senilis species group, but with the affi nis species group.In the light of this confl icting classifi cation independent studies with additional material on the systematics of the genus Gambusia appear to be timely and necessary.
The genus Gambusia is distributed widely over continental and coastal habitats, from eastern North  Measurements were made by vernier calipers, reading to 0.1 mm.Measurements and counts follow standard practice (MILLER, 1948).The length of distal tip of gonopodium ray 4a and 3 is measured on a horizontal line from the distal tip of ray 4p to distal tip of gonopodium hook.The determination of relative gonopodial serra length (SEL/SEW) follows the method of PEDEN (1973).The number of specimens for all counts is greater or equal to 5. The total gillraker 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 are 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 components of the gonopodial system follow ROSEN & GORDON (1953), ROSEN & KALLMAN (1959) and ROSEN & BAILEY (1963).Ge nomic DNA was isolated from dorsal fi n clips according to ALTSCHMIED et al. (1997).Mitochondrial sequences were amplifi ed by PCR with primers H16100 (5'ATGTAGGGTTACAYTACTTTAAATGG3') and L15513 (5'CTRGGAGACCCNGAAAACTT3').The PCR was done under the following conditions: denaturation 95 °C for 120 sec, 35 cycles of denaturation 95 °C for 30 sec annealing 49 °C for 30 sec, extension 72 °C for 40 sec, followed by a fi nal extension step at 72 °C for 120 sec.PCR cycles were run from less than 100 ng genomic DNA.In each case a single PCR product was obtained and sequenced directly.Nucleotide sequences were analysed using programs of the MEGA4.0 program package (TAMURA et al., 2007).Multiple sequence alignments were generated and phylogenetic trees were constructed using maximum parsimony, minimum evolution and neighbour-joining methods.Robustness of the trees was tested by bootstrap analyses using 100 replicas or in the case of neighbour-joining trees 1000 replicas.Pair-wise distances were calculated with the program M4 as part of MEGA4.0 conducted on the basis of the Maximum Composite Likelihood method using all distance correction methods implemented in the program.All DNA sequences generated in this study are deposited in GenBank under accession numbers GU 583794, GU 593795.

Subgenus Arthrophallus HUBBS, 1926
The subgenus Arthrophallus is characterized by the derived character (S1) sections 4b to 6a and 6b to

Diagnosis
Gambusia zarskei is a small sized species of the subgenus Arthrophallus (SL in males usually not exceeding 25 mm and in females 35 mm), which is distinguished from all other species of the senilis species group by the following characters: shafts of distal spines of gonopodium ray 3 much prolonged, lateral projection on elbow weekly developed, vs. well developed in  The cephalic sensory canals system consists of open canals and neuromasts.Supraorbital canal well developed, sections 1+2a, 2b -4a, 4b -6a, 6b -7 represented as open grooves; preopercular 7 neuromasts; preorbital 7 neuromasts; mandibular with 4 neuromasts.
Gonopodium short and compact, 2.9 to 3.2 times in SL; ray 3 broadly expanded, terminating in a well  Vertebrate Zoology ■ 60 (1) 2010 developed spine series; distal spines of ray 3 very long, angular and projected beyond rays 4 to 5, 6 to 7 angulate spines long and pointed anterodistally, base of ray 3 somewhat tapered; ray 4a very short reaching to the middle of the large rounded terminal hook on ray 5a, ray 4a with an anterior swelling (elbow), elbow segments usually not fused, major elbow segment straight and not very long; ray 4p longer than 4a and terminating with a large rounded hook, 5 to 8 retrorse serrae, the lengths much longer than the widths of their bases; ray 5a shorter than ray 4p and longer than ray 4a.Ray 6 and 7 straight, ray 6 thickened distally.
Dorsal fi n with 8, rarely 9 rays (fi rst 2 rays simple, all others branched), origin of dorsal fi n posterior to the insertion of anal fi n; caudal fi n with 31 to 33 (14 -16 branched) rays; anal fi n 9 rays (fi rst 3 rays simple, all others branched); pectoral fi n with 14 to 15 rays (top 2 and bottom 3 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 urogenital papilla and in males reaching to the base of the gonopodium, fi rst ray thin and bent distally.Adult males with not very prominent keel, starting at the edge of caudal peduncle and ending near base of gonopodium.Males and females without sex specifi c coloration .Body color of adult females and males greyish blue; lower posterior body sides intensely orange-yellow; iris black; short subocular dark bar leading posteroventrally across cheek from lower border of the eye.Border of dorsal fi n black and subbasal row of a dark spot series present on lower third of dorsal fi n, more intense in males.All fi ns hyaline and often light yellowish.Adult females with a black anal spot, the melanophores being mainly concentrated between the anus and urogenital papilla.

Etymology:
We name this species after Dr. AXEL ZAR-SKE in recognition of his valuable contributions to discussions on the conservation biology and problems of endangered fi shes such as G. zarskei.
Comparison and relationships: RAUCHENBERGER (1989) gives the following derived characters for the senilis species subgroup: medial anal spot in females and transversely enlarged segment distal to serrae on ray 4p.A third derived character for the senilis species group is the reduction of gonopodium ray 4a segments distal from the elbow.All these characters are present in G. zarskei.On the basis of these synapomorphies, G. zarskei is herein unequivocally attached to the senilis species group.The species G. geiseri, G. longispinis and G. atrora are not referred herein to the senilis species group because no synapomorphies could be found that unite them with G. alvarezi, G. amistadensis, G. gaigei, G. hurtadoi, G. senilis or G. zarskei.
By morphological criteria G. zarskei is most closely related to G. amistadensis and G. hurtadoi.There are several synapomorphies that unite G. amistadensis, G. hurtadoi with G. zarskei, namely: shafts of distal spines of gonopodium ray 3 much prolonged, lateral projection on elbow developed.However, G. zarskei is recognized as a separate species, because it does not share the following synapomorphies between G. hurtadoi: distal tip of gonopodium long.On the other hand there is one autapomorphy of G. zarskei: terminal spines of gonopodium ray 3 prolonged.G. zarskei is further distinguished from G. hurtadoi, G. alvarezi and G. senilis by fewer spines of gonopodium ray 3 (6 -7 vs. 7 -8), from G. senilis by the absence of an anal spot in females and a much shorter main segment of the gonopodium elbow, and from G. alvarezi and G. hurtadoi by fewer dorsal fi n rays (8 vs. 9) and fewer anal fi n rays (9 vs. 10).
A molecular phylogenetic analysis was performed using mitochondrial sequences (fi g. 6, tab.2).The resulting data set consists of 343 bases from the 3'part of the cytochrome b gene for each species.The genetic distance calculations revealed that there is an obvious closest genetic relationship of G. zarskei with G. hurtadoi.
The molecular data were analyzed with maximum parsimony, minimal evolution and neighbour-joining methods, which yielded almost identical phylogenetic results.The topology of the resulting trees was always the same.Two major branches supported by high bootstrap values were resolved, which unite G. zarskei with G. hurtadoi in one group and G. vittata with G. atrora in the other.The new species is clearly separated from G. hurtadoi similar to the split between G. vittata and G. atrora.Although the region that was analyzed is relatively short the phylogenetic information is robust and the separation of the species in percent changes is Unexpectedly, the sequence of the 5' part of the cytochrome b gene of G. zarskei could not be used for the phylogenetic analysis, although this part of the mitochondrial genome has proven to be very useful for analysing phylogenetic relationships on the species level in all our previous studies of poeciliid fi shes (MEYER & SCHARTL, 2002;MEYER & SCHARTL, 2003;MEYER, SCHNEIDER, RADDA, WILDE & SCHARTL, 2004;SCHARTL, MEYER & WILDE, 2006).In this region the cytochrome b sequence of G. zarskei has several deletions and aminoacid changes at highly conserved positions (data not shown), which would result in a significantly altered protein whose functionality would have to be questioned.Given the conservation of the primary sequence of b-type cytochromes this is a puzzling result that cannot be explained without further studies.At least, this highly aberrant cytochrome b sequence is another molecular discriminator for the new species, as all other Gambusia species analyzed so far have the expected aminoacid conservation of cytchrome b.
Distribution.Gambusia zarskei is only known from the upper Rio Conchos system in the Sierra San Diego area, east of San Diego de Alcalá, Chihuahua, Mexico.
Habitat notes.Gambusia zarskei inhabits the waters of small warm springs and their outlets at the foot of Sierra San Diego on the northwestern mountain side.The collection sites are small outlets, approx.0.5 to 2.0 m wide and 0.60 to 1.00 m deep, that fl ow into the Rio Chuviscar drainage, a tributary of the Rio Con-  chos, 2 km east of San Diego de Alcalá on an unpaved road.At the type locality (N 28° 35' 36.2",W 105° 33' 00.0") on March, 18 th , 2006 before noon the water had a temperature of 24 to 26 °C, a conductivity of 1486 mS, pH 7.85, and a total hardness of 9.The water was crystal clear, fast fl owing and without aquatic vegetation.The substratum consisted mainly of gravel and sand.The brook fl ows in an open area.Accompanying fi shes were Gambusia senilis and Cyprinodon spec.Gambusia zarskei was mainly found in water depth of 0.05 to 0.10 m along the water edges, where the water temperature might rises to 34 °C near the hot spring.

Fig. 6 .
Fig. 6.Phylogram of Gambusia zarskei and related Gambusia species based on mitochondrial DNA sequences.50 % majority rule consensus tree rooted on Belonexos belizanus as outgroup.Average bootstrap values obtained using different types of analysis (see Material and Methods) are indicated above the branches.Bootstrap values below 50 % are not shown.
America to northwestern South America, including the Antilles.The new species is found at an altitude of 1135 m and is thereby one of several endemic northern Gambusia species which live in thermal springs in the uplands of northern Mexico and southern USA.The present paper describes a new species of Gambusia from the headwaters of the Rio Conchos, Chihuahua, Mexico.

Table 1 .
Measurements (in mm) of holotype and paratypes of Gambusia zarskei sp.n.
Tab. 2. Pairwise distance matrix of Gambusia species and Belonesox belizanus mitochondrial sequences using the Maximum Composite Likelihood method.