Introduction

The family Profundulidae is one of few families of freshwater fishes endemic to the Mesoamerican region. It has been suggested that its history goes back to the Pliocene, or even the Miocene (^{Miller, 1955}). Currently, most species in the family exhibit low abundances, and their distribution is restricted to the headwaters and spring-fed-tributaries of rivers in southern Mexico, El Salvador, Honduras and Guatemala, from both Pacific and Atlantic versants. Species diversity in the family is fairly low, consisting of 8 species in 2 genera: Profundulus and Tlaloc (^{Morcillo, Ornelas-García, Alcaraz, Matamoros, & Doadrio, 2015}). In the genus Tlaloc , 4 species are currently recognized: T. candalarius , T . hildebrandi , T. labialis and T. portillorum , all of which exhibit limited distributions. The genus Profundulus also has 4 species, namely P. punctatus , P. oaxacae , P. guatemalensis and P. kreiseri , but has a decidedly wider distribution range.

In an early study dealing with systematics of Profundulidae, ^{Miller (1955)} pointed out a wide degree of morphological variation, being more evident within the P. punctatus "group", in which more than one species might be recognized. Years later, within the P. punctatus "group" ^{Miller (2005)} corroborated the validity of P. oaxacae (^{Meek, 1902}), whose distribution was restricted to altitudes above 1,550 m in the Río Atoyac - Verde Basin, Oaxaca, in southern Mexico (^{Martínez-Ramírez, Doadrio-Villarejo, & de Sostoa-Fernández, 2004}). Furthermore, in a genetic population analysis of the subgenus Profundulus based on allozymes (^{Doadrio, Carmona, Martínez, & De Sostoa, 1999}), at least 5 different lineages were recovered. Of these, 2 distinct lineages were found in the Mixteca region and were well differentiated from P. punctatus . One of them corresponded to P. oaxacae and the other one included a clade named P. sp. nov. 1, which corresponded to populations from Mixteca headwaters (with an elevation over 700 m) and showed a high degree of molecular divergence from the rest of species of the subgenus Profundulus (Nei's distances D = 0.165 − 0.845). In fact, based on the aforementioned analysis, P. sp. nov. 1 was recovered as a sister clade to the rest of species in the P. punctatus "group", where P. oaxacae is included.

As a consequence of the striking morphological differences between P. oaxacae and P. punctatus , Miller recognized the former as a valid species (^{Miller, 2005}). These differences include the number of scales around the body (25-28 in P. punctatus vs . 30 in P. oaxacae ), and the lateral color pattern (prominent round spots in P. punctatus vs . narrow weak and vertical marks in P. oaxacae ).

Another aspect clearly separating this pair of species is their distribution, in which P. oaxacae is restricted to the upper part of the río Verde basin, Oaxaca while P. punctatus inhabits the lower part of the same basin. P. sp. nov. 1 also inhabits the upper part of the río Verde basin, but shows a high genetic distinctiveness regarding both, P. oaxacae and P. punctatus (^{Doadrio et al., 1999}). Consequently, in this study we evaluated the morphological distinctiveness between P. oaxacae and P. sp. nov. 1, and determined whether morphological variation corresponds to the genetic differentiation previously documented by ^{Doadrio et al. (1999)}.

Materials and methods

Sample collection and taxonomic determination

A total of 5 localities within the Mixteca region were surveyed using cast nets and electrofishing (Fig. 1). A total of 50 specimens were collected, with a range of individuals per locality from 5 to 15 (Table 1). Fin clips were obtained and individuals tagged. Fin clips and voucher specimens were preserved in 95% ethanol and deposited in Colección Nacional de Peces, Instituto de Biología, Universidad Nacional Autónoma de México (CNPE-IBUNAM), Colección de Peces, El Colegio de la Frontera Sur (ECOSC), Museo Nacional de Ciencias Naturales de Madrid, España (MNCN) and Colección de Peces del Centro de México Dr. Edmundo Díaz Pardo, Universidad Autónoma de Querétaro, Mexico (CPCMEDP-UAQ). Localities for P. sp. nov. 1 were determined based on a previous molecular study (Table 1; ^{Doadrio et al., 1999}). For P. oaxacae , we measured 15 specimens from Rio Chiquito, Tlaxiaco, in the Mixteco-Balsas basin. P. oaxacae were identified using Miller's keys (^{Miller, 2005}).

Figure 1 Map showing sampling points for the species: P. mixtlanensis sp. nov. (black circles) and P. oaxacae (white circles). 

Table 1 Sampling localities, number of localities and number of individuals that were used in the morphologic analysis. Taxonomic designation is according to our study, and to ^{Miller (1955}, ²⁰⁰⁵⁾. 

Map	Locality	N	Basin	Species proposed	Species by Miller (1955, 2005)	UTM-X	UTM-Y
1	Río Grande, Santiago Juxtlahuaca, Oaxaca, México	5	Mixteco-Balsas	P. mixtlanensis	P. punctatus	605394	1910523
2	Spring, Sola de Vega, Oaxaca. México.	-	Atoyac	P. oaxacae	P. oaxacae	715433	1826989
3	Río Cucharas, El Carmen Tuxtitlán, Putla de Guerrero, Oaxaca, México	7	Atoyac	P. mixtlanensis	P. punctatus	615930	1884461
4	Río Chiquito, Tlaxiaco, Oaxaca, México	15	Mixteco-Balsas	P. oaxacae	P. oaxacae	643961	1905758
5	Spring, Aldama, Oaxaca, México	15	Atoyac	P. mixtlanensis	P. punctatus	648638	1875597
6	Spring, Chalcatongo, Oaxaca, México	-	Atoyac	P. mixtlanensis	P. punctatus	650543	1884349
7	Spring, Santiago Yosondúa, Oaxaca. México	8	Atoyac	P. mixtlanensis	P. punctatus	651751	1866154
8	Río de las Grutas, San Sebastián de las Grutas, Oaxaca, México	-	Atoyac	P. oaxacae	P. oaxacae	718060	1839603

Description

Profundulus mixtlanesis Ornelas-García, Martínez & Doadrio, new species (Fig. 2)

Figure 2 Morphological comparison of the species of Profundulus: (a) Profundulus (P.) mixtlanensis sp. nov. and P. oaxacae individuals; (b) lateral view of the head of both species; (c) dorsal head view of both species; (d) holotype of P. mixtlanensis sp. nov. 

Results

Morphometric analyses showed a separation between the two species groups tested. The two-way ANOVA using 17 morphometric measurements showed significant differences between species for most of the comparisons (p ≤ 0.001, Table 2). Analyses however, did not disclose any significant differences in sex (p > 0.05), or sex × species interaction (p > 0.05, data not shown). Because results did not support the existence of sexual dimorphism within or between species, all posterior analyses were performed without sex separations. Ordination using PCA allowed for identification of two separate clusters of points. Each cluster corresponded to P. oaxacae and to P. sp. nov. 1 (Fig. 3). PCI explained 91.73% of the variance, where the most important variables in the formation of the groups were SL, PrOL and ED in the positive side of PCI, and PrPD, PrPA and PrPV in the negative side. PCII explained 2.79% of the variance, in which the most important variables were PrPD, PrPA, PrPP and BD, whereas PCIII explained 1.70% of the variance, with the most important variables PrPP, HL, PVL and VAL (Eigenvalues in Supplementary material 4).

Figure 3 Bivariate plot of PCA results from morphometric measurements after allometric regression. Solid circles correspond to P. mixtlanensis sp. nov. and white to P. oaxacae. PC1 explains 91.73% of the variance, whereas PC2 explains 2.79% of the variance. 

DFA also showed significant discrimination between species. All individuals assigned to P. oaxacae were correctly classified whereas assignation to P. sp. nov. 1 was 97.14% correct (Fig. 4a, F (16, 33) = 7.0769 p < 0.0001; posterior probabilities are shown in Supplementary materials 2 and 3). Linear discriminant factor from the DFA showed a contrasting distribution between the two groups of cases (Fig. 4). The root factor was ordered mostly according to the following variables: PrPD and AFL on the positive side and PrPA, DFL and BLD on the negative side.

Figure 4 Results from the discriminant function analysis (DFA): (a) proportions of correctly assigned individuals: 97% for P. mixtlanensis sp. nov. (individuals 1–35 in the graph) and 100% for P. oaxacae (individuals 36–50); (b) distribution frequencies of root factor from the DFA between species. 

Results of the Mann-Whitney U test applied to meristic variables were also significant for three of five variables tested between species (Table 2), and when applied to proportions, comparisons were significant for 10 of 16 tested (Table 3). Highly significant differences were found in the following proportions: PrPA/SL, PrPP/SL, BD/SL, ADL/SL, BLD/SL, APL/SL, HL/SL, PrOL/HL and ED/SL.

Even when there is slight overlap, significant differences were found between species in the following meristic variables (Table 3; p < 0.001): number of lateral scales, with a mean of 33 and range of 30-35 in P. mixtlanensis , and 31 mean and range of 29-32 in P. oaxacae ; and with a lower number of anal-fin rays for P. oaxacae (12-14), while for P. mixtlanensis the anal-fin rays range from 12 to 21 (Fig. 5). Given the previous results, we conclude that P. mixtlanensis fulfills the criteria to be considered a different taxonomic unit, using both morphological and molecular (^{Doadrio et al., 1999}) criterion.

Figure 5 Frequency distribution of 5 meristic characters for P. mixtlanensis sp. nov. (black squares) and P. oaxacae (white squares). 

Discussion

P. mixtlanensis and P. oaxacae occur at geographically distant locations in the Mixteco basin, and as we observed in the present description, the two species are easily distinguishable by their body shapes and coloration patterns. P. oaxacae shows a characteristic spotty pattern along the lateral part of the body while in P. mixtlanensis only shows a humeral spot. The molecular distinctiveness of P. mixtlanensis with respect to the other species within the Profundulus genus has been demonstrated by an allozyme study (^{Doadrio et al., 1999}). In addition, phylogenetic analysis carried out by the authors showed that both, P. oaxacae and P. mixtlanensis depict a high degree of divergence from the rest of the species within the subgenus Profundulus (Dp = 10.5 ± 5.9 mtDNA and Dp = 1.7 ± 0.2 nDNA, respectively, data not shown).

Appendix A Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.rmb.2015.10.001.

Appendix A Supplementary data

The following are the supplementary data to this article: 1870-3453-rmbiodiv-86-04-00926-ss01.docx