Copeia, 2005(2), pp. 320±339 Historical Biogeography of the New-World Pup®sh Genus Cyprinodon (Teleostei: Cyprinodontidae) ANTHONY A. ECHELLE,EVAN W. CARSON,ALICE F. ECHELLE,R.A.VAN DEN BUSSCHE, THOMAS E. DOWLING, AND AXEL MEYER Analysis of mtDNA sequence variation (2,548 bp from ND2, cytb, and part of the control region) indicates that the genus Cyprinodon began diverging in the Late Mio- cene from a common ancestor with Megupsilon, a monotypic genus on the Mesa del Norte of Mexico. The geographic pattern of mtDNA variation, with estimates of divergence time, suggests that by the end of the Miocene Cyprinodon occurred from the Atlantic Coast and West Indies to near the western margin of North America via ancestral Rio Grande and Colorado River systems. Phylogeographic structure within the major mtDNA complexes supports a variety of hypotheses from geology and previous phylogenetic analyses for Late Neogene connections among basins in south- western North America now separated by formidable barriers to dispersal. Com- parison of the mtDNA tree with previous phylogenetic inferences from allozymes indicates that reticulate evolution involving divergent lineages probably was impor- tant in the history of Cyprinodon. El anaÂlisis de variacioÂn de secuencias de ADNmt (ND2, cytb, y parte de la regioÂn reguladora; 2,548 pb) indica que el geÂnero Cyprinodon empezo a divergir en el mio- ceno tardõÂo de un antepasado comuÂn con Megupsilon,ungeÂnero monotõÂpico de la Mesa del Norte de MeÂxico. El patroÂn geogra®co de la variacioÂn de ADNmt, con estimadas de los tiempos divergencias, sugiere que al llegar al ®n del mioceno Cypri- nodon ocurrio desde la costa AtlaÂntica y las Antillas hasta casi el margen oeste de NorteameÂrica por los sistemas pluviales antiguos del rõÂo Bravo y del rõÂo Colorado. La estructura ®logeogra®ca entre los grupos principales de ADNmt apoya a una variedad de hipoÂtesis de la geologõÂa y de los anaÂlisis ®logeneÂticos anteriores para conexiones del neogeno tardõÂo entre cuencas del suroeste de NorteameÂrica que ahora estaÂn separadas por barreras imponentes contra la dispersioÂn. La compara- cioÂn del aÂrbol de ADNmt con deducciones ®logeneÂticos anteriores de alozimas indica que la evolucioÂn reticulada que incluye lõÂneas divergentes probablemente fue importante en la historia del Cyprinodon. ISH distributions and systematics have con- warm deserts and southern Great Plains of F tributed importantly to a large body of hy- southwestern North America (Miller, 1981). As potheses for the paleohydrology and associated a group, cyprinodontids of this region inhabit biogeography of arid regions of North America, lowland waters where they are less likely than most of which have emphasized relatively recent many other ®shes to cross basin divides and, geological/climatological events of Pleistocene therefore, more likely to preserve ancient pat- times (Hubbs and Miller, 1948; Miller, 1948; re- terns of geographic variation (but see Minckley views in Hocutt and Wiley, 1986). In contrast, et al., 2002, for a possible exception in the Mex- Minckley et al. (1986) suggested, primarily from ican highlands). regional plate-tectonics, that the relevant time- Approximately 30 of the 50 or so species of frame for understanding the biogeography of Cyprinodon occur in arid regions of southwest- extant western ®shes might extend to Middle ern North America where they are primarily dis- Tertiary. Correspondingly, a review of molecular tributed allopatrically as endemics to relatively phylogenetic studies of ®shes in the Great Basin small bodies of water, often single streams or region suggested that many lineages are much spring-systems in isolated valley ¯oors (Miller, older than generally appreciated (Smith et al., 1981). The remaining 20 or so species occur as 2002). local endemics in the West Indies-Yucatan Pen- In this paper, we use mitochondrial DNA var- insula region or as wide-ranging species with al- iation to assess the historical biogeography of lopatric distributions in coastal or coastal-plain the pup®sh genus Cyprinodon, the most wide- waters from Massachusetts to Venezuela (Smith spread ®sh genus in a region comprising the et al., 1990; Wildekamp, 1995). q 2005 by the American Society of Ichthyologists and Herpetologists ECHELLE ET AL.ÐPUPFISH BIOGEOGRAPHY 321 The paleohydrology of southwestern North America suggests many opportunities for sec- ondary contact among previously allopatric ®sh lineages (Hubbs and Miller, 1948; Minckley et al., 1986; Smith and Miller, 1986). This and the present, primarily allopatric occurrences of pup®shes imply that, subsequent to most in- stances of secondary contact, the number of lin- eages in basins of concern was reduced to one by hybridization and genetic introgression or by extinctions leaving no genetic remnant. Studies of reproductive compatibility among extant pup®shes suggest that ancient contact between divergent pup®shes generally would have led to genetic introgression. Laboratory experiments with three members of a small spe- cies ¯ock on the Yucatan Peninsula revealed in- terfertility for all combinations and indicated pre-mating reproductive isolation for only one of the three (Strecker and Kodric-Brown, 2000). Experiments with allopatric pup®shes show lit- tle evidence of pre- or post-mating isolation be- tween divergent species (Turner and Liu, 1977; Cokendolpher, 1980; Villwock, 1982). In the wild, hybridization is indicated in all three mor- Fig. 1. Distribution of samples and basins. DV 5 phologically well-studied instances of contact 5 5 between species (Minckley, 1969; Humphries Death Valley System, GB GuzmaÂn Basin, TB Tu- larosa Basin, CC 5 Cuatro CieÂnegas Basin, RM 5 RõÂo and Miller, 1981; Minckley and Minckley, 1986), Mezquital, SPB 5 Sandia and Potosi Basins. Basins not and genetic introgression exists in all known sit- labeled are Devils River (7), Alamito Creek (8), RõÂo uations where endemic species were exposed to Florido (9), and Laguna Santiaguillo (16). Localities anthropogenic introduction of a non-native 4 and 22 both comprise two sites in close proximity pup®sh (Echelle and Echelle, 1994, 1996; (Material Examined). Childs et al., 1996), including one instance of introgression over more than 400 river-kilome- ters in less than ®ve years (Echelle and Connor, mately 20 species (some undescribed) known 1989). from coastal Atlantic and Gulf of Mexico region Hybridization confounds attempts to recover and the West Indies. One of the excluded spe- phylogenetic relationships, but it can, at the cies (C. salvadori) is a recently described form same time, inform reconstructions of biogeo- from the RõÂo Conchos basin in Chihuahua (Lo- graphic history because it might represent the zano, 2002) that is similar to C. eximius and an- only evidence of past contact between lineages other (C. latifasciatus) has been extinct since the (e.g., DeMarais et al., 1992). In this study, sev- 1950s (Miller, 1964). The three remaining, arid- eral such instances are indicated by the present land species excluded from the analysis (C. ce- geography of pup®shes and con¯icting phylo- ciliae, C. inmemoriam, and C. longidorsalis) repre- genetic inferences from allozyme variation sent a monophyletic group of four similar spe- (Echelle and Echelle, 1992, 1993a, 1998) and cies (Lozano and Contreras, 1993; Echelle et al., the mtDNA results presented here. 1995) from the Sandia Basin, Nuevo Leon, one of which, C. veronicae, is included here. This group was recently extirpated in the wild and is MATERIALS AND METHODS extinct except for C. longidorsalis and C. veroni- Sampling.ÐWe obtained collections of 26 spe- cae, which are being maintained in various cies of Cyprinodon from 38 localities (Material aquarium facilities (Contreras and Lozano, Examined, Fig. 1). We also used GenBank se- 1996; pers. comm.). quences for nine additional species not includ- The remaining species not examined are con- ed in our collections (see below). The complete sidered close relatives of C. variegatus (Miller, dataset includes 24 of the 29 historically extant 1962; Smith et al., 1990; Lozano and Contreras, species of Cyprinodon in arid regions of south- 1999): C. bobmilleri from a Gulf of Mexico drain- western North America and 11 of the approxi- age in Nuevo Leon, C. hubbsi from inland Flori- 322 COPEIA, 2005, NO. 2 da, two undescribed species from Cuba (M. 0.8% agarose gels, and visualized by autoradi- Smith, pers. comm.), C. laciniatus and an unde- ography. This identi®ed 19 composite haplo- scribed species ¯ock (Holtmeier, 2001) from the types: C. variegatus 5 9, C. elegans 5 2, C. rubro- Bahamas, and C. higuey and C. nichollsi from His- ¯uviatilis 5 4, C. pecosensis 5 2, and C. bovinus paniola. Surveys of mtDNA variation in pup®sh 5 2. We sequenced one sample of each com- from the Bahamas, Hispaniola, other sites in the posite RFLP haplotype, and, for the remaining West Indies, and the Atlantic and Gulf of Mexico collections, two specimens each except the fol- coasts (Bunt, 2001; L. Fuselier et al., unpubl. lowing (n 5 1): one of the two collections of C. data) indicate that our collections capture the artifrons, one of the three collections of C. exi- major clades of mtDNA variation in this region. mius, two pup®shes of the western clade (C. ma- Except for C. dearborni from Venezuela, spe- cularius and C. nevadensis), the two Hispaniolan cies of Cyprinodon with large or fragmented geo- species, and the three genera, Megupsilon, Cua- graphic ranges were collected from multiple lo- lac, and Jordanella. calities: six for the wide-ranging C. variegatus A total of 65 specimens were sequenced for (three each from the Atlantic and Gulf of Mex- the entire NADH dehydrogenase subunit 2 ico coasts), three encompassing the extremes of (ND2; 1047 bp) and cytochrome b (cytb; 1140 the range of C. eximius, two for the coastal form bp) genes and a 361-bp section of the control on the Yucatan Peninsula (C. artifrons), and one region adjacent to tRNAThr. The primers ND2B- for each of two isolated populations of C.