Hydrobiologia (2007) 579:317–335 DOI 10.1007/s10750-006-0473-9 PRIMARY RESEARCH PAPER Genetic and morphologic variation of the Pecos assiminea, an endangered mollusk of the Rio Grande region, United States and Mexico (Caenogastropoda: Rissooidea: Assimineidae) Robert Hershler Æ Hsiu-Ping Liu Æ B. K. Lang Received: 23 May 2006 / Revised: 1 November 2006 / Accepted: 11 November 2006 / Published online: 13 January 2007 Ó Springer Science+Business Media B.V. 2007 Abstract Assiminea pecos is an endangered of A. pecos, which was consistently depicted as a species of amphibious gastropod that occupies monophyletic unit nested within or sister to the four widely separated portions of the Rio Grande shallowly structured group comprised of Ameri- region in the southwestern United States (Pecos can members of this species. Consistent with our River basin) and northeastern Mexico (Cuatro findings, we describe the Mexican population as a Cienegas basin). Our statistical and discriminant new species, which is provisionally placed in the function analyses of shell variation among the large, worldwide genus Assiminea pending fur- disjunct populations of this species indicate that ther study of the phylogentic relationships of the Mexican specimens differ in their morphometry North American assimineids. Our molecular data from those of the United States and can be suggest that the Rio Grande region assimineids, diagnosed by several characters. We also ana- which are among the few inland members of the lyzed variation in the mitochondrial genome by otherwise estuarine subfamily Assimineinae, di- sequencing 658 bp of mitochondrial COI from verged from coastal progenitors in the late Mio- populations of A. pecos, representatives of the cene, with subsequent Pleistocene vicariance of other three North American species of Assiminea, Mexican and American species perhaps associ- and several outgroups. Our results indicated ated with development of the modern, lower substantial divergence of the Mexican population course of the Rio Grande. Handling editor: K. Martens Keywords Assiminea Á Gastropoda Á North America Á Systematics Á Biogeography Á & R. Hershler ( ) Conservation Department of Invertebrate Zoology, Smithsonian Institution, NHB W-305, MRC 163, P.O. Box 37012, Washington, DC 20013-7012, USA Introduction e-mail: [email protected] H.-P. Liu The Assimineidae is a diverse family of small, Department of Biological Sciences, University of amphibious and terrestrial caenogastropods found Denver, Denver, CO 80208, USA in tropical and temperate regions throughout much of the world (Abbott, 1958; Ponder & DeKeyzer, B. K. Lang New Mexico Department of Game and Fish, 1998). The North American fauna consists of four One Wildlife Way, Santa Fe, NM 87507, USA amphibious species currently assigned to the large, 123 318 Hydrobiologia (2007) 579:317–335 cosmopolitan genus Assiminea, and referable to Pecos assiminea, although several additional the informal nitida-complex (sensu Abbott, 1958). populations have been reported from the Roswell Two of these species share the brackish coastal and Ft. Stockton areas, and another isolated habitats typical of other members of the subfamily segment of Pecos River drainage near Balmo- Assimineinae (Fukuda & Ponder, 2003) and rhea, Texas (USFWS, 2002, 2005). have extremely broad ranges along the Pacific As currently envisaged, the Pecos assiminea is (A. californica [Tryon]) and Atlantic-Gulf of composed of four groups of populations separated Mexico (A. succinea [Pfeiffer]) margins (Abbott, from each other by ca. 75–760 km (Fig. 1). Given 1974). The other two live in spring-riparian settings its tight linkage with aquatic habitats, A. pecos is in Death Valley (A. infima Berry) and the Rio likely incapable of dispersing long distances within Grande region (A. pecos Taylor) and are among the highly fragmented drainage of the arid Rio the few representatives of this subfamily associ- Grande region and consequently its broadly dis- ated with inland aquatic habitats (Fukuda & junct populations are probably not exchanging Ponder, 2003). Both of these ‘‘land-locked’’ spe- genes at present. The fossil record of the Pecos cies are thought to have been derived from coastal assiminea consists of a few Holocene collections progenitors that dispersed inland during the late from sites closely proximal to extant colonies Tertiary (Berry, 1947; Taylor, 1985) and conse- (Taylor, 1987) and does not provide insight into quently are of biogeographic interest. They have the timing of population isolation. Taylor (1985), also become a focus of conservation attention however, speculated that this snail (which was then because of threats to their fragile, groundwater- undescribed) evolved from Gulf Coastal progen- reliant habitats – both have critically imperiled itors that dispersed along a hypothesized brackish (G1) global heritage rankings (NatureServe, 2006) paleodrainage in the Rio Grande Valley, with and A. pecos was recently listed as endangered by isolation of inland, salt-adapted populations occur- the USWFS (2005). However, despite the compel- ring upon inception of the modern, freshwater ling and important features of the North American lower Rio Grande during the Pleistocene. If this assimineids, these snails are poorly known taxo- hypothesis is correct, Mexican (Cuatro Cienegas) nomically and additional studies are needed to and American (Pecos River basin) populations better clarify their species limits and assess their separated by the putative Rio Grande barrier have phylogenetic relationships. long been isolated and may represent divergent Assiminea pecos, commonly known as the conspecific subunits, or separate species. Pecos assiminea (Turgeon et al., 1998), was In this paper we survey morphological and described from a small number of sites in the mitochondrial DNA variation of A. pecos popu- upper Pecos River basin near Roswell, New lations, and assess their evolutionary relationships Mexico (including the type locality); lower seg- using molecular evidence. We use our results to ment of this watershed near Fort Stockton, Texas; re-evaluate the current taxonomic treatment of and Cuatro Cienegas basin, Coahuila (Mexico), these populations as a single species and test which drains (in part) to the Rio Grande (Taylor, Taylor’s (1985) hypothesis of the biogeographic 1987). Taylor (1987) clearly differentiated A. history of assimineids in the Rio Grande region. pecos from its North American congeners on Additionally, we discuss the implications of our the basis of its strongly rounded shell whorls, findings for ongoing efforts to recover the endan- deep suture, absence of a subsutural thread, and gered Pecos assiminea (NMDGF, 2004). broad umbilicus. However, conspecificity of the widely separated populations assigned to A. pecos was not convincingly demonstrated in this study Materials and methods as the only (shell) morphological data provided were from topotypes and all but one of the Genetics figured specimens were also from the type locality (Taylor, 1987: Table 1, Fig. 2a–d). There have Samples from seven populations of A. pecos been no subsequent taxonomic studies of the spread across its entire geographic range were 123 Hydrobiologia (2007) 579:317–335 319 Fig. 1 Map showing the four disjunct areas in the Rio Grande region, United States and Mexico, which comprise the geographic range of Assiminea pecos collected between 2000–2004, preserved in 90% species. Locality and other data for all samples ethanol, and used for sequencing of mitochon- are provided in Table 1. drial DNA. Two to seven animals were analyzed Genomic DNA was isolated from individual for each population, totaling 30 specimens. We snails using a CTAB protocol (Bucklin, 1992). Six also analyzed small samples (three-five speci- hundred fifty-eight base pairs (bp) of mitochon- mens) of each of the other three North American drial cytochrome c oxidase subunit I were ampli- species of Assiminea. Inasmuch as the phyloge- fied and sequenced with primers COIL1490 and netic relationships of the North American assi- COIH2198 (Folmer et al., 1994) following proto- mineids have never been studied, we used as cols of Liu et al. (2003). Sequences were deter- outgroups two members of the family from mined for both strands and then edited and geographically distant Taiwan—Paludinella tai- aligned using SequencherTM version 3.1.1. New wanensis Habe, Pseudomphala latericea (H. & A. sequences were deposited in GenBank (Acces- Adams)—and rooted our trees with the latter sion numbers DQ533841–533866). 123 320 123 Table 1 Samples used for sequencing of COI Species Code Area Locality and voucher Haplotypes GenBank Accession # Sample size (if available) Assiminea pecos A9, A16 Roswell Impoundment #7, northwest VI DQ533848 5 corner, Bitter Lake NWR, Chaves Co., NM; USNM 1007246, USNM 1069820 – A10 Roswell Sago Springs, Bitter Lake NWR, VI DQ533847 3 Chaves Co., NM; USNM 1011495 – A5 Ft. Stockton Monsanto Spring, Diamond Y II, III DQ533842 DQ533843 2 Draw, Pecos Co., TX – A6, A17 Ft. Stockton Diamond Y Spring, ca. 200– VI, VII, VIII, IX DQ533849 DQ533850 7 250 m downflow from source, DQ533851 Diamond Y Draw, Pecos Co., DQ533852 TX; USNM 1069823 – A8, A31 Ft. Stockton ‘‘Johns Hole,’’ ca. 0.8 km west IV, V, VI DQ533844 DQ533845 7 of TX Hwy 18, Diamond Y DQ533846 Draw, Pecos Co., TX; USNM 1007143, USNM 1085823 – A18 Balmorhea East Sandia Spring, Reeves Co., VI, IX DQ533853 DQ533854 3 TX; USNM 1069829 – A3 Cuatro Cienegas Spring–marsh complex just west I DQ533841 3 of Hwy 30, ca. 2.5 km north