Identity of Freshwater Shrimp Populations (Palaemon Weber, 1795) from Northern Mexico: Genetic Variation at Local and Regional Scales

JOURNAL OF CRUSTACEAN BIOLOGY, 34(4), 481-493, 2014

IDENTITY OF FRESHWATER SHRIMP POPULATIONS (PALAEMON WEBER, 1795) FROM NORTHERN MEXICO: GENETIC VARIATION AT LOCAL AND REGIONAL SCALES

Fernando Álvarez 1, Carlos Pedraza-Lara 1,2,∗, and José Luis Villalobos 1

1 Colección Nacional de Crustáceos, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-153, México 04510, D.F., México 2 Colección Nacional de Insectos, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-153, México 04510, D.F., México

ABSTRACT The freshwater genus Palaemon is widely distributed in north-eastern Mexico, where six species have been recognized. Of special interest is the area of the Cuatro Cienegas Valley (CCV) and the Salado and Bravo Rivers basins in central and northern Coahuila, where interconnections, ancient and contemporary, have created a mosaic of populations of species belonging to Palaemon that cannot unequivocally be assigned to one species. We seek to encompass the species determination in a phylogenetic framework by reconstructing phylogenetic relationships of the aforementioned populations and 4 of the species occurring in México. We collected information of three genetic fragments (COI, 12S, and H3) and performed maximum likelihood and Bayesian inference phylogenetic analyses. Also, through the analysis of a partial sequence of the cytochrome oxidase subunit I gene (COI) from individuals coming from 22 populations, we explored phylogeographic patterns from the three basins. Relaxed molecular clock were carried out focused on dating the cladogenesis of all species, while mismatch and Bayesian Skyline Plots analyses were used to test for possible demographic changes in populations from CCV. Gene-separated and concatenated phylogenetic analyses supported the monophyly of the species described from Mexico, but did not show their inclusion in one monophyletic clade, rather depicting a structure congruent with multiple invasions to freshwater. Dating analysis provided long-term temporal framework for cladogenesis. Three different lineages were found in the CCV, conﬁrming the high diversity of this region. One of them is identiﬁed with P. sutkussi, and two are possible new species to science. Haplotype analyses provided insight from recent population processes and are congruent with a scenario where despite keeping signatures of past cladogenesis, more recent genetic structure reveals surprisingly higher connectivity between basins associated to the Bravo river system and CCV. KEY WORDS: distribution, drought, freshwater shrimp, genetics, protected areas, systematics DOI: 10.1163/1937240X-00002248

INTRODUCTION paludosus (Gibbes, 1850) (Baja California), and P. suttkusi Palaemon has a cosmopolitan distribution and after its (Strenth, 1976) (Coahuila). The identity of each of these taxonomic re-appraisal, includes 83 marine, estuarine and species in Mexico seemed to be a resolved matter until re- freshwater species (De Grave and Ashelby, 2013). The cently, when more intensive sampling was conducted in the taxonomy of the genus is complex because of the reduced Cuatro Ciénegas Valley (CCV) and the neighboring Sabinas number of useful characters to discriminate among species, River basin, northern Coahuila. Captured shrimp from these and because these characters have a limited extent of trips showed differing morphology to that of P. suttkusi, variation. The dentition of the rostrum and the spinulation which is the species described for the region and could not of the appendix masculina, both with a small range of be assigned to this species unequivocally. In a recent paper variation, are the main examples of taxonomically important on the phylogeography of P. suttkusi from the CCV, Chaves- characters for this genus and related palaemonids (Ashelby Campos et al. (2011) explored the relationship between the et al., 2012). Typically, small morphological variations populations of P. suttkusi from the CCV and the Río Sal- associated to disjunct or isolated distributions, have been ado, which is a neighbouring basin to the CCV. They iden- used to describe and differentiate species (Ashelby et al., tified two different lineages based on 27 COI haplotypes; 2012; De Grave and Ashelby, 2013). however, they could not decide whether the observed vari- In regards to habitat type, six freshwater species of Palae- ation could support the description of a second species for mon (formerly included in the junior synonym Palaemon- the region. The two lineages were identified both inside and etes) occur in northern Mexico: P. hobbsi (Strenth, 1994) outside the CCV making it difficult to provide an accurate (Tamaulipas), P. kadiakensis (Rathbun, 1902) (Coahuila), P. taxonomic framework in which define proper conservation lindsayi (Villalobos-Figueroa and Hobbs, 1974) (San Luis measures of these shrimps in the CCV. Evidence emerging Potosi), P. mexicanus (Strenth, 1976) (San Luis Potosi), P. from the aforementioned issues with species discrimination

∗ Corresponding author; e-mail: [email protected]

© The Crustacean Society, 2014. Published by Brill NV, Leiden DOI:10.1163/1937240X-00002248 482 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 4, 2014 using morphology data, together with the genetic variabil- sequences were deposited in GenBank and alignments are available from ity detected, made it plausible to suspect on the presence of the authors upon request. cryptic diversity in several populations from the CCV. Our Phylogeny: COI Genealogy and Concatenated Analyses work here attempts to attain taxonomic certainty concern- After trimming of possible primer sites was done, all data sets were ing these populations, which is of primary importance for aligned using MUSCLE (Edgar, 2004). For COI data, recommendations conservation within the protected areas scheme in Mexico to detect the occurrence of nmtDNA were carried out for each sequence, (Cabral and Cruz, 2007). which included the identification of stop codons and a high frequency of non-synonymous substitutions as well as unusual levels of genetic Our study examines the genetic identity of the populations divergence in samples coming from one population (Song et al., 2008; of Palaemon from the CCV in order to answer the following Buhay, 2009). As outlined before, we carried out an initial evaluation of questions: 1) do the populations of CCV belong to one or phylogenetic structure using data from COI only. From the data set I (153 several species? 2) what is the current distribution of P. individuals, 601 bp), mitochondrial COI sequences were collapsed into suttkusi unique haplotypes using DnaSP v. 5 (Librado and Rozas, 2009), and a ? 3) do the CCV and the basin of the Sabinas River phylogenetic inference was carried out with this haplotype data, which function as separate systems or is it a common basin? and we term data set II (62 individuals and 601 bp). Subsequently, a second 4) what is the degree of divergence among populations when phylogenetic analysis was carried out with representatives from COI mt- the comparison includes three other species, P. kadiakensis, lineages only, conforming a concatenated matrix together with 12S and P. lindsayi and P. mexicanus? H3 fragments, which we term data set III (14 individuals and 1298 bp, see Table 1). Phylogenetic procedures here described were applied to data set II and III. In order to identify the most appropriate evolutionary MATERIALS AND METHODS model by the Akaike corrected information criterion (AICc), the program jModeltest v. 0.1.1 (Posada, 2008) was used. Phylogenetic analyses were Acquisition of Samples conducted using maximum likelihood (Ml) with the computer program A total of 153 COI-sequences, which included 53 newly generated PhyML (Guindon and Gascuel, 2003). We assessed confidence in branches sequences, and accounted for four of the five species naturally occurring using 1000 nonparametric bootstrap replicates under the most accurate in Mexico, were processed for the present analysis (Table 1, Fig. 1). Our evolutionary model. This model was also used to carry out Bayesian sampling was conducted between 2010 and 2012 in six trips to the CCV and inference (BI) of phylogeny as implemented in MrBayes v. 3.1.2 (Ronquist the Sabinas River basin. Palaemon mexicanus and P. lindsayi were collected and Huelsenbeck, 2003), applying a Monte Carlo Markov Chain (MCMC) in their type localities: Santa Anita stream near Ciudad Valles and Media procedure after 5 million generations, and partitioned by codon position for Luna Lagoon, both in San Luis Potosi, respectively. Palaemon kadiakensis data set I and additionally by gene for data set II. Convergence between was collected in several localities along the Bravo River from La Amistad the different parameters in paired simultaneous analyses (4 chains by run) and run length were adjusted considering an adequate sampling based on Dam to Piedras Negras, Coahuila. Seeking to cover most of the genetic average standard deviation of split frequencies being <0.01 (Huelsenbeck variation, the analysis also considered 100 COI identified as P. suttkusi in and Ronquist, 2005). The burn-in period was determined as the set of trees GenBank from a previous study (Chaves-Campos et al., 2011a), which were saved prior to log likelihood stabilization and convergence as estimated especially useful to describe patterns of variability inside the CCV. In total, using Tracer 1.4 (Rambaut and Drummond, 2007). Tracer v. 1.4.1 was used 22 populations assigned to different species of Palaemon were included. to check for convergence between chain runs and optimal values of run Previous works have found that instead of being monophyletic, several parameters. The use of the molecular clock approach requires corroboration palaemonid genera share evolutionary histories according to its region of of the constant evolutionary rate of branch lengths. origin (Ashelby et al., 2012; De Grave and Ashelby, 2013). Consequently, A log-likelihood ratio test was used to examine the clock-like eight sequences of the genera Macrobrachium (Spence Bate, 1868), 2 Cryphiops (Dana, 1852) and Alpheus (Fabricius, 1798), together with two evolution of sequences in the data set by calculating a χ statistic based 2 = × of the estuarine P. vulgaris (Say, 1818) from Genbank were included on ML values with and without rate constancy enforced (χ 2 − − − = as out-groups in phylogenetic analyses (see Table 1). Consequently, out- (( ln L clock constrained) ( ln L unconstrained));df number of ter- − groups were chosen between species inhabiting water basins or sea regions minal nodes 2) (Felsenstein, 1981). Following this, we implemented a closest to the study area. Only sequences from P. vulgaris were included dating scheme for cladogenesis using a Bayesian relaxed molecular clock for concatenated analyses, as it was the only species for which the three estimation on COI, as for this marker a range of mutation rates are available fragments were available from Genbank. All samples are deposited in from previous estimates in crustaceans, which is desirable in dating analy- the National Crustacean Collection (CNCR), of the Institute of Biology, ses. This allowed to obtain mean node ages and their 95% highest posterior UNAM, Mexico. densities (HPDs) as implemented in BEAST ver. 1.8 (Drummond et al., 2012). A uniform Yule tree prior was specified, as appropriate for hierarchi- DNA Sequencing cal rather than reticulate relationships. An uncorrelated relaxed lognormal molecular clock was applied to model rate variation across branches. As Genomic DNA was isolated from one pereiopod from each individual using an approximate average divergence rate reported for crustaceans, a range the QIAGEN BioSprint 15DNA bloodKit. Tissues were previously held in based on three COI rates from other crustaceans groups was used: 1.66% digestion for 6 hours at 55°C. A preliminary phylogenetic analysis with COI divergence per million years (pmy) determined for grapsid crabs (Knowl- identified the presence of several lineages in the samples. Consequently, ton and Weigt, 1998), and 2.6% pmy (Knowlton et al., 1993) and 1.4% we sequenced two more genetic markers for 14 specimens representatives pmy (Cook et al., 2008) obtained for snapping shrimps. Analyses were run of the identified mt-lineages. We analyzed a 601-bp segment of the for 20 million generations with a sampling frequency of 2000 generations. cytochrome oxidase subunit I gene (COI), which has proved to be useful Tracer 1.4 was used to determine the appropriate burn-in by monitoring run for species determination in other crustacean groups (Costa et al., 2007). parameters. Primers used were LCO1490: 5-GGTCAACAAATCATAAAGATATTGG and HCO2198: 5-TAAACTTCAGGGTGACCAAAAAATCA (Folmer et Genetic Diversity and Demography al., 1994). Additionally, 366 bp of 12S and 331 bp of H3 fragments All analyses for genetic diversity and demography were carried out with were amplified, using the primers and PCR cycling conditions specified in data set I. Mitochondrial sequences were analyzed in DnaSP v. 5 (Librado (Pedraza-Lara et al., 2012). PCR amplifications were carried out in 25 μl and Rozas, 2009) to generate a haplotype distribution. Also, mitochondrial × reactions containing: 1 PCR buffer, 0.5 μM of each primer, 0.2 mM of haplotype diversity (Hd) and nucleotide diversity (π) were calculated each dNTP, 1.5 mM MgCl2,1UTaq polymerase (Biotools) and about 50 ng in DnaSP (Librado and Rozas, 2009) for each basin and population of template DNA. Cycling profile for PCR amplifications was 3 min at 94°C considered. Relationships between haplotypes were inferred using the (1 cycle), 30 s at 94°C, 30 s at 48°C and 60 s at 72°C (30 cycles), followed Median Joining algorithm with the software Network 4.611 (Bandelt by a final extension of 4 min at 72°C. PCR products were visualized in 1.0% et al., 1999). This algorithm allows inferring relationships and possible agarose gels (1 × TBE) stained with SYBR-Safe (Invitrogen). Fragments connections obtaining the most parsimonious arrangement of haplotypes. were sequenced in an ABI 3730 DNA Analyzer. Sequences were aligned To test for possible structuring between haplotype groups, st values were using the computer program Muscle (Edgar, 2004) and verified by eye. All calculated using Arlequin v. 3.5 (Excoffier and Lischer, 2010). ÁLVAREZ ET AL.: GENETICS OF PALAEMON FROM MÉXICO 483 a IBUNAM: CNCR Data from the present work and that of b GenBank accession No. Voucher KJ769065-KJ769066 – – 26103 KJ769067-KJ769069, –HQ324479-HQ324496 KJ769045 KJ769033 26295 KJ769035-KJ769044, KJ769008KJ769048, KJ769012KJ769059, KJ769060 KJ769021 HQ324516-HQ324520 KJ769025 26510-26512 KJ769050 KJ769047 KJ769077, KJ769078 KJ769080, HQ324497-HQ324515 COI 12S H3 Data from the work of Chaves-Campos et al. (2011). a ) N SUT(2) group ( SUT(3), KAD(2) RCC(5), Population collected from type locality. ∗ CC MEZ RCC(1), CH(1) KJ769034 KJ769007, KJ769020, 25865 Bravo – KAD(1), Bravo – KAD(2) KJ769061-KJ769062 KJ769018 KJ769031 25866 CC MEZ RCC(17), CCCC ECC WCC CH(1), SUT(1) KJ769079 CH(12), – – – studied. b b Palaemon b into the Poza Azul San Judas Tadeo, COA dam, COA MA Maris spring, MO El Moral, COA Bravo – KAD(2) KJ769063-KJ769064 KJ769019 KJ769032 26056 PAM La Amistad METC Río Mesquites Tio Candido CC MEZ CH(3) KJ769074-KJ769076 – – 27167 LM Lamadrid Salado – CH(2) KJ769046, – – 26106 TETI Santa Tecla La Teclita CC ECC CC ECC SUT(2) SUT(1) KJ769056-KJ769057, KJ769011 KJ769058 KJ769024 26522 – – 26523 TB Tierra Blanca sp. PA Chanel flowing sp.sp. GA AN El Garabatal Antiojo CC CC MEZ WCC CH(3) CH(1) KJ769081-KJ769083 KJ769049, – – KJ769013 KJ769026 26521 – sp. CH Churince Chaves-Campos et al. (2011). ECC, Eastern Cuatro Ciénegas; MEZ, Mezquites river; WCC, western Cuatro Ciénegas. P. P. suttkusi P. suttkusi P. P. P. suttkusi P. suttkusi P. suttkusi P. kadiakensis P. kadiakensis P. suttkusi Table 1. Locality data for the populations of SpeciesP. kadiakensis Code Locality Basin Region in CC Haplotype P. 484 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 4, 2014 a IBUNAM: CNCR GenBank accession No. Voucher KJ769087KJ769085 KJ769015KJ769052 KJ769030 KJ769028 KJ769010 KJ769023 HQ324451-HQ324469 – – COI 12S H3 ) N group ( SUT(1) Pánuco –Pánuco – CH(2)Salado – – KJ769086,CCCCCC – – KJ769016, KJ769017 KJ769084, MEZSalado KJ769029, MEZ ECC 24904 RCC(18) SUT(2) KJ769051, RCC(11), KJ769014, SUT(19) HQ324432-HQ324450 HQ324521-HQ324438 – HQ324470-HQ324478 – KJ769027, KJ769009, – 25864 KJ769022, 25874 – – – a a a ∗ ∗ ∗ a –––––– – – – – – – – – – – – – – – – – – – AB250550 EU787489 EU005035 EF539194 JN874538 GQ995518 – – – – – – – – – – – – ––– – – – – – – – – – JQ306029 FN668979 FJ581833 – – – – – – stream, Ciudad Valles, SLP spring, Rio Verde, SLP Nadadores River, near Las Hermanas Águila PMEX Santa Anita CEPL Celemania Playitas Salado – CC ECC SUT(3) SUT(2) KJ769053-KJ769055 – KJ769072-KJ769073 – – – 26058 26664 PLIN La Media Luna SAL Salado de sp.sp.sp. JS ME PZA Juan Santos Mojarral East Pozas Azules sp. SJA San José del P. suttkusi P. mexicanus Table 1. (Continued.) SpeciesP. suttkusi Code Locality Basin Region in CC Haplotype P. lindsayi P. suttkusi P. P. P. Macrobrachium asperulum Macrobrachium australiense Macrobrachium faustinum Macrobrachium inflatum Macrobrachium nipponense Macrobrachium rosenbergii Alpheus pachychyrus Cryphiops caementarius Palaemon vulgaris P. ÁLVAREZ ET AL.: GENETICS OF PALAEMON FROM MÉXICO 485

Fig. 1. Map showing sampling localities for populations of Palaemon. A, northeastern Mexico; B, the Cuatro Cienegas Valley. PAM, La Amistad Dam; MA, Maris spring; MO, El Moral; SAL, Salado River; SJA, San José del Águila; GA, El Garabatal; CE, Celemania; LM, Lamadrid; CH, Churince; TE, Santa Tecla; TI, Santa Teclita; TB, Tierra Blanca; JS, Juan Santos; ME, Mezquites River; PA, Poza azul; PL, Playitas; PZA, Pozas azules. This ﬁgure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/1937240x.

To detect possible signatures of demographic changes or selection in and Rambaut, 2007). In each case, MCMC chains were run for 5 × the recent history of groups of haplotypes, we tested for deviations from 107 generations. Convergence was assessed using Tracer v. 1.5 and a model of mutation-drift equilibrium using the two most powerful tests uncertainty in parameter estimates was reﬂected in values of the 95% to detect deviations from neutrality due to population expansion: Fu’s FS highest probability density (HPD). (Fu, 1997) and R2 (Ramos-Onsins and Rozas, 2002) tests of neutrality (using 1000 pseudo-replications in DnaSP). Fu’s FS is especially sensitive to an excess of rare haplotypes (Fu, 1997; Ramos-Onsins and Rozas, 2002). RESULTS Neutrality was also assessed by the probability of obtaining raggedness Phylogenetic Relationships values of r less than the observed, calculated using the coalescent algorithm in DnaSP over 1000 pseudo-replications with a random seed and no The selected model of nucleotide evolution for all analyses recombination. Tajima’s D (Tajima, 1989) and F ∗ of Fu and Li (Fu and Li, is outlined in Table 2. All, the individual COI and H3 1993) tests of neutrality were also used to assess changes in demographic genealogy, as well as the concatenated analyses showed history. In addition, we performed a mismatch analysis in each haplotype group, all species from Mexico included in an unsupported group which plots the distribution of the number of differences between pairs of (Fig. 2). This is the reason why all phylogenetic analyses sequences, in DnaSP ver. 5. Population expansion would appear as a “wave” showed two clades containing P. kadiakensis from the Bravo in the mismatch distribution, while stable population sizes produce ragged River and P. lindsayi from the Media Luna Lagoon at the multi-modal distributions (Rogers and Harpending, 1992b; Harpending, base of the rest of clades. One clade with moderately high 1994). We used initial values of θ0 = 0, and θ1 = 99,999. To obtain an approximate time corresponding to a possible expansion event leading to support in all analyses (<80 bootstrap values and <90 PP) actual populations of Palaemon we used the expansion parameter τ = 2μt obtained in the analysis, where μ is the mutation rate and t is the time Table 2. Substitution model and phylogenetic performance of each gene in generations since expansion. The τ parameter is an estimate of time fragment. after expansion (t) in mutational units. Thus, if the divergence rate per = nucleotide and year (τ 2μ,whereμ is the substitution rate per lineage) Gene/pos. Size (bp) Substitution model Variable sites PI %PI and the number of nucleotides of the fragment analysed (l) are known, it is possible to calculate the age when the expansion occurred using the COI 601 – 201 125 20.8 expression τ = 2μlt (modiﬁed from Harpending et al., 1993). As an 1st 200 TrN + I442622 approximate average divergence rate reported for crustaceans, three COI 2nd 200 TrN 18 4 20 rates from other crustacean groups were considered as explained above. rd + As previously reported, we considered a generation time of half a year for 3 201 HKY G 139 95 47.2 species of Palaemon and equal sex ratio. 12S 366 HKY 80 34 9.2 Finally, to evaluate possible changes in effective population size (under H3 331 HKY 32 21 6.3 neutral evolution) in the different haplotype groups, we inferred a Bayesian All 1298 – 313 180 13.8 skyline plot (BSP) for each group in BEAST v. 1.5.3 (Drummond 486 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 4, 2014

Fig. 2. Phylogenetic trees of the species of Palaemon and populations from northeastern México. A, genealogy of COI data; B, concatenated data; C, gene genealogy using H3 data only. Bootstrap support from ML (above) and Posterior Probabilities from Bayesian Inference (below) are indicated on each node. Major haplotype groups are identiﬁed by colours and names. Red squares show individuals potentially representing hybrids or large-distance contacts, as seen from incongruence between nuclear-mitochondrial information (individual TB1) or by coming from different basins (individual MA2). grouped P.mexicanus with the clades containing populations Log-likelihood scores with the molecular clock enforced of P. suttkusi from the type locality and those from the CCV. and not enforced were −78.453 and −78.767, respectively. These are grouped in two main clades, one of them with As the likelihood ratio test rejected the null hypothesis of a populations inhabiting along the Salado River basin and the global molecular clock (x2, P = 0.001), the sequences ana- central and eastern portions of the CCV, which we call here lyzed did not evolve at a homogenous rate along all branches as the rare Cuatro Ciénegas-clade (RCC). This lineage was and we proceeded to use a relaxed molecular clock (Fig. 3). mostly composed by sequences from a previous study, and Ages from the dating analysis were recovered with consis- only contained one sequence from the present work (Fig. 2). tency through repetitions (Fig. 3). The crown age for the tree A second closely related clade contains populations from was 16.8 million years ago (Mya) (95% HPD interval for the western portion of the CCV and the headwaters of the node heights/ages: 10.9-24.5 Mya), which corresponds to Mesquites River (Churince-clade). the split between the most recent common ancestor (MRCA) ÁLVAREZ ET AL.: GENETICS OF PALAEMON FROM MÉXICO 487

Fig. 3. Estimate of splitting times among the major clades for the species of Palaemon and populations from northeastern Mexico (numbers above nodes are dates in million years, while bars show 95% HPD intervals). This figure is published in colour in the online edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/1937240x. of the out-group (P. vulgaris) and the species of Palaemon group includes mostly individuals from the west side of the occurring in Mexico. This analysis estimated an approxi- CCV (Churince-group); and a fourth one containing popu- mate age of 10.8 Myr for the splitting between P. kadiak- lations widely scattered in the CCV (RCC-group), contain- ensis and the rest of the species/populations of Palaemon ing mostly sequences of a previous work and only one se- (6.9-15.5 Mya, 95% HPD). Time for the splitting of the quence from the present study from La Poza Azul chan- MRCA of P. lindsayi and the rest of species was approxi- nel. The RCC-group contained the most diverse group of mately 7.15 million years ago (4.6-10.4 Mya, 95% HPD). haplotypes followed by the Suttkusi-group (Table 2). Ge- The next clade, the one representing MRCA between P. m ex - netic diversity values were lower for the Churince- and icanus and populations from the CCV and Salado River, Kadiakensis-groups. In general, most st values between was estimated at 5.17 Mya (3.3-7.6 Mya, 95% HPD), close haplotype groups show a slight to moderate degree of dif- date to the one for the MRCA of the rest of the populations ferentiation (Table 3). The highest values were found be- (4.1 Mya, 2.5-5.9 Mya, 95% HPD). tween the species from the southern part of the distribution and the rest of groups (st = 0.252-0.500), while lower val- Haplotype and Nucleotide Diversity ues were found between the Suttkusi-group and some hap- Haplotype analysis of COI matrix showed 50 haplotypes lotype groups from the CCV (st = 0.044-0.094) (Table 3). found in the populations from northern Mexico (Table 2). Several significant values were obtained in comparisons in- The analysis of haplotypes focused on the populations from volving the Churince- and RCC-haplotype-groups. the Salado and Bravo Rivers, excluding the haplotypes found for P. mexicanus and P. lindsayi (Fig. 4). Four hap- Demography lotype groups can be distinguished: one corresponding to A neutrality test applied to the data showed no significant P. kadiakensis (Kadiakensis-group) located in the Bravo deviation from neutrality for most of the haplotype groups River; another corresponding to P. suttkusi (Suttkusi-group), except for the Churince-group, which showed significant which includes the population from the type locality; a third values for Fu’s F (−3.084, P<0.05) and R2 (0.108, 488 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 4, 2014

Fig. 4. Haplotype network inferred from populations of Palaemon and its corresponding geographical location. A, northeastern Mexico; B, the Cuatro Cienegas Valley.

P<0.05) tests (Table 2). Despite this, the other tests 1997; Ramos-Onsins and Rozas, 2002). The Churince-group did not show signiﬁcant deviations from neutrality, although was also the only one for which a unimodal mismatch Tajima’s D showed negative values for the Churince (D = distribution was found (data not shown), which is commonly −1.73, 0.10 >P >0.05) and Suttkusi (D =−0.96, 0.10 > inferred for populations that have undergone expansion P>0.05) groups. The two tests show signiﬁcant effects (Rogers and Harpending, 1992a; Harpending, 1994). Given for the Churince-population, which could be interpreted as the mutation rates considered, the resulting value of π = deviations from neutrality due to population expansion (Fu, 0.24 for the Churince-group suggests an expansion between ÁLVAREZ ET AL.: GENETICS OF PALAEMON FROM MÉXICO 489

Fig. 5. Bayesian Skyline Plot of haplotype groups inside the CCV, showing effective population size over time under neutral evolution.

Table 3. Mitochondrial COI genetic diversity and neutrality indexes to different geographical areas inhabited by populations of Palaemon in México. ∗ Signiﬁcant values (P<0.05), indicative of deviations from neutrality. RCC, rare haplotype group in Cuatro Ciénegas; n, number of individuals; h, number of haplotypes; S, number of polymorphic sites; Hd, haplotype diversity; k, average number of polymorphic sites; π, nucleotide diversity.

2 Region/group nhS Hd kπRFu’s F RCC 93 21 61 0.827 (0.000) 19.295 0.0328 0.157 8.093 Churince 25 12 3 0.23 (0.012) 0.24 0.0045 0.108∗ −3.084∗ Suttkusi 25 18 52 0.94 (0.001) 10.747 0.018 0.106 −3.408 Kadiakensis 7 4 3 0.857 (0.010) 1.524 0.0025 0.254 −0.655 P. mexicanus 2 1 7 1.000 (0.000) 0 0 – – P. lindsayi 2 2 7 0.845 (0.12) 7 0.0414 – –

∗ Table 4. Pairwise st comparisons between groups of populations of Palaemon (bottom-left). Statistically signiﬁcant after sequential Bonferroni correction (P = 0.0005). K2P between-group mean distances of COI are given (top-right).

Churince RCC Kadiakensis P. lindsayi P. mexicanus Suttkusi Churince – 0.046 0.111 0.096 0.048 0.044 RCC 0.14824∗ –0.112 0.097 0.064 0.071 Kadiakensis 0.12755∗ 0.16204∗ –0.145 0.121 0.134 P. lindsayi 0.08852 0.13529 0.10283 – 0.106 0.115 P. mexicanus 0.2904 0.31862∗ 0.35052 0.5–0.069 Suttkusi 0.08833∗ 0.09946∗ 0.09508 0.04495 0.2527∗ – 490 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 4, 2014

22,697 and 14,491 years before present (YBP) for the 1.66% basin in northern central México has been found to bear dif- and 2.6% divergence rates, and about 26,911 YBP for the ferent lineages of fauna in other freshwater groups, as cray- average rate of 1.4%. fish (Hobbs, 1984) and a number of fish groups (Ornelas- The effective population sizes and demographic trends es- García et al., 2008; Schönhuth et al., 2008, 2012), which timated from the BSP analysis similarly showed no apparent has been interpreted as a consequence of a long and com- drastic movements in all haplotype groups (Fig. 5). Certain plex biogeographic history with its neighbouring basins. The increase is shown however by the Churince- and Suttkusi- monophyletic clade grouping P. mexicanus and the CCV- groups, where slightly higher effective population numbers populations would correspond to one freshwater adaptation are observed. and its distribution is consistent with the biogeography if the region. Strong affinities between some parts of the Bravo and DISCUSSION Pánuco basins have been argued for other freshwater organisms (Miller and Smith, 1986; Schönhuth et al., 2008), and Taxonomic Implications this has been used to argue in favour of past connections The phylogenetic analyses showed an unsupported assem- between both regions. Our dates from divergence analysis blage that contained all species from Mexico included in show a time for MRCA of the populations now found in this study. Individually, most of the previously recognized the CCV-Salado River and Pánuco basins around 5.17 Mya, species (P. kadiakensis, P. mexicanus, P. lindsayi) were re- and are partially congruent with data which date the split be- covered as monophyletic clades, thus confirming their valid- tween freshwater fauna from Pánuco and CCV-Salado basins ity. Our phylogenetic results confirm the strong distinction (Concheiro Pérez et al., 2007; Hulsey et al., 2010; Schönhuth found in previous works between P. suttkusi (which was de- et al., 2012; De la Maza-Benignos et al., 2014). A posterior scribed for the Río Salado basin) and populations from CCV event would separate the lineage leading to the P. suttkusi- (Chaves-Campos et al., 2011a). The finding in this work of clade. This could correspond to a separation of the CCV- a distinct lineage (Churince) and the location of P. suttkusi Salado basins from the rest of the basins of the northern part in phylogenetic analyses allow new taxonomic insights for of the Central Plateau around 4.1 Mya (2.6-5.9 Mya), which CCV.The two endemic clades from Churince and RCC show is close to dates estimated for the splitting of the endemic as high COI K2P distances as those commonly observed fish Herychthys minckley, dated around 5 Mya (Hulsey et between crustacean species (>4%, see Table 4) (Breinholt al., 2010). et al., 2009). Its possible consideration as an undescribed Phylogenetic structure shows two mostly endemic lin- species will depend upon the recognition of additional ev- eages to the CCV, corresponding to the RCC- and Churince- idence based on morphological characters, and will be the clades. According to our phylogenetic and dating analyses, subject of future work. these lineages could correspond to one cladogenetic event in CCV whose dates (3.2 Mya, 1.9-4.7 Mya), are inside the Phylogeny and Biogeography dates range shown by proposed cladogenesis in other fresh- The group containing the species of Palaemon occurring in water endemics such as the snail Mexispyrgus churinceanus Mexico was only weakly supported (Fig. 2). This is con- (Johnson, 2005). Together, these two lineages would encom- sistent with results from other studies that showed the pa- pass other findings of at least two genetic groups roughly raphyletic nature of the genus, as several species were re- identified with the west and east sides of the Sierra de covered as sister taxa of distinct palaemonid genera in a San Marcos for different faunal groups, e.g., several snails, world-wide scale (Ashelby et al., 2012). Evidence obtained fish and aquatic turtles (Johnson, 2005; Carson and Dowl- here supports that the group formed by the species of Palae- ing, 2006; Chaves-Campos et al., 2011b; McGaugh, 2012; mon in México is likely the result of several independent Coghill et al., 2013). The Churince-lineage could be iden- events of freshwater adaptation from marine/estuarine an- tified with the west component of other organisms, and its cestors, which has been proposed as the plesiomorphic form distribution partially match the pattern of connection be- of life (Ashelby et al., 2012). These results contrast with tween the north-west (Antiojo) and the southwest (Churince) suggestions made by Strenth (1976), who considered that a through the Garabatal River, and confirms previous propos- series of shared larval characters and the presence of large als of inter-basin relationships (Minckley, 1969; Carson and eggs in the North American members of Palaemon were Dowling, 2006; McGaugh, 2012). evidence of a common origin when compared to its ma- Closure of the CCV has been proposed to be periodically rine/estuarine species. There are two most basal clades of affected by desiccation, uplift of anticlinal structures, and this group, which correspond to P. kadiakensis and P. lind- by accompanying warping of the plateau in general (Kellum sayi, which is coherent with a hypothesis of origin for the et al., 1936). Such phenomena could have affected the Mexican species linked to ancestral groups occupying ma- isolation and cladogenesis in repeated times in the CCV rine or estuarine ranges at south-eastern US and northern and surrounding regions. The proposed changes in rainfall Mexico, at the northern part of the Central plateau and the regime associated to climatic oscillation in the CCV to some gulf drainages. Specifically, the finding of P. lindsayi, which degree, could easily rise or diminish surface water levels inhabits the Media Luna Lagoon in the Pánuco basin, as a connecting or isolating parts of the basin and potentially different lineage to the one including the other species from providing enough time to affect cladogenesis in low water the same basin (P. mexicanus) and the closer relationship of level periods (Minckley, 1969), and could be related to the latter to the CCV populations of Palaemon would cor- cladogenesis of the endemic lineages observed today in respond to an independent freshwater invasion. The Pánuco CCV. In this way, P. suttkusi, RCC- and Churince-clades ÁLVAREZ ET AL.: GENETICS OF PALAEMON FROM MÉXICO 491 would be secondarily joined in recent times, probably by previous findings with populations of Palaemon within and the effect of human-built channels, as has been observed outside CCV, even in such short periods of time, in which in other studies (Johnson, 2005). It is possible, as seen case P. suttkusi would be modifying substantially the genetic in other studies, that channels have mediated the contact landscape of the endemic genomes. The time period com- between otherwise-separated gene pools (Chaves-Campos prised between the field sampling conducted for the Chaves- et al., 2011a). This can be the effect behind the finding Campos et al. (2011) study in 2009 and our fieldwork done of sample of TB1 (from Tierra Blanca, CCV, see Fig. 2), from 2010 to 2012, roughly three years, was sufficient to possessing a mitochondrial haplotype of P. kadiakensis,but change the haplotypic landscape of populations of Palae- nuclear information of the RCC-clade. This is most probably mon inside the CCV. A striking result in the present study due to introgression of the mitochondrial genome of P. is the finding of the Churince-haplotype-group, which have kadiakensis into that of RCC, and could be a more extended not been found previously (Chaves-Campos et al., 2011a). process going on in CCV. Even when hybridization has been This could be related to our more extensive sampling effort recorded for several organisms, to our knowledge, this is in the western portion of the basin. The Suttkusi-group, con- the first time that contact between populations involving taining P. suttkusi from the type locality, is widespread out- CCV-species is recorded for such long distances (Carson side the CCV as it would be expected, but it is also widely and Dowling, 2006). The effect of the human-mediated distributed inside it, especially in the eastern part. All this channelization in shaping the current genetic landscape evidence suggests that a high rate of haplotype turnover is in the CCV where these lineages co-occur today needs occurring in this region, probably related to introgression of to be re-evaluated in future studies using evidence from suitable genetic markers for these time scales. Another P. suttkusi into endemic haplotype groups. In addition, the possible event of introgression would the one behind the finding aforementioned of one individual with a P. kadiak- discovery of one individual from the Bravo River showing a ensis haplotype in the CCV and another one of P. suttkusi in P. suttkusi-haplotype (sample MA2). These examples show the Bravo River is interpreted by us as additional evidence the importance of including a wide phylogenetic sampling in supporting a high degree of interconnection between distant future works aiming to interpret genetic dynamics of species populations in the whole Bravo River basin. inhabiting CCV, especially with freshwater shrimp. The evidence of a sudden expansion of the Churince- haplotype-group around 14,491-26,911 YBP could be the Phylogeography and Demography in the CCV result of a climatic process in which this part of the basin Even when closely related in a phylogenetic context, phy- could have experienced a rising in water levels. This could logeography of populations of the CCV show a high degree have favoured conditions for the observed expansion in of genetic differentiation between the haplotype groups in- the western part of the CCV. It is reasonable to think habiting this region, as seen from the significant st val- that the presence of P. suttkusi haplotypes in the CCV ues (see Table 3). In addition, relative abundance of hap- corresponds to a more recent event, as the CCV and Salado lotypes found in this study is considerably different from River basins were barely connected in the past centuries, that found by Chaves-Campos et al. (2011). The most abun- until channelization started at the end of the 19th century dant haplotype group obtained in the aforementioned study to transport water outside of the CCV (Minckley, 1969). was the one assigned here to RCC, while we just found one Under this scenario, interconnection between the previous individual assigned to this group (PA1, see Figs. 2 and 4). haplotype groups from CCV (Churince and RCC) would In contrast, the most abundant haplotype groups found by have been seriously affected due to the recent contact with us corresponded to the Suttkusi- and Churince-groups, even the Suttkusi-group. when three populations are shared between the present study In this way, the biogeographic history of the populations and the one by Chaves-Campos et al. (2011a). A possible of Palaemon in the Bravo River basin is complex and reflects explanation for this change in haplotype frequencies is a previous splitting events, driven by long term climatic high rate of haplotype turnover, a pattern commonly seen changes, which would have affected the three lineages in situations where a high degree of geographic interconnec- observed today, but more recent events of interconnection tion is present (Fitzpatrick et al., 2012). Also, future works between the previously isolated areas could be mixing up could assess the extent to which introgression of P. sutkussi this structure. The role of recent connections among the into endemic CCV-genomes is happening, which is evident freshwater bodies in the region will have to be evaluated in when examining the nuclear genealogy (Fig. 2). This process the near future, considering that natural processes are also could also explain the scarce occurrence of the RCC-group in the sampling for the present work, and the relatively high acting upon the hydrology of the region. frequency of P. suttkusi mitochondrial haplotypes. While for some organisms it has not been important (McGaugh, 2012), ACKNOWLEDGEMENTS high rates of introgression between genomes of different species have been shown to determine genetic structure of We thank the WWF-FCS (Fundación Carlos Slim) Alliance for their other species in the CCV, as a result of a complex history generous support of the field work in the Cuatro Ciénegas region, and Dr. of hydrologic dynamics (Carson and Dowling, 2006). Con- Valeria Souza for inviting us to participate in this important conservation siderable introgression has been observed between popula- effort. We are also grateful to Dr. Frederick Schram and two anonymous reviewers for very fruitful suggestions made to previous versions of tions inhabiting the CCV in other freshwater species (Car- this manuscript. F.A. gratefully acknowledges the support from DGAPA- son and Dowling, 2006) and could also be the case for the PAPIIT UNAM grant IN214910. This work was completed as a part of a discrepancy in haplotype frequencies between our study and postdoctoral fellowship granted to CPL by CSIC-UNAM. 492 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 34, NO. 4, 2014

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